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NKX2-1/TITF1/TTF-1-Induced ROR1 is required to sustain EGFR survival signaling in lung adenocarcinoma
Abstract
We and others previously identified NKX2-1, also known as TITF1 and TTF-1, as a lineage-survival oncogene in lung adenocarcinomas. Here we show that NKX2-1 induces the expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn sustains a favorable balance between prosurvival PI3K-AKT and pro-apoptotic p38 signaling, in part through ROR1 kinase-dependent c-Src activation, as well as kinase activity-independent sustainment of the EGFR-ERBB3 association, ERBB3 phosphorylation, and consequential PI3K activation. Notably, ROR1 knockdown effectively inhibited lung adenocarcinoma cell lines, irrespective of their EGFR status, including those with resistance to the EGFR tyrosine kinase inhibitor gefitinib. Our findings thus identify ROR1 as an "Achilles' heel" in lung adenocarcinoma, warranting future development of therapeutic strategies for this devastating cancer.
... NKX2-1, a lineage-specific transcription factor, has been noted to be essential for the development of peripheral parts of the lung and in morphogenesis, and it is overexpressed in lung adenocarcinomas [24,76,77]. NKX2-1 induced ROR1 transcription and was critically involved in the maintenance of a balance between the pro-apoptotic p38 pathway and the pro-survival PI3K/AKT signaling pathway [16]. Furthermore, activated ROR1 binds to SRC and activates it. ...
... An association between EGFR mutations and NKX2-1 expression has been described in lung adenocarcinoma [78]. ROR1 has also been shown to form heterodimers with EGFR, promoting the maintenance of lung cancer cell survival [16,79]. High ROR1 expression in EGFR T790M NSCLC patients was related to an inferior progression-free survival in erlotinib-treated patients compared to those with low ROR1 expression, substantiating a role of ROR1 in the disease pathobiology and supporting that the inhibition of ROR1 might add to the therapeutic effect [79]. ...
... High ROR1 expression in EGFR T790M NSCLC patients was related to an inferior progression-free survival in erlotinib-treated patients compared to those with low ROR1 expression, substantiating a role of ROR1 in the disease pathobiology and supporting that the inhibition of ROR1 might add to the therapeutic effect [79]. Overall, the data indicate a role of ROR1 expression in lung adenocarcinoma, which may be independent of ROR1 kinase activity [16]. ...
The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 (p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1+ group than in the p53 positive non-squamous NSCLC patients (p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1+ NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.
... Since its identification in 1992, a pro-tumourigenic role for ROR1 signalling has been established in a growing list of both haematological malignancies (including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and acute lymphoblastic leukemia (ALL)), and solid tumours (including ovarian, endometrial, lung and chemotherapyresistant breast cancer [4][5][6][7][8][9][10]. Several therapeutic strategies targeting ROR1 have reached Phase I/II clinical trials, which include a monoclonal antibody (zilovertamab), antibodydrug conjugates (ADC; NBE-002 and VLS-101), chimeric antigen receptor (CAR) T cell therapy and bispecific antibody (BiTE) to ROR1 and CD3 (NVG-111), for an array of malignancies [11]. ...
... This is based on the bioinformatic prediction of the principal variant of ROR1 as a cell surface receptor as well as studies that have demonstrated cell surface expression of ROR1. However, numerous studies have consistently demonstrated cytoplasmic rather than cell surface expression of ROR1 by immunohistochemistry on both frozen and formalin-fixed, paraffin-embedded tumour samples [8,15,[17][18][19][20][21]. This was confirmed by Balakrishnan et al., who developed a monoclonal antibody with careful consideration to specifically address ambiguities in previous studies [22]. ...
... ROR1-v3 lacks an SP that ROR1-v1 contains for cell surface localisation ( Figure 1B and Table 2). This small difference could have profound implications: the protein product of ROR1-v3 may be expressed within the cell and is a possible explanation for numerous studies employing different antibodies reporting cytoplasmic staining for ROR1 [8,15,[17][18][19][20]. ROR1-v2 was presumed to be responsible for the cytoplasmic localisation [22]; however, our analysis demonstrates that this variant also has an SP for membrane expression ( Figure 1B and Table 2). ...
ROR1/2 are putative druggable targets increasing in significance in translational oncology. Expression of ROR1/2 mRNA and transcript variants has not been systematically examined thus far. ROR1/2 transcript variant sequences, signal peptides for cell surface localisation, and mRNA and transcript variant expression were examined in 34 transcriptomic datasets including 33 cancer types and 54 non-diseased human tissues. ROR1/2 have four and eight transcript variants, respectively. ROR1/2 mRNA and transcript variant expression was detected in various non-diseased tissues. Our analysis identifies predominant expression of ROR1 transcript variant ENST00000545203, which lacks a signal peptide for cell surface localisation, rather than the predicted principal variant ENST00000371079. ENST00000375708 is the predominantly expressed transcript variant of ROR2. ROR1/2 expression in healthy human tissues should be carefully considered for safety assessment of targeted therapy. Studies exploring the function and significance of the predominantly expressed ROR1 transcript variant ENST00000545203 are warranted.
... Since its identification in 1992, a pro-tumourigenic role for ROR1 signalling has been established in a growing list of both haematological malignancies (including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and (1;19) acute lymphoblastic leukemia (ALL)), and solid tumours (including ovarian, lung and chemotherapy resistant breast cancer [3][4][5][6][7][8]. A number of therapeutic strategies targeting ROR1 have reached Phase I/II clinical trials, which include a monoclonal antibody (Cirmtuzumab), antibody drug conjugates (ADC; NBE-002 and VLS-101), chimeric antigen receptor (CAR) T cell therapy and bispecific antibody (BiTE) to ROR1 and CD3 (NVG-111), for an array of malignancies [9]. ...
... This is based on bioinformatic prediction of the principal variant of ROR1 as a cell surface receptor as well as studies that have demonstrated cell surface expression of ROR1. However, numerous studies have consistently demonstrated cytoplasmic rather than cell surface expression of ROR1 by immunohistochemistry on both frozen and formalin-fixed, paraffin-embedded tumour samples [7,10,[12][13][14][15]. This was confirmed by Balakrishnan et al., who developed a monoclonal antibody with careful consideration to specifically address ambiguities in previous studies [16]. ...
... ROR1-v3 lacks a SP that ROR1-v1 contains for cell surface localisation ( Fig. 1B and Table 2). This small difference could have profound implications: the protein product of ROR1-v3 may be expressed within the cell and is a possible explanation for numerous studies employing different antibodies reporting cytoplasmic staining for ROR1 [7,10,[12][13][14][15]. ROR1-v2 was presumed to be responsible for the cytoplasmic localisation [16]; however, our analysis demonstrates that this variant also has a SP for membrane expression (Fig. 1B and Table 2). ...
Background
ROR1/2 are putative druggable targets increasing in significance in translational oncology. Expression of ROR1/2 mRNA and transcript variants has not been systematically examined thus far.
Methods
ROR1/2 transcript variant sequences, signal peptides for cell surface localisation, and mRNA and transcript variant expression were examined in 34 transcriptomic datasets including 33 cancer types and 54 non-diseased human tissues.
Results
ROR1/2 have four and eight transcript variants respectively. ROR1/2 mRNA and transcript variant expression was detected in various non-diseased tissues. Our analysis identifies predominant expression of ROR1 transcript variant ENST00000545203, which lacks a signal peptide for cell surface localisation, rather than the predicted principal variant ENST00000371079. ENST00000375708 is the predominantly expressed transcript variant of ROR2.
Conclusion
ROR1/2 expression in healthy human tissues should be carefully considered for safety assessment of targeted therapy. Studies exploring the function and significance of the predominantly expressed ROR1 transcript variant ENST00000545203 are warranted.
... Induced ROR1 expression in the ROR1 negative MSTO-211H lung adenocarcinoma cell line increased cell growth in xenografts similar to that of the ROR1 positive NCI-H1975 lung cancer cell line. The data suggest that ROR1 kinase activity was required to fully confer growth advantage (Yamaguchi et al. 2012). However, kinase activity of ROR1 may differ between cell lines, normal cells, and tumor cells but ROR1 expression was important for survival and migration of tumor cells ). ...
... Gene profiling of CLL cells revealed a 45-fold increased expression of ROR1 as compared to normal mature B-cells (Rosenwald et al. 2001;Klein et al. 2001). ROR1 expression was also noted in diffuse large B-cell (DLBCL) (Ghaderi et al. 2020), mantle cell lymphoma (MCL) (Mellstedt et al. 2019), Hodgkin lymphoma (Velentza et al. 2018), renal cell carcinoma (Rabbani et al. 2010), acute lymphocytic leukemia (ALL) (Shabani et al. 2007(Shabani et al. , 2008, lung adenocarcinoma (Yamaguchi et al. 2012), breast cancer (Zhang et al. 2012a), neuroblastoma (Dave et al. 2019), glioma (Chai et al. 2020), melanoma (Hojjat-Farsangi et al. 2013a), gastric (Chang et al. 2015), and several other malignancies (Zhang et al. 2012a;Hojjat-Farsangi et al. 2013a, b;Daneshmanesh et al. 2008;Shabani et al. 2007Shabani et al. , 2011O'Connell et al. 2013). ROR1 phosphorylation was also noted in melanoma cell lines (Hojjat-Farsangi et al. 2013a) and specific anti-ROR1 mAbs induced apoptosis (Hojjat-Farsangi et al. 2013a). ...
... Other kinases may also be involved (Yamaguchi et al. 2012;Cui et al. 2013a). ROR1 expression has also been studied in ALL as well as the proliferative ability of ROR1 positive cells in the bone marrow of B-and T-ALL (Diamanti et al. 2019). ...
Receptor tyrosine kinases (RTKs) are frequently dysregulated in malignancies and important for the malignant characteristics of tumor cells. RTKs are attractive structures for drug targeting of cancer. The RTK ROR1 is of significance during embryogenesis but downregulated in post-partum tissues. However, ROR1 is overexpressed in several hematological and solid tumors and important for tumor cell proliferation, survival, migration, and metastasis. WNT5a is a main ligand for ROR1. Several clinical trials are ongoing using anti-ROR1 antibody based drugs directed against the external domain (monoclonal antibodies, BiTE, CAR-T). We have produced small molecules (KAN834/1571c) fitting to the ATP pocket of the intracellular tyrosine kinase (TK) domain of ROR1 (TK inhibitor, TKI). These inhibitors of ROR1 prevented ROR1 phosphorylation and inactivated the WNT/β-catenin independent as well as WNT/β-catenin dependent pathways. ROR1-TKI induced apoptosis of ROR1 positive fresh patient derived tumor cells and appropriate cell lines and a dose and time dependent tumor reduction in animal models. In combination with other clinically relevant targeting drugs as venetoclax a synergistic apoptotic effect was seen. Two other small molecules (ARI-1 and strictinin) bound also to ROR1 and inhibited tumor growth. Development of small molecule ROR1 inhibitors is warranted to include this novel therapeutic approach for cancer therapy.
... ROR1 was also found at high levels in lung cancer cells, which serves as a prognostic biomarker in patients with lung adenocarcinoma. Silencing of ROR1 led to growth inhibition in cell lines representing human lung adenocarcinoma (44,45). In ovarian cancer, tumor cells with high expression of ROR1 exhibited stem cell-like gene-expression signatures and had a greater capacity to engraft immunodeficient mice (46). ...
... Expression levels of ROR1 are tightly correlated with the progression of CLL, making it a good biomarker for prognosis (32). In lung cancer, ROR1 is involved in the activation of c-Src and MET, causing inhibition of tumor cell apoptosis (44,52). However, these studies did not define whether ROR1 actively phosphorylated c-Src and MET or merely served as their substrate. ...
... The group led by Dr. Takashi Takahashi investigated the role of ROR1 in lung adenocarcinoma in a series of studies and found that ROR1 supported pro-survival signaling in both kinase-dependent and kinase-independent manner. The kinase activity of ROR1 was shown to be required for the regulation of HIF-1a expression (65), repression of the ASK1-p38 axis, oxidative stress-induced cell death (66), and phosphorylation of c-Src (44). On the other hand, ROR1 can serve as a scaffold protein in a kinase-independent manner to facilitate cavin-1/ caveolin-1 interaction (53, 67) and binding to HSP90a (67). ...
Receptor tyrosine kinase ROR1 plays an essential role in embryogenesis and is overexpressed in many types of malignant tumors. Studies have demonstrated that it plays an important role in oncogenesis by activating cell survival signaling events, particularly the non-canonical WNT signaling pathway. Antibody-based immunotherapies targeting ROR1 have been developed and evaluated in preclinical and clinical studies with promising outcomes. However, small molecule inhibitors targeting ROR1 are underappreciated because of the initial characterization of ROR1 as a peusdokinase. The function of ROR1 as a tyrosine kinase remains poorly understood, although accumulating evidence have demonstrated its intrinsic tyrosine kinase activity. In this review, we analyzed the structural and functional features of ROR1 and discussed therapeutic strategies targeting this kinase.
... In the absence of catalytic activity, ROR1 and ROR2 can interact with other RTKs such as MET, epidermal growthfactor receptor (EGFR), and muscle-specific kinase (MuSK) or intracellular kinases such as Src and casein kinase CK1ε and become trans-phosphorylated [35,[59][60][61][62]. The majority of signaling events taking place at the receptor level were shown to be mediated by the Ser/Thr and Pro-rich regions of ROR1/2 that play a scaffolding role for downstream effectors [34][35][36][37]. ...
... Rights reserved. signaling, as it is known that both receptors can undergo trans-phosphorylation [35,[59][60][61][62]. ...
ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.
... It induces EMT and metastasis in several cancer types, including GC (Ran et al., 2018;Gu and Hu, 2019). ROR1 is a transcriptional target of NKX2-1, which maintains a sustained balance between pro-survival and pro-apoptotic signaling by inducing ROR1 expression (Yamaguchi et al., 2012;Borcherding et al., 2014). However, the role of ROR1, ROR2, and their associated molecules Wnt5A, NKX2-1, and FOXF1 have not fully been explored in GC. ...
... miR-27b-3p inhibits GC cell proliferation, colony formation in soft agar, and xenograft tumor formation (Tao et al., 2015). Furthermore, ROR1 has been identified as a transcriptional target of homeobox protein NKX-2, which is required for sustained EGFR signaling in lung cancer patients and serves as an independent prognostic predictor of OS (Yamaguchi et al., 2012;Menck et al., 2021). Indeed, in our study, NKX2-1 showed a high prognostic value in GC patients, correlating with ROR1 expression. ...
Background:
Gastric cancer (GC) is diagnosed at advanced stages and has high mortality rates. Surgical resection and adjuvant chemotherapy are the main therapeutic approaches for GC. Despite curative resection, recurrence and metastasis contribute to a high mortality rate in patients with GC. The receptor-tyrosine-kinase-like orphan receptors 1/2 (ROR1/2) are transmembrane proteins belonging to the receptor tyrosine kinase (RTK) family. ROR1 and ROR2 are known to overexpress in the tumor tissues from several types of cancer patients. However, the role of RORs in the prognosis has not been understood.
Methods:
This study aimed to determine the association of mRNA expression of ROR1, ROR2, and their signaling components WNT5A, NKX2-1, and FOXF1, with the survival outcome of GC patients. We performed Kaplan-Meir survival analysis on publicly available 'The Cancer Genome Atlas (TCGA)' data sets using 'Kaplan-Meir Plotter.'
Results:
High mRNA expression of ROR1, ROR2, NKX2-1, and FOXF1 was significantly correlated with worse overall survival (OS) of GC patients. Interestingly ROR1 and ROR showed a prognostic role in the intestinal subtype, but not in the diffuse subtype of GC. Furthermore, ROR1 was positively correlated with regulatory T cells and M2-type macrophages and negatively correlated with Th17 and natural killer T cells in the tumor stroma of patients with GC.
Conclusion:
We conclude that the expression of ROR1, ROR2, and their associated genes correlate with worst prognosis of GC patients, particularly in the intestinal type.
.
... Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a receptor tyrosine kinase-like protein that has been implicated to play a role in many different types of cancer [3][4][5][6][7][8] . ROR1 has oncofetal expression, as it is an embryonic protein that is not normally expressed in differentiated cells, but can be re-expressed in cancer cells 9 . ...
... Thus, ROR1-targeted therapy could be tumor specific, and a recent study has demonstrated that ROR1 expression is correlated with worsened patient outcomes in lung adenocarcinomas 5 . ROR1 inhibition has been shown to lead to cell death in pancreatic, leukemia, and lung cancer cells, suggesting that it is a viable therapeutic target 4,7,[10][11][12][13] . ...
Small cell lung cancer (SCLC) remains a deadly form of cancer, with a 5-year survival rate of less than 10 percent, necessitating novel therapies. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein that is emerging as a therapeutic target and is co-expressed with BCL2 in multiple tumor types due to microRNA coregulation. We hypothesize that ROR1-targeted therapy is effective in small cell lung cancer and synergizes with therapeutic BCL2 inhibition. Tissue microarrays (TMAs) and formalin-fixed paraffin-embedded (FFPE) SCLC patient samples were utilized to determine the prevalence of ROR1 and BCL2 expression in SCLC. Eight SCLC-derived cell lines were used to determine the antitumor activity of a small molecule ROR1 inhibitor (KAN0441571C) alone and in combination with the BCL2 inhibitor venetoclax. The Chou-Talalay method was utilized to determine synergy with the drug combination. ROR1 and BCL2 protein expression was identified in 93% (52/56) and 86% (48/56) of SCLC patient samples, respectively. Similarly, ROR1 and BCL2 were shown by qRT-PCR to have elevated expression in 79% (22/28) and 100% (28/28) of SCLC patient samples, respectively. KAN0441571C displayed efficacy in 8 SCLC cell lines, with an IC50 of 500 nM or less. Synergy as defined by a combination index of <1 via the Chou-Talalay method between KAN0441571C and venetoclax was demonstrated in 8 SCLC cell lines. We have shown that ROR1 inhibition is synergistic with BCL2 inhibition in SCLC models and shows promise as a novel therapeutic target in SCLC.
... In vitro experiments found that NKX2-1 transactivates the receptor tyrosine kinase-like orphan receptor 1 (ROR1), which in turn promotes EGFR-induced ERBB3-dependent activation of the PI3K pathway (46). ERBB3 also regulates the expression of PD-L1 through the activation of the PI3K/PDK1/RSK/CREB signaling axis (47). ...
... ROR1 is a key carcinoembryonic glycoprotein that maintains the balance of pro-survival and pro-apoptotic signaling in lung adenocarcinoma [158,159]. Wallstabe et al. [160] developed a microphysiological three-dimensional (3D) model of lung and breast cancer that provides better characterization of the solid tumours that are encountered by CAR-T cells. The system was used to evaluate the anti-tumour effect of ROR1CAR-T cells composed of a ROR-1-specific single-chain antibody, an IgG4-Fc-derived hinge, the CD28TM domain, and 4-1BB-CD3ζ or CD28-CD3ζ signaling modules [160]. ...
Immune microenvironment and immunotherapy have become the focus and frontier of tumor research, and the immune checkpoint inhibitors has provided novel strategies for tumor treatment. Malignant pleural effusion (MPE) is a common end-stage manifestation of lung cancer, malignant pleural mesothelioma and other thoracic malignancies, which is invasive and often accompanied by poor prognosis, affecting the quality of life of affected patients. Currently, clinical therapy for MPE is limited to pleural puncture, pleural fixation, catheter drainage, and other palliative therapies. Immunization is a new direction for rehabilitation and treatment of MPE. The effusion caused by cancer cells establishes its own immune microenvironment during its formation. Immune cells, cytokines, signal pathways of microenvironment affect the MPE progress and prognosis of patients. The interaction between them have been proved. The relevant studies were obtained through a systematic search of PubMed database according to keywords search method. Then through screening and sorting and reading full-text, 300 literatures were screened out. Exclude irrelevant and poor quality articles, 238 literatures were cited in the references. In this study, the mechanism of immune microenvironment affecting malignant pleural effusion was discussed from the perspectives of adaptive immune cells, innate immune cells, cytokines and molecular targets. Meanwhile, this study focused on the clinical value of microenvironmental components in the immunotherapy and prognosis of malignant pleural effusion.
... Cellular differentiation, growth, and metastasis are all significantly influenced by receptor tyrosine kinases (RTKs) [36,37]. Many functional findings show that ROR1 is linked to noncanonical WNT-signaling to promote cancer cell survival, growth, and invasion, and ROR1 exerts cellular signaling in both kinasedependent and independent mechanisms [38]. ...
Breast cancer (BC) patient who receives chemotherapy for an extended length of time may experience profound repercussions in terms of metastases and clinical outcomes due to the involvement of the epithelial-to-mesenchymal transition (EMT) mechanism and enriched cancer stem cells (CSCs). BC cells that express high levels of lncRNA deleted in lymphocytic leukemia-2 (lncRNA DLEU2) and type I tyrosine kinase-like orphan receptor ROR1 (ROR1) may play roles in the enhanced ability of the activation EMT and CSC induction. Here we find that lncRNA DLEU2 and ROR1 are specifically upregulated in tumor tissues compared to their normal counterparts in TCGA, PubMed GEO datasets, and samples from archived breast cancer tumor tissues. Following chemotherapy, lncRNA DLEU2 and ROR1 were enhanced in BC tumor cells, coupled with the expression of CSCs, EMT-related genes, and BMI1. Mechanistically, ROR1 and lncRNA DLEU2 overexpression led to enhanced tumor cell proliferation, inhibition of apoptosis, cell-cycle dysregulation, chemoresistance, as well as BC cell’s abilities to invade, migrate, develop spheroids. These findings imply that the role of lncRNA DLEU2 and ROR1 in BC therapeutic failure is largely attributed to EMT, which is intricately linked to enriched CSCs. In conclusion, our findings indicate that a lncRNA DLEU2 and ROR1-based regulatory loop governs EMT and CSC self-renewal, implying that targeting this regulatory pathway may improve patients’ responses to chemotherapy and survival.
... Moreover, ROR1 has been shown to cancer cell survival and proliferation in various solid tumors. High expression of ROR1 was associated with EMT and tumor metastasis in breast cancer cells [16,19] and showed poor prognosis in lung adenocarcinoma [10]. Additionally, high ROR1 expression in ovarian cancer cells showed stem cell-like gene-expression profiles [21]. ...
Background and Objectives: Receptor tyrosine kinase-like orphan receptor type 1 (ROR1) plays a critical role in embryogenesis and is overexpressed in many malignant cells. These characteristics allow ROR1 to be a potential new target for cancer treatment. The aim of this study was to investigate the role of ROR1 through in vitro experiments in endometrial cancer cell lines. Materials and Methods: ROR1 expression was identified in endometrial cancer cell lines using Western blot and RT-qPCR. The effects of ROR1 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) markers were analyzed in two endometrial cancer cell lines (HEC-1 and SNU-539) using either ROR1 silencing or overexpression. Additionally, chemoresistance was examined by identifying MDR1 expression and IC50 level of paclitaxel. Results: The ROR1 protein and mRNA were highly expressed in SNU-539 and HEC-1 cells. High ROR1 expression resulted in a significant increase in cell proliferation, migration, and invasion. It also resulted in a change of EMT markers expression, a decrease in E-cadherin expression, and an increase in Snail expression. Moreover, cells with ROR1 overexpression had a higher IC50 of paclitaxel and significantly increased MDR1 expression. Conclusions: These in vitro experiments showed that ROR1 is responsible for EMT and chemoresistance in endometrial cancer cell lines. Targeting ROR1 can inhibit cancer metastasis and may be a potential treatment method for patients with endometrial cancer who exhibit chemoresistance.
... NKX2.1 is generally regarded as the master regulator during the fatal lung development stage. Since the lung specification of foregut, NKX2.1 is widely expressed in lung epithelial cells (Yamaguchi et al., 2012). Our results indicated that 25 μg/mL PM2.5 significantly downregulated NKX2.1 in AFE and LPOs stage (Figs. 2 and 4). ...
Exposure to air pollution has been proven to be associated with impaired fetal lung development. However, due to the lack of reliable human source models, it is still challenging to deeply understand the human fetal lung development under PM2.5 exposure. Here, we utilized human embryonic stem cell (hESC) line H9 to generate lung bud tip progenitor organoids (LPOs), a process that mimics early stages of fetal lung development including definitive endoderm (DE) formation, anterior foregut endoderm (AFE) differentiation and lung progenitor cell specification, to evaluate potential pulmonary developmental toxicity of PM2.5. We demonstrated that PM2.5 exposure the entire LPOs induction from hESCs significantly affected cellular proliferation of LPOs, and altered the expression of lung progenitor cell markers NKX2.1, SOX2 and SOX9, which are canonically defined subsequently proximal-distal airways specification. To explore the dynamic influences of PM2.5 exposure at different stages of LPOs specification, we also found that PM2.5 exposure significantly affected the expression of several transcriptional factors that are important for the differentiation of DE and AFE. Mechanistically, we suggested PM2.5-induced developmental toxicity to LPOs was partially linked with the Wnt/β-catenin signaling pathway. Therefore, our findings further emphasize the substantial health risks in the development of respiratory system associated with prenatal exposure to PM2.5.
... ROR1 suppresses melanoma cell invasion but increases epithelial-mesenchymal transition and breast cancer cell metastasis. ROR1 levels beyond a certain threshold were linked to pancreatic cell death (Yamaguchi et al., 2012). This disparity shows that ROR1 may have different functions in different forms of cancer, and its relevance in EC is currently unknown. ...
Objective:
To asses miR-379-5p expression in endometrial cancer (EC) and its correlation with ROR1 expression and to investigate the relation between miR-379-5p and ROR1 expressions and the clinicopathological picture of EC.
Methods:
Fifty female of EC were joined to this study. The gene expression of miR-379-5p (by quantitative real time-PCR) and ROR1 (by quantitative real time-PCR and immunohistochemistry) were studied in EC and normal nearby endometrial tissue.
Results:
The gene expression of miR-379-5p was significantly downregulated while that of ROR1 was significantly upregulated in EC tissues compared to adjacent normal endometrial tissues. Furthermore, miR-379 and ROR1 expressions significantly associated with tumor stage (P< 0.045), grade (P< 0.001), myometrial invasion (P <0.001) and LN metastasis (P< 0.034). In addition, miR-3795p and ROR1 gene expression were negatively correlated (r = -0.746, P < 0.001).
Conclusions:
In EC, miR-379-5p can be used as a diagnostic marker, and ROR1 could be a potential target of miR-379-5p.
... [18][19][20] These findings suggest that expression of Ror1 and subsequent Ror1-mediated signaling in GSCs might contribute to the malignant progression of glioblastomas. It has been reported that expression of Ror1 in lung adenocarcinoma cells is mediated by NKX2-1/TITF1/TTF-1, 21 while expression of Ror1 in chronic lymphocytic leukemia and gastric cancer cells can be mediated by STAT3. 22,23 However, the molecular mechanism regulating expression of Ror1 in glioblastomas is still unclear. ...
Ror1 plays a crucial role in cancer progression by regulating cell proliferation and migration. Ror1 is expressed abundantly in various types of cancer cells and cancer stem-like cells. However, the molecular mechanisms regulating expression of Ror1 in these cells remain largely unknown. Ror1 and its putative ligand Wnt5a are expressed highly in malignant gliomas, especially in glioblastomas, and the extents of Ror1 expression are correlated positively with poorer prognosis in patients with gliomas. We show that Ror1 expression can be up-regulated in glioblastoma cells under spheroid culture, but not adherent culture conditions. Notch and hypoxia signaling pathways have been shown to be activated in spheroid-forming glioblastoma stem-like cells (GSCs), and Ror1 expression in glioblastoma cells are indeed suppressed by inhibiting either Notch or hypoxia signaling. Meanwhile, either forced expression of Notch intracellular domain (NICD) in or hypoxic culture of glioblastoma cells result in enhanced expression of Ror1 in the cells. Consistently, we show that both NICD and HIF1α bind to upstream regions within the Ror1 gene more efficiently in GSCs under spheroid culture conditions. Furthermore, we provide evidence, indicating that binding of Wnt5a to Ror1, up-regulated by Notch and hypoxia signaling pathways in GSCs, might promote their spheroid-forming ability. Collectively, these findings indicate for the first time that Notch and hypoxia signaling pathways can elicit Wnt5a-Ror1 axis through transcriptional activation of Ror1 in glioblastoma cells, thereby promoting their stem cell-like property.
... Overexpression of ROR1 in patients was first described in chronic lymphocytic leukemia (CLL) by applying gene expression profiling [3]. ROR1 has also been shown to be overexpressed in several other hematological malignancies, including mantle cell lymphoma (MCL), as well as in solid tumors [4][5][6][7][8][9][10][11][12]. Small molecules and monoclonal antibodies have been used to target dysregulated RTKs in malignancies in a therapeutic approach [13,14]. ...
The receptor tyrosine kinase orphan receptor 1 (ROR1) is absent in most normal adult tissues but overexpressed in various malignancies and is of importance for tumor cell survival, proliferation, and metastasis. In this study, we evaluated the apoptotic effects of a novel small molecule inhibitor of ROR1 (KAN0441571C) as well as venetoclax (BCL-2 inhibitor), bendamustine, idelalisib (PI3Kδ inhibitor), everolimus (mTOR inhibitor), and ibrutinib (BTK inhibitor) alone or in combination in human MCL primary cells and cell lines. ROR1 expression was evaluated by flow cytometry and Western blot (WB). Cytotoxicity was analyzed by MTT and apoptosis by Annexin V/PI staining as well as signaling and apoptotic proteins (WB). ROR1 was expressed both in patient-derived MCL cells and human MCL cell lines. KAN0441571C alone induced significant time- and dose-dependent apoptosis of MCL cells. Apoptosis was accompanied by decreased expression of MCL-1 and BCL-2 and cleavage of PARP and caspase 3. ROR1 was dephosphorylated as well as ROR1-associated signaling pathway molecules, including the non-canonical WNT signaling pathway (PI3Kδ/AKT/mTOR). The combination of KAN0441571C and ibrutinib, venetoclax, idelalisib, everolimus, or bendamustine had a synergistic apoptotic effect and significantly prevented phosphorylation of ROR1-associated signaling molecules as compared to KAN0441571C alone. Our results suggest that targeting ROR1 by a small molecule inhibitor, KAN0441571C, should be further evaluated particularly in combination with other targeting drugs as a new therapeutic approach for MCL.
... TTF-1 induced receptor tyrosine kinase-like orphan receptor 1, which regulates the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, and activation of PI3K-AKT can promote PD-L1 expression. 44,45 On the contrary, TTF-1-negative lung adenocarcinoma cell lines overexpressed serglycin, which was reported to upregulate the PD-L1 expression. 46 Thus, the mechanism of the association between TTF-1 and PD-L1 is still unclear and requires further investigation. ...
Background:
We aimed to identify the relationship between thyroid transcription factor-1 (TTF-1) expression of lung adenocarcinoma and the efficacy of immune-checkpoint inhibitor (ICI) therapy.
Methods:
This retrospective multicenter study comprised patients with advanced lung adenocarcinoma treated with ICI monotherapy. We collected clinical medical records including data on TTF-1 expression and analyzed the relationship between TTF-1 expression and programmed death-ligand 1 tumor proportion score (PD-L1 TPS), objective response rate (ORR), progression-free survival (PFS), and overall survival (OS).
Results:
In total, 108 patients with lung adenocarcinoma were analyzed. The rate of TPS ≥1% and ≥50% in patients with positive TTF-1 expression was significantly higher than that in patients with negative TTF-1 expression (88% vs. 60%, p < 0.001; 65% vs. 24%, p < 0.001). The ORR was significantly higher in TTF-1 positive patients than in TTF-1-negative patients (38% vs. 8%, p = 0.003). Among patients with TPS ≥50% and 1%-49%, the ORR in TTF-1 positive and negative patients was 48% (26/54) versus 17% (1/6) (p = 0.21), and 32% (6/19) versus 11% (1/9) (p = 0.37), respectively. The ORR for patients with TPS <1% was 0% in both the TTF-1 negative and positive cases. The median PFS and OS was significantly longer in TTF-1-positive patients than in TTF-1-negative patients (5.4 vs. 1.6 months, p < 0.001; 18.2 vs. 8.0 months, p = 0.041). Multivariate analysis revealed that TTF-1-negative status was an independent unfavorable prognostic factor for PFS.
Conclusion:
Patients with TTF-1-positive status receiving ICI monotherapy showed better outcomes than those with TTF-1-negative lung adenocarcinoma.
... 46 NKX2-1can influence some anti-tumorigenic genes to make them become mutation and become lung cancer without lifestyle change. 47 Binding of NKX2-1 to varied gene locus are usually in downstream, proximal promotor (upstream) and intron which also induces lung adenocarcinomas. NKX2-1 can influence EGFR gene become mutation and stimulate lung tumorigenesis. ...
Introduction:
Rheumatoid arthritis is associated with various cancers. Many studies have investigated physical exercise interventions as health improvements to ameliorate the risk of cancer during rheumatoid arthritis diagnosis. Recently, microRNAs were used as biomarkers for health assessment and cancer prediction in rheumatoid arthritis patients.
Methods:
The effects of exercise interventions on serum microRNAs were investigated in pristane-induced arthritis (PIA) rat models. Twelve Sprague-Dawley male rats were divided into 4 groups including non-exercise without PIA (N-EX), non-exercise with PIA (N-EX + PIA), exercise without PIA (EX) and exercise with PIA (EX + PIA). Blood samples were collected at the end of the study period to analyze miRNA biomarkers and target cancer gene predictions.
Results:
Four significant Rattus norvegicus (rno-microRNAs) may purpose as tumor suppressors were identified as potential target cancer gene candidate expressions within the 4 comparative interventional exercise groups. One rno-microRNA and target cancer gene candidate was up-regulated and 3 rno-microRNAs and their target cancer genes were down-regulated.
Conclusions:
Exercise interventions affected rno-miRNAs regulated target cancer gene candidates ITPR3, SOCS6, ITGA6, and NKX2-1 as biomarkers for cancer prognosis in rheumatoid arthritis diagnosis.
... Amplification or overexpression of TITF1 (thyroid transcription factor 1) on chromosome 14q13.3 can enhance lung cancer cell proliferation via inducing the expression of the ROR1 (receptor tyrosine kinase-like orphan receptor 1) [29,30]. Overexpression of CCND1 (Cyclin D1) promoted NSCLC proliferation and progression through regulating the cell cycle [31]. ...
Background
Pharmacogenomics is crucial for individualized drug therapy and plays an increasingly vital role in precision medicine decision-making. However, pharmacogenomics-based molecular subtypes and their potential clinical significance remain primarily unexplored in lung adenocarcinoma (LUAD).
Methods
A total of 2065 samples were recruited from eight independent cohorts. Pharmacogenomics data were generated from the profiling of relative inhibition simultaneously in mixtures (PRISM) and the genomics of drug sensitivity in cancer (GDSC) databases. Multiple bioinformatics approaches were performed to identify pharmacogenomics-based subtypes and find subtype-specific properties.
Results
Three reproducible molecular subtypes were found, which were independent prognostic factors and highly associated with stage, survival status, and accepted molecular subtypes. Pharmacogenomics-based subtypes had distinct molecular characteristics: S-Ⅰ was inflammatory, proliferative, and immune-evasion; S-Ⅱ was proliferative and genetics-driven; S-III was metabolic and methylation-driven. Finally, our study provided subtype-guided personalized treatment strategies: Immune checkpoint blockers (ICBs), doxorubicin, tipifarnib, AZ628, and AZD6244 were for S-Ⅰ; Cisplatin, camptothecin, roscovitine, and A.443654 were for S-Ⅱ; Docetaxel, paclitaxel, vinorelbine, and BIBW2992 were for S-III.
Conclusion
We provided a novel molecular classification strategy and revealed three pharmacogenomics-based subtypes for LUAD patients, which uncovered potential subtype-related and patient-specific therapeutic strategies.
... Several kinases have been indicated to phosphorylate ROR receptors, leading to their activation. These include the glycogen synthase kinase 3 (GSK3), hepatocyte growth factor receptor (MET), proto-oncogene tyrosine protein kinase (SRC) and ROR itself (Akbarzadeh et al., 2008;Bainbridge et al., 2014;Gentile et al., 2011;Gentile et al., 2011;Gentile et al., 2014;Yamaguchi et al., 2012;Yamamoto et al., 2007). ...
Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signalling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled (DVL). During canonical (β-catenin dependent) signalling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its DIX domain, which are cross-linked by its DEP domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signalling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signalling. In the first chapter of this thesis work I used biophysical techniques, nuclear magnetic resonance spectroscopy (NMR) and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved serine phosphorylation site upstream of the DEP domain (S418 in human DVL2) that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. I also characterized the human DVL2 S435 conserved phosphorylation site located in the DEP hinge loop, a sensitive region for DEP domain swapping. Mutations of S418 and its cognate surface on the reciprocal DEP core or in S435 attenuate DEP-dependent dimerization of Dishevelled and its canonical signalling activity in cells, without impeding its binding to Frizzled. DVL2 S418 and S435 are crucial residues that could be employed to switch off canonical Wnt signalling. In the second chapter I focused on the study of Diversin. The protein Diversin plays a positive role in noncanonical Wnt signalling such as planar cell polarity and has been reported to play an inhibitory role on canonical Wnt signalling. Reports indicate that Dishevelled DEP domain and Diversin ANK domain interact and that this interaction is crucial for the role of Diversin on noncanonical outputs. Using cell-bases assays, I concluded that overexpression of Diversin inhibits canonical Wnt signalling, consistent with previous results. Moreover, I observed that Dishevelled and Diversin interact and that DEP and ANK are required for this interaction. However, I could not observe a direct interaction between monomeric DEP and ANK, suggesting that the interaction between Dishevelled and Diversin depends on extra regions, different protein conformation or that accessory factors are required for their interaction.
... ROR1 has been reported earlier to interact with the members of the Src kinase family (Yamaguchi et al., 2012;Gentile et al., 2014). In order to test if this holds true for ROR1 and Lyn, we overexpressed both proteins in HEK-293T cells and performed immunoprecipitation experiments. ...
Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies characterized by the dependence on B-cell receptor (BCR) signaling and by the high expression of ROR1, the cell surface receptor for Wnt-5a. Both, BCR and ROR1 are therapeutic targets in these diseases and the understanding of their mutual cross talk is thus of direct therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src family participating in BCR signaling, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 in its kinase domain and aids the recruitment of the E3 ligase c-CBL. We show that ROR1 surface dynamics in migrating primary CLL cells as well as chemotactic properties of CLL cells were inhibited by Lyn inhibitor dasatinib. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of crosstalk between BCR and ROR1 signaling pathways.
... Recently, receptor tyrosine kinase-like orphan receptor 1-specific (ROR1-specific) CAR T cells were also evaluated preclinically [19,20]. ROR1 protein is highly expressed in some hematologic and epithelial cancer cells, including lung, breast, colon, pancreas, renal, and ovarian cancers [21], and presumably plays a role in malignant transformation [22]. It has been demonstrated that ROR1 expression is limited in normal adult tissues, suggesting that immunotherapies targeting ROR1 could be feasible [23]. ...
Cancer immunotherapy is becoming more important in the clinical setting, especially for cancers resistant to conventional chemotherapy, including targeted therapy. Chimeric antigen receptor (CAR)-T cell therapy, which uses patient’s autologous T cells, combined with engineered T cell receptors, has shown remarkable results, with five US Food and Drug Administration (FDA) approvals to date. CAR-T cells have been very effective in hematologic malignancies, such as diffuse large B cell lymphoma (DLBCL), B cell acute lymphoblastic leukemia (B-ALL), and multiple myeloma (MM); however, its effectiveness in treating solid tumors has not been evaluated clearly. Therefore, many studies and clinical investigations are emerging to improve the CAR-T cell efficacy in solid tumors. The novel therapeutic approaches include modifying CARs in multiple ways or developing a combination therapy with immune checkpoint inhibitors and chemotherapies. In this review, we focus on the challenges and recent advancements in CAR-T cell therapy for solid tumors.
... ROR1 is a crucial oncofetal glycoprotein that can sustain prosurvival and pro-apoptotic signaling in lung adenocarcinomas (95,96). It has been proposed as a targeted antigen in CAR-T cell therapy as the overexpression of ROR1 protein has been observed in various malignancies, including lung cancer (97,98). ...
Chimeric antigen receptor T (CAR-T) cell therapy has exhibited a substantial clinical response in hematological malignancies, including B-cell leukemia, lymphoma, and multiple myeloma. Therefore, the feasibility of using CAR-T cells to treat solid tumors is actively evaluated. Currently, multiple basic research projects and clinical trials are being conducted to treat lung cancer with CAR-T cell therapy. Although numerous advances in CAR-T cell therapy have been made in hematological tumors, the technology still entails considerable challenges in treating lung cancer, such as on−target, of−tumor toxicity, paucity of tumor-specific antigen targets, T cell exhaustion in the tumor microenvironment, and low infiltration level of immune cells into solid tumor niches, which are even more complicated than their application in hematological tumors. Thus, progress in the scientific understanding of tumor immunology and improvements in the manufacture of cell products are advancing the clinical translation of these important cellular immunotherapies. This review focused on the latest research progress of CAR-T cell therapy in lung cancer treatment and for the first time, demonstrated the underlying challenges and future engineering strategies for the clinical application of CAR-T cell therapy against lung cancer.
... In terms of mechanism, studies have shown that TTF-1 can regulate the growth and metastasis of lung cancer cells through a variety of downstream target genes, including Selenbp1, EGFR, Foxa2, CDX2, and DDB1. [36][37][38][39][40]. Yamaguchi et al. [41] further identified ROR1 as a direct transcriptional target of TTF-1, which induces receptor tyrosine kinase like receptor expression and ERBB3 phosphorylation via ROR1 kinase-dependent c-Src activation, maintaining the balance between the prosurvival PI3K-Akt and proapoptotic p38 signaling pathways. In virtue of the possibility of TTF-1 as a diagnostic or prognostic marker, a large number of retrospective studies have been conducted on the prognosis of non-small-cell lung cancer (NSCLC), especially lung adenocarcinoma. ...
Thyroid transcription factor-1 (TTF-1/NKx2.1) is a member of the NKx2 tissue-specific transcription factor family, which is expressed in thyroid follicle, parathyroid gland, alveolar epithelium, and diencephalon which originated from ectoderm, and participates in the differentiation, development, and functional maintenance of the above organs. Recent studies have shown that the abnormal expression of TTF-1 is closely related to the occurrence of a variety of human diseases and can be used as a potential new target for the diagnosis and treatment of related diseases. In this article, in order to strengthen the systematic understanding of TTF-1 and promote the progress of related research, we reviewed the structure, expression regulation, biological functions of TTF-1, and its role in the occurrence and development of human-related clinical diseases. Meanwhile, we prospect the future research direction of TTF-1, which might ultimately contribute to the understanding of the pathogenesis of related clinical diseases and the development of new prevention and treatment strategies.
... The aberrantly expressed ROR1 has been extensively suggested to play a pivotal role in the process of different varieties of cancers or malignant cells, including gastric cancer (Ikeda et al., 2020), breast cancer (Fultang et al., 2020;Stuber et al., 2020), ovarian carcinoma (Wu et al., 2019), and pancreatic cancer (Xu et al., 2018). In terms of molecular function in tumors, ROR1 has the important role of activating PI3K-AKT and MEK-ERK (Daneshmanesh et al., 2015;, signal transducer and activator of transcription 3 (STAT3) (Li et al., 2010), cellularmesenchymal (c-Met) (Gentile et al., 2014), epidermal growth factor receptor (EGFR) (Yamaguchi et al., 2012), and B-cell antigen receptor (BCR) (Karvonen et al., 2017). What is more, Zhang R. et al. (2019) demonstrated that the XIST/miR-30a-5p/ROR1 ceRNA network could be perceived as useful markers deciphering colorectal cancer, which was consistent with our speculation of ceRNA's regulatory function in tumorigenesis. ...
Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human malignancies with poor prognosis. However, the underlying mechanisms of ATC remain to be elucidated. Recently, increasing studies have focused on competitive endogenous RNA (ceRNA) to discover valuable biomarkers for the diagnosis of ATC. The present study identified 705 differentially expressed mRNAs and 47 differentially expressed lncRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were also conducted. Additionally, an lncRNA/miRNA/mRNA network was constructed which included 1103 regulatory relations. The upregulation of RP11-395G23.3 in ATC cells was confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). In the loss of function assays, results suggested silencing of RP11-395G23.3 inhibited cell proliferation and induced cell apoptosis. Mechanically, RP11-395G23.3 could increase ROR1 via sponging miR-124-3p as a ceRNA. Moreover, ROR1 expression was decreased with the downregulation of RP11-395G23.3, but was rescued by the co-transfection of the miR-124-3p inhibitor in ATC cells. Our research suggested that the RP11-395G23.3/miR-124-3p/ROR1 axis potentially acted as a potential target for the diagnosis of ATC.
... When highly expressed, NKX2-1 is thought to function through EGFR signaling to promote pro-survival signaling in cancer cells (Yamaguchi, Yanagisawa et al., 2012). When NKX2-1 expression is lost in patients with lung adenocarcinoma, epithelial cells undergo a mucinous transformation associated with increased invasion of cancer cells and a worse prognosis for patients (Barletta, Perner et al., 2009, Berghmans, Paesmans et al., 2006. ...
Lineage transcription factors mark, promote, and maintain multiple distinct cell types originating from a common progenitor. Despite their essential role, how such factors function and bind genome wide to orchestrate the epigenetic changes necessary to form and maintain these identities in vivo is unclear. One lineage transcription factor NK Homeobox 2-1 (NKX2-1) is expressed throughout the lung epithelium during development and was thought to be lost in the extraordinarily thin cell type required for gas exchange– the alveolar type 1 (AT1) cell. Complementing precise genetic knockouts with cell type-specific ChIP-seq, ATAC-seq, and scRNA-seq, our study shows that AT1 and AT2 cells both express and require Nkx2-1 for their development and maintenance through NKX2-1 mediated regulation of cell type-specific genes. Furthermore, NKX2-1 is guided by cell type-specific transcription factors, binding to regulatory elements unique to AT1 or AT2 cells. In the absence of AT1 cell type-specific transcription cofactors in AT1 cells, NKX2-1 reverts to AT2 cell type-specific binding resulting in an AT1 to AT2 cell fate conversion. This remarkable cellular plasticity was further exemplified by loss of Nkx2-1, whereupon both AT1 and AT2 cells first lose chromatin accessibility at their respective cell type-specific and lineage Nkx2-1 binding sites and gain chromatin accessibility at NKX2-1 sites specific to the alternate cell fate within the alveolar epithelium, followed by a final shift to gastrointestinal lineage identity. These data suggest that in vivo, lineage transcription factors positively establish and maintain lineage and cell type identity with the aid of cell type-specific transcription factors, while repressing alternative cellular identities. This study not only demonstrates how a lineage transcription factor regulates the development, and maintenance of distinct cell epigenomes, but also establishes an experimental paradigm to further investigate lineage transcription factors in vivo.
... Similarly, TTF-1 expression has been reported as a favorable prognostic biomarker in NSCLCs [11,12,[38][39][40]. Transcriptional activation of TTF-1 has been shown to be necessary for EGFR downstream signaling in EGFR mutant tumors [41], and TTF-1 has been suggested to correlate with EGFR oncogene addiction in these tumors [28]. Tumors showing dual positivity for EGFR mutations and TTF-1 IHC have shown the best outcomes, while those negative for both carried the worst outcomes [12,21,28]. ...
Background:
Thyroid transcription factor (TTF-1) is a diagnostic marker expressed in 75%-85% of primary lung adenocarcinomas (ACs). Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene is the most common targetable driver alteration in lung AC. Previous studies have shown a positive correlation between TTF-1 and EGFR mutation status. We aimed to determine the predictive value of TTF-1 immunoexpression for underlying EGFR mutation status in a large Indian cohort.
Materials and methods:
This retrospective designed study was conducted with medical record data from 2011 to 2020. All cases of primary lung AC and non-small cell lung carcinoma not otherwise specified (NSCLC, NOS) with known TTF-1 expression diagnosed by immunohistochemistry using 8G7G3/1 antibodies and EGFR mutation status diagnosed by quantitative polymerase chain reaction were retrieved, reviewed, and the results were analyzed.
Results:
Among 909 patient samples diagnosed as lung AC and NSCLC, NOS, TTF-1 was positive in 76.8% cases (698/909) and EGFR mutations were detected in 29.6% (269/909). A strong positive correlation was present between TTF-1 positivity and EGFR mutation status (odds ratio, 3.61; p < .001), with TTF-1 positivity showing high sensitivity (90%) and negative predictive value (87%) for EGFR mutation. TTF-1 immunoexpression did not show significant correlation with uncommon/dual EGFR mutations (odds ratio, 1.69; p = .098). EGFR-tyrosine kinase inhibitor therapy was significantly superior to chemotherapy among EGFR mutant cases irrespective of TTF-1 status; however, no significant differences among survival outcomes were observed.
Conclusion:
Our study confirms a strong positive correlation between TTF-1 expression and common EGFR mutations (exon 19 deletion and exon 21 L858R) in advanced lung AC with significantly high negative predictive value of TTF-1 for EGFR mutations.
... Likewise, previous reports [27] along with these data show that reduced expression of TTF-1 is significantly associated with unfavourable prognosis in patients with LAD, indicating a tumour suppressive function of NKX2-1/TTF-1 in lung tumourigenesis. However, loss-of-function and gain-of-function studies in human lung carcinoma and transformed cells support a role of NKX2-1 as an oncogene [7,[30][31][32][33]. Furthermore, haploinsufficiency or conditional knockout of Nkx2-1/ Ttf-1 in a transgenic mouse model leads to enhanced development of Kras-mutated lung tumours, in contrast to suppression of Egfr-mutated lung tumours [26]. ...
There are multiple transcription start sites (TSSs) in agreement with multiple transcript variants encoding different isoforms of NKX2-1/TTF-1 (thyroid transcription factor 1); however, the clinicopathological significance of each transcript isoform of NKX2-1/TTF-1 in lung adenocarcinoma (LAD) is unknown. Herein, TSS-level expression of NKX2-1/TTF-1 isoforms was evaluated in 71 LADs using bioinformatic analysis of cap analysis of gene expression (CAGE)-sequencing data, which provides genome-wide expression levels of the 5′-untranslated regions and the TSSs of different isoforms. Results of CAGE were further validated in 664 LADs using in situ hybridisation. Fourteen of 17 TSSs in NKX2-1/TTF-1 (80% of known TSSs in FANTOM5, an atlas of mammalian promoters) were identified in LADs, including TSSs 1–13 and 15; four isoforms of NKX2-1/TTF-1 transcripts (NKX2-1_001, NKX2-1_002, NKX2-1_004, and NKX2-1_005) were expressed in LADs, although NKX2-1_005 did not contain a homeodomain. Among those, six TSSs regulated NKX2-1_004 and NKX2-1_005, both of which contain exon 1. LADs with low expression of isoforms from TSS region 11 regulating exon 1 were significantly associated with poor prognosis in the CAGE data set. In the validation set, 62 tumours (9.3%) showed no expression of NKX2-1/TTF-1 exon 1; such tumours were significantly associated with older age, EGFR wild-type tumours, and poor prognosis. In contrast, 94 tumours, including 22 of 30 pulmonary invasive mucinous adenocarcinomas (IMAs) exhibited exon 1 expression without immunohistochemical TTF-1 protein expression. Furthermore, IMAs commonly exhibited higher exon 1 expression relative to that of exon 4/5, which contained a homeodomain in comparison with EGFR-mutated LADs. These transcriptome and clinicopathological results reveal that LAD use at least 80% of NKX2-1 TSSs and expression of the NKX2-1/TTF-1 transcript isoform without exon 1 (NKX2-1_004 and NKX2-1_005) defines a distinct subset of LAD characterised by aggressive behaviour in elder patients. Moreover, usage of alternative TSSs regions regulating NKX2-1_005 may occur in subsets of LADs.
... Importantly, such studies have revealed that NKX2-1 functions as a tumor promoter or a tumor suppressor in a context-dependent fashion. For example, NKX2-1 promotes EGFR-mutant lung tumorigenesis while it suppresses KRAS-mutant lung tumorigenesis [23][24][25][26][27][28][29] . Among the downstream target genes induced by NKX2-1, MYBPH, LMO3, and CD274 (also known as PD-L1) are of particular interest since they directly influence lung tumorigenesis 24,28,30 . ...
The transcription factor NKX2-1/TTF-1 is involved in lung pathophysiology, including breathing, innate defense and tumorigenesis. To understand the mechanism by which NKX2-1 regulates genes involved in such pathophysiology, we have previously performed ChIP-seq and identified genome-wide NKX2-1-binding sites, which revealed that NKX2-1 binds to not only proximal promoter regions but also multiple intra- and inter-genic regions of the genes regulated by NKX2-1. However, the roles of such regions, especially non-proximal ones, bound by NKX2-1 have not yet been determined. Here, using CRISPRi (CRISPR/dCas9-KRAB), we scrutinize the functional roles of 19 regions/sites bound by NKX2-1, which are located in genes involved in breathing and innate defense ( SFTPB, LAMP3 , SFTPA1, SFTPA2 ) and lung tumorigenesis ( MYBPH, LMO3, CD274/PD-L1 ). Notably, the CRISPRi approach reveals that a portion of NKX2-1-binding sites are functionally indispensable while the rest are dispensable for the expression of the genes, indicating that functional roles of NKX2-1-binding sites are unequally yoked.
Ror-family receptors, Ror1 and Ror2, are type I transmembrane proteins that possess an extracellular cysteine-rich domain, which is conserved throughout the Frizzled-family receptors and is a binding site for Wnt ligands. Both Ror1 and Ror2 function primarily as receptors or co-receptors for Wnt5a to activate the β-catenin-independent, non-canonical Wnt signaling, thereby regulating cell polarity, migration, proliferation, and differentiation depending on the context. Ror1 and Ror2 are expressed highly in many tissues during embryogenesis but minimally or scarcely in adult tissues, with some exceptions. In contrast, Ror1 and Ror2 are expressed in many types of cancers, and their high expression often contributes to the progression of the disease. Therefore, Ror1 and Ror2 have been proposed as potential targets for the treatment of the malignancies. In this review, we provide an overview of the regulatory mechanisms of Ror1/Ror2 expression and discuss how Wnt5a-Ror1/Ror2 signaling is mediated and regulated by their interacting proteins.
Change within the intratumoral microbiome is a common feature in lung and other cancers and may influence inflammation and immunity in the tumor microenvironment, affecting growth and metastases. We previously characterized the lung cancer microbiome in patients and identified Acidovorax temperans as enriched in tumors. Here, we instilled A. temperans in an animal model driven by mutant K-ras and Tp53. This revealed A. temperans accelerates tumor development and burden through infiltration of proinflammatory cells. Neutrophils exposed to A. temperans displayed a mature, pro-tumorigenic phenotype with increased cytokine signaling, with a global shift away from IL-1β signaling. Neutrophil to monocyte and macrophage signaling upregulated MHC II to activate CD4⁺ T cells, polarizing them to an IL-17A⁺ phenotype detectable in CD4⁺ and γδ populations (T17). These T17 cells shared a common gene expression program predictive of poor survival in human LUAD. These data indicate bacterial exposure promotes tumor growth by modulating inflammation.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most refractory cancers with the worst prognosis. Although several molecules are known to be associated with the progression of PDAC, the molecular mechanisms underlying the progression of PDAC remain largely elusive. The Ror‐family receptors, Ror1 and Ror2, which act as a receptor(s) for Wnt‐family ligands, particularly Wnt5a, are involved in the progression of various types of cancers. Here, we show that higher expression of Ror1 and Wnt5b , but not Ror2 , are associated with poorer prognosis of PDAC patients, and that Ror1 and Wnt5b are expressed highly in a type of PDAC cell lines, PANC‐1 cells. Knockdown of either Ror1 or Wnt5b in PANC‐1 cells inhibited their proliferation significantly in vitro, and knockout of Ror1 in PANC‐1 cells resulted in a significant inhibition of tumor growth in vivo. Furthermore, we show that Wnt5b‐Ror1 signaling in PANC‐1 cells promotes their proliferation in a cell‐autonomous manner by modulating our experimental setting in vitro. Collectively, these findings indicate that Wnt5b‐Ror1 signaling might play an important role in the progression of some if not all of PDAC by promoting proliferation.
肺微小脑膜上皮样结节（minute pulmonary meningothelial-like nodules, MPMNs）是一种与脑膜上皮有相似病理学特征的肺部良性小病灶，和恶性肿瘤有相似的影像学表现，可在临床上导致误诊。关于MPMNs的发病机制尚未达成共识，有观点认为MPMNs可能是一种反应性增生，也有观点认为MPMNs与中枢神经系统脑膜瘤有共同的起源和分子机制。了解MPMNs的特征，深入研究其发病机制，有助于提高对该病的认识和诊断水平。本文就MPMNs的临床、病理、影像学特点以及鉴别诊断和发病机制进行综述，并全面分析了其发病机制的研究进展，对进一步探索提出展望。
Amplified oncogene expression is a critical and widespread driver event in cancer, yet our understanding of how amplification-mediated elevated dosage mediates oncogenic regulation is limited. Here, we find that the most significant focal amplification event in lung adenocarcinoma (LUAD) targets a lineage super-enhancer near the NKX2-1 lineage transcription factor. The NKX2-1 super-enhancer is targeted by focal and co-amplification with NKX2-1 , and activation or repression controls NKX2-1 expression. We find that NKX2-1 is a widespread dependency in LUAD cell lines, where NKX2-1 pioneers enhancer accessibility to drive a lineage addicted state in LUAD, and NKX2-1 confers persistence to EGFR inhibitors. Notably, we find that oncogenic NKX2-1 regulation requires expression above a minimum dosage threshold—NKX2-1 dosage below this threshold is insufficient for cell viability, enhancer remodeling, and TKI persistence. Our data suggest that copy-number amplification can be a gain-of-function alteration, wherein amplification elevates oncogene expression above a critical dosage required for oncogenic regulation and cancer cell survival.
Highlights
The most significant amplification event in LUAD targets a lineage super-enhancer that controls expression of the NKX2-1 lineage oncogene.
NKX2-1 is a dosage dependency in most NKX2-1(+) LUAD cell lines
NKX2-1 remodels lineage enhancer accessibility to drive a lineage addicted state and confer persistence to EGFR targeted therapy
NKX2-1 oncogenic regulation requires a minimum oncogenic dosage, which dictates NKX2-1 regulation of enhancer remodeling, TKI persistence, and cancer cell viability
Rho in filopodia (Rif), a member of the Rho family of small GTPases, induces filopodia formation primarily on the dorsal surface of cells; however, its function remains largely unclear. Here, we show that Rif interacts with Ror1, a receptor for Wnt5a that can also induce dorsal filopodia. Our immunohistochemical analysis revealed a high frequency of coexpression of Ror1 and Rif in lung adenocarcinoma. Lung adenocarcinoma cells cultured on Matrigel established front–rear polarity with massive filopodia on their front surfaces, where Ror1 and Rif were accumulated. Suppression of Ror1 or Rif expression inhibited cell proliferation, survival, and invasion, accompanied by the loss of filopodia and cell polarity in vitro, and prevented tumor growth in vivo. Furthermore, we found that Rif was required to activate Wnt5a-Ror1 signaling at the cell surface leading to phosphorylation of the Wnt signaling pathway hub protein Dvl2, which was further promoted by culturing the cells on Matrigel. Our findings reveal a novel function of Rif in mediating Wnt5a-Ror1-Dvl2 signaling, which is associated with the formation of polarized filopodia on 3D matrices in lung adenocarcinoma cells.
The concept of multi-targeted immunotherapeutic systems has propelled the field of cancer immunotherapy into an exciting new era. Multi-effector molecules can be designed to engage with, and alter, the patient's immune system in a plethora of ways. The outcomes can vary from effector cell recruitment and activation upon recognition of a cancer cell, to a multipronged immune checkpoint blockade strategy disallowing evasion of the cancer cells by immune cells, or to direct cancer cell death upon engaging multiple cell surface receptors simultaneously. Here, we review the field of multi-specific immunotherapeutics implemented to treat B cell malignancies. The mechanistically diverse strategies are outlined and discussed; common B cell receptor antigen targeting strategies are outlined and summarized; and the challenges of the field are presented along with optimistic insights for the future.
Tumor-initiating cells are major drivers of chemoresistance and attractive targets for cancer therapy, however, their identity in human pancreatic ductal adenocarcinoma (PDAC) and the key molecules underlying their traits remain poorly understood. Here, we show that a cellular subpopulation with partial epithelial-mesenchymal transition (EMT)-like signature marked by high expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) is the origin of heterogeneous tumor cells in PDAC. We demonstrate that ROR1 depletion suppresses tumor growth, recurrence after chemotherapy, and metastasis. Mechanistically, ROR1 induces the expression of Aurora kinase B (AURKB) by activating E2F through c-Myc to enhance PDAC proliferation. Furthermore, epigenomic analyses reveal that ROR1 is transcriptionally dependent on YAP/BRD4 binding at the enhancer region, and targeting this pathway reduces ROR1 expression and prevents PDAC growth. Collectively, our findings reveal a critical role for ROR1high cells as tumor-initiating cells and the functional importance of ROR1 in PDAC progression, thereby highlighting its therapeutic targetability.
Breast cancer (BC) is the most frequently occurring cancer type seriously threatening the lives of women worldwide. Clinically, the high frequency of diverse resistance to current therapeutic strategies advocates a demand to develop novel and effective approaches for the efficient treatment of BC. The chimeric antigen receptor T (CAR-T) cells therapy, one of the immunotherapies, has displayed powerful capacity to specifically kill and eliminate tumors. Due to the success of CAR-T therapy achieved in treating hematological malignancy, the effect of CAR-T cells therapy has been tested in various human diseases including breast cancer. This review summarized and discussed the landscape of the CAR-T therapy for breast cancer, including the advances, challenge and countermeasure of CAR-T therapy in research and clinical application. The roles of potential antigen targets, tumor microenvironment, immune escape in regulating CAR-T therapy, the combination of CAR-T therapy with other therapeutic strategies to further enhance therapeutic efficacy of CAR-T treatment were also highlighted. Therefore, our review provided a comprehensive understanding of CAR-T cell therapy in breast cancer which will awake huge interests for future in-depth investigation of CAR-T based therapy in cancer treatment.
ROR1 and Aurora kinase were overexpressed in various cancers and essential for cell proliferation, survive and metastasis. Pharmaceutical inhibition of ROR1 and Aurora kinase abrogated the activation of downstream signaling and induced cancer cell apoptosis. Hence, ROR1 and Aurora kinase considered as attractive therapeutic targets for the development of anticancer drugs. In the present work, three series of novel 6-(imidazo[1,2-a] pyridin-6-yl)-quinazolin-4(3H)-one derivatives were designed and synthesized via bioisosterism and scaffold-hopping strategies guided by FLF-13, an Aurora kinase inhibitor we discovered earlier. Most of compounds in series 2 and series 3 showed submicromolar to nanomolar inhibitory activity against multiple cancer cell lines. More importantly, compounds 12d and 12f in series 3 showed nanomolar inhibitory activity against all test cancer cells. The most promising compound 12d exhibited potent inhibitory activity against Aurora A and Aurora B with IC50 values of 84.41 nM and 14.09 nM, respectively. Accordingly, compounds 12d induced G2/M phase cell cycle arrest at 24 h and polyploidy at 48 h. It's worth noting that 12d also displayed inhibitory activity against ROR1 and induce cell apoptosis. Furthermore, 12d could significantly inhibit the tumor growth in SH-SY5Y xenograft model with tumor growth inhibitory rate (IR) up to 46.31 % at 10 mg/kg and 52.66 % at 20 mg/kg. Overall, our data suggested that 12d might serve as a promising candidate for the development of therapeutic agents for cancers with aberrant expression of ROR1 and Aurora kinases by simultaneously targeting ROR1 and Aurora kinase.
Ror1 signaling regulates cell polarity, migration, proliferation, and differentiation during developmental morphogenesis, and plays an important role in regulating neurogenesis in the embryonic neocortices. However, the role of Ror1 signaling in the brains after birth remains largely unknown. Here, we found that expression levels of Ror1 in the mouse neocortices increase during the postnatal period, when astrocytes mature and start expressing GFAP. Indeed, Ror1 is highly expressed in cultured post-mitotic mature astrocytes. RNA-Seq analysis revealed that Ror1 expressed in cultured astrocytes mediates upregulated expression of genes related to fatty acid (FA) metabolism, including the gene encoding carnitine palmitoyl-transferase 1a (Cpt1a), the rate-limiting enzyme of mitochondrial fatty acid β-oxidation (FAO). We also found that Ror1 promotes the degradation of lipid droplets (LDs) accumulated in the cytoplasm of cultured astrocytes after oleic acid loading, and that suppressed expression of Ror1 decreases the amount of FAs localized at mitochondria, intracellular ATP levels, and expression levels of peroxisome proliferator-activated receptor α (PPARα) target genes, including Cpt1a. Collectively, these findings indicate that Ror1 signaling promotes PPARα-mediated transcription of FA metabolism-related genes, thereby facilitating the availability of FAs derived from LDs for mitochondrial FAO in the mature astrocytes. This article is protected by copyright. All rights reserved.
The receptor tyrosine kinase orphan receptor 1 (ROR1) is a receptor for WNT5A and related Wnt proteins, that play an important role during embryonic development by regulating cell migration, cell polarity, neural patterning, and organogenesis. ROR1 exerts these functions by transducing signals from the Wnt secreted glycoproteins to the intracellular Wnt/PCP and Wnt/Ca++ pathways. Investigations in adult human cells, particularly cancer cells, have demonstrated that besides these two pathways, the WNT5A/ROR1 axis can activate a number of signaling pathways, including the PI3K/AKT, MAPK, NF-κB, STAT3, and Hippo pathways. Moreover, ROR1 is aberrantly expressed in cancer and was associated with tumor progression and poor survival by promoting cell proliferation, survival, invasion, epithelial to mesenchymal transition, and metastasis. Consequently, numerous therapeutic tools to target ROR1 are currently being evaluated in cancer patients. In this review, we will provide a detailed description of the signaling pathways regulated by ROR1 in cancer and their impact in tumor progression.
TTF-1-expressing non-small cell lung cancer (NSCLC) is one of the most prevalent lung cancer types worldwide. However, theparadoxical activity of TTF-1 in both lung carcinogenesis and tumor suppression is believed to be context-dependentwhich calls for a deeper understanding about the pathological expression of TTF-1. In addition, the expression circuitry of TTF-1-target genes in NSCLC has not been well examined which necessitates to revisit the involvement of TTF-1- in a multitude of oncologic pathways. We used RNA-seq and clinical data of patients from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), including ChIP-seq data from different NSCLC cell lines, and mapped the proteome of NSCLC tumor. Our analysis showed significant variability in TTF-1 expression among NSCLC,and further clarified that this variability is orchestrated at the transcriptional level. We also found that high TTF-1 expression could negatively influence the survival outcomes of stage 1 LUAD which may be attributed to growth factor receptor-driven activation of mitogenic and angiogenic pathways. Mechanistically, TTF-1 may also control the genes associated with pathways involved in acquired TKI drug resistance or response to immune checkpoint inhibitors. Lastly, proteome-based biomarker discovery in stage 1 LUAD showed that TTF-1 positivity is potentially associated with the upregulation of several oncogenes which includes interferon proteins, MUC1, STAT3, and EIF2AK2. Collectively, this study highlights the potential involvement of TTF-1 in cell proliferation, immune evasion, and angiogenesis in early-stage NSCLC.
The present study investigated the expression and role of ROR2 in small cell lung cancer (SCLC). To examine the expression of ROR2, 27 surgically resected SCLC tissue samples were immunostained for ROR2. Sixteen tissue samples were positive and some showed intratumor heterogeneity in staining intensity. The heterogeneity of ROR2 expression was also observed in tumor tissues from a PDX model of SCLC, in which there were cells with high ROR2 expression (ROR2high cells) and without its expression (ROR2low cells). These cells were subjected to a RNA sequence analysis. GSEA was performed and the results obtained revealed the enrichment of molecules such as G2M checkpoint, mitotic spindle, and E2F targets in ROR2high cells. The rate of EdU incorporation was significantly higher in ROR2high cells than ROR2low cells from the PDX model and the SCLC cell lines. Cell proliferation was suppressed in ROR2 KO SBC3 cells in vitro and in vivo. Comparisons of down-regulated differentially expressed genes in ROR2 KO SBC3 cells with up-regulated DEG in ROR2high cells from the PDX model revealed 135 common genes. After a Metascape analysis of these genes, we focused on Aurora kinases. In SCLC cell lines, the knockdown of ROR2 suppressed Aurora kinases. Therefore, ROR2 appears to regulate the cell cycle through Aurora kinases. The present results reveal a role for ROR2 in SCLC and afford a candidate system (ROR2-Aurora kinase) accompanying tumor heterogeneity in SCLC.
Targeted molecular therapy provides a new opportunity for treating different types of cancers. Cellular protein expression is vital for such targeted therapies. Receptor tyrosine kinases (RTKs) are the most potential targets for cancer therapies. Transmembrane RTKs such as receptor tyrosine kinase-like orphan receptors (ROR) are absent in normal tissues but found in human cancers, contributing to tumor growth, migration, and invasion. Specific and tumor-restricted expression profiles of these receptors offer an exceptionally unique therapeutic targeting approach for human cancer. Targeting evolutionarily conserved ROR1 and ROR2 by small molecule inhibitors and monoclonal antibodies serves as a promising cancer therapeutic target. ROR1 and ROR2 are involved in noncanonical Wnt signaling as Wnt5a receptors in metastatic cancers and organogenesis. ROR1 is the hallmark for many cancers and is critically linked with solid and blood cancer progression. Current evidence reveals the furthermore involvement of ROR2 as a Wnt ligand-receptor in canonical Wnt signaling. High expression is correlated with the most aggressive cancer. Further studies to understand the structure of the tyrosine kinase domain of ROR1 and the newly discovered nontyrosine kinase functioning of ROR2 are required to explore possible targeting strategies.
The Ror-family proteins, Ror1 and Ror2, act as receptors or co-receptors for Wnt5a and its related Wnt proteins to activate non-canonical Wnt signaling. Ror1 and/or Ror2-mediated signaling plays essential roles in regulating cell polarity, migration, proliferation and differentiation during developmental morphogenesis, tissue-/organo-genesis and regeneration of adult tissues following injury. Ror1 and Ror2 are expressed abundantly in developing tissues in an overlapping, yet distinct manner, and their expression in adult tissues is restricted to specific cell types such as tissue stem/progenitor cells. Expression levels of Ror1 and/or Ror2 in the adult tissues are increased following injury, thereby promoting regeneration or repair of these injured tissues. On the other hand, disruption of Wnt5a-Ror2 signaling is implicated in senescence of tissue stem/progenitor cells that is related to the impaired regeneration capacity of aged tissues. In fact, Ror1 and Ror2 are implicated in age-related diseases, including tissue fibrosis, atherosclerosis (or arteriosclerosis), neurodegenerative diseases, and cancers. In these diseases, enhanced and/or sustained (chronic) expression of Ror1 and/or Ror2 is observed, and they might contribute to the progression of these diseases through Wnt5a-dependent and -independent manners. In this article, we overview recent advances in our understanding of the roles of Ror1 and Ror2-mediated signaling in the development, tissue regeneration and age-related diseases, and discuss their potential to be therapeutic targets for chronic inflammatory diseases and cancers.
Lung cancer that exhibits epidermal growth factor receptor (EGFR) gene mutation is sensitive to EGFR-tyrosine kinase inhibitors (TKIs), such as osimertinib. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) may be involved in overcoming EGFR-TKI resistance. Growth inhibition, colony formation, apoptosis, and mRNA/protein levels in four osimertinib-sensitive and resistant cell lines transfected with small interfering RNA (siRNA) targeting ROR1 (siROR1) were evaluated. Cell growth and colony formation were suppressed and apoptosis was increased in all cell lines treated with siROR1. Although EGFR, AKT, and ERK phosphorylation were not suppressed in all cell lines, TGF-β2, AXL, CDH2, PARP1, PEG10, and TYMS mRNA expression levels were reduced. The combination of osimertinib with siROR1 was effective for the four cell lines, particularly in the two osimertinib-sensitive lines. In conclusion, targeting ROR1 in combination with osimertinib in EGFR mutant lung cancer may be a novel therapeutic option.
Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is highly expressed in a wide variety of hematological and solid cancers, but is low or absent in adult tissues. Here, we show that ROR1 is released with exosomes from ROR1-positive cancer cells. We also developed a simple dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) to detect cancer-derived ROR1-positive exosomes, which are captured by two anti-ROR1 antibodies and detected by the fluorescence of free chelating europium. This new DELFIA method can detect cancer-derived ROR1-positive exosomes in the cell supernatant and serum with a wide range and rapidly compared with the conventional western blot assay. This method may be useful as a companion diagnostics for ROR1-positive cancers.
In the pathological diagnosis of lung cancer, the expression of certain transcription factors is important as differentiation markers and for predicting underlying molecular abnormalities. TTF-1 is a master regulator of lung differentiation, and TTF-1-positive lung adenocarcinomas frequently harbor mutually exclusive driver mutations, such as EGFR and ALK. Furthermore, some studies have reported that TTF-1 itself is important for the survival of cancers. HNF4α, which is involved in gastrointestinal epithelial differentiation, is mutually exclusive with TTF-1 in lung cancer, and HNF4α-positive lung adenocarcinomas frequently harbor a TTF-1 gene-inactivating mutation/hypermethylation and KRAS mutation. SALL4, an embryonic tumor marker, is highly expressed in high-grade fetal adenocarcinoma. SALL4 is attracting attention as a new oncogene and a target for molecular therapy. In EMT-type lung cancer, the loss of chromatin remodeling factors, such as SMARCA4 and SMARCA2, has been observed, and in recent years, a new disease concept known as SMARCA4-deficient dedifferentiated tumor has been proposed. It was recently reported that small-cell carcinomas can be classified into four molecular subtypes depending on four transcription factors: ASCL1, NEUROD1, POU2F3, and YAP1. Based on my own pathological research on lung cancer, I will briefly explain these transcription factors and regulators related to the pathological diagnosis.
Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110- catalytic subunit (encoded by PIK3CA)1. They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110- mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110- H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan–PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography–computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.
Lung cancers with neuroendocrine (NE) features are often very aggressive but the underlying molecular mechanisms remain elusive. The transcription factor ASH1/ASCL1 is a master regulator of pulmonary NE cell development that is involved in the pathogenesis of lung cancers with NE features (NE-lung cancers). Here we report the definition of the microRNA miR-375 as a key downstream effector of ASH1 function in NE-lung cancer cells. miR-375 was markedly induced by ASH1 in lung cancer cells where it was sufficient to induce NE differentiation. miR-375 upregulation was a prerequisite for ASH1-mediated induction of NE features. The transcriptional coactivator YAP1 was determined to be a direct target of miR-375. YAP1 showed a negative correlation with miR-375 in a panel of lung cancer cell lines and growth inhibitory activities in NE-lung cancer cells. Our results elucidate an ASH1 effector axis in NE-lung cancers that is functionally pivotal in controlling NE features and the alleviation from YAP1-mediated growth inhibition.
Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzumab-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance.
TTF-1 [thyroid transcription factor-1; also known as Nkx2.1, T/EBP (thyroid-specific-enhancer-binding protein) or TITF1] is a homeodomain-containing transcription factor essential for the morphogenesis and differentiation of the thyroid, lung and ventral forebrain. TTF-1 controls the expression of select genes in the thyroid, lung and the central nervous system. In the lung, TTF-1 controls the expression of surfactant proteins that are essential for lung stability and lung host defence. Human TTF-1 is encoded by a single gene located on chromosome 14 and is organized into two/three exons and one/two introns. Multiple transcription start sites and alternative splicing produce mRNAs with heterogeneity at the 5' end. The 3' end of the TTF-1 mRNA is characterized by a rather long untranslated region. The amino acid sequences of TTF-1 from human, rat, mouse and other species are very similar, indicating a high degree of sequence conservation. TTF-1 promoter activity is maintained by the combinatorial or co-operative actions of HNF-3 [hepatocyte nuclear factor-3; also known as FOXA (forkhead box A)], Sp (specificity protein) 1, Sp3, GATA-6 and HOXB3 (homeobox B3) transcription factors. There is limited information on the regulation of TTF-1 gene expression by hormones, cytokines and other biological agents. Glucocorticoids, cAMP and TGF-beta (transforming growth factor-beta) have stimulatory effects on TTF-1 expression, whereas TNF-alpha (tumour necrosis factor-alpha) and ceramide have inhibitory effects on TTF-1 DNA-binding activity in lung cells. Haplo-insufficiency of TTF-1 in humans causes hypothyroidism, respiratory dysfunction and recurring pulmonary infections, underlining the importance of optimal TTF-1 levels for the maintenance of thyroid and lung function. Recent studies have implicated TTF-1 as a lineage-specific proto-oncogene for lung cancer.
Lung cancer with epidermal growth factor receptor (EGFR)-activating mutations responds favorably to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. However, 25% to 30% of patients with EGFR-activating mutations show intrinsic resistance, and the responders invariably acquire resistance to gefitinib. Here, we showed that hepatocyte growth factor (HGF), a ligand of MET oncoprotein, induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations by restoring the phosphatidylinositol 3-kinase/Akt signaling pathway via phosphorylation of MET, but not EGFR or ErbB3. Strong immunoreactivity for HGF in cancer cells was detected in lung adenocarcinoma patients harboring EGFR-activating mutations, but no T790M mutation or MET amplification, who showed intrinsic or acquired resistance to gefitinib. The findings indicate that HGF-mediated MET activation is a novel mechanism of gefitinib resistance in lung adenocarcinoma with EGFR-activating mutations. Therefore, inhibition of HGF-MET signaling may be a considerable strategy for more successful treatment with gefitinib.
Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.
The thyroid-specific enhancer-binding protein (T/ebp) gene was disrupted by homologous recombination in embryonic stem cells to generate mice lacking T/EBP expression. Heterozygous animals developed normally, whereas mice homozygous for the disrupted gene were born dead and lacked the lung parenchyma. Instead, they had a rudimentary bronchial tree associated with an abnormal epithelium in their pleural cavities. Furthermore, the homozygous mice had no thyroid gland but had a normal parathyroid. In addition, extensive defects were found in the brain of the homozygous mice, especially in the ventral region of the forebrain. The entire pituitary, including the anterior, intermediate, and posterior pituitary, was also missing. In situ hybridization showed that the T/ebp gene is expressed in the normal thyroid, lung bronchial epithelium, and specific areas of the forebrain during early embryogenesis. These results establish that the expression of T/EBP, a transcription factor known to control thyroid-specific gene transcription, is also essential for organogenesis of the thyroid, lung, ventral forebrain, and pituitary.
The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.
The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.
Activation of Akt/PKB by growth factors requires multiple phosphorylation events. Phosphorylation of Thr308 and Ser473 of Akt by its upstream kinase(s) or autophosphorylation is critical for optimal activation of its kinase activity. Here,
we present evidence that tyrosine phosphorylation is required for Akt activation. Epidermal growth factor treatment induces
tyrosine phosphorylation of Akt in COS1 and PC3M cells, which is abrogated by PP2, a selective inhibitor for Src family tyrosine
kinases. Elevated Akt activity is observed in v-Src transformed NIH3T3 cells, which is accompanied with increased tyrosine
phosphorylation of Akt. Akt activity induced by growth factors is significantly reduced in SYF cells lacking Src, Yes, and
Fyn, which can be restored by introducing c-Src, but not the kinase-inactive Src, back to these cells. Furthermore, we have
identified two tyrosine residues near the activation loop of Akt that are important for its activation. Substitution of these
residues with phenylalanine abolishes Akt kinase activity stimulated by growth factors. These two YF mutants fail to block
Forkhead transcription factor activity in 293 cells and are unable to prevent apoptosis induced by matrix detachment. Our
data suggest that, in addition to phosphorylation of Thr308 and Ser473, tyrosine phosphorylation of Akt may be essential for its biological function.
Activation of Akt by growth factors is a multistep process. Here, we provide evidence that tyrosine kinase Src is directly associated with Akt through the interaction between its SH3 domain and a conserved proline-rich motif (PXXP) in the C-terminal regulatory region of Akt. Substitution of the proline residues Pro-424 and Pro-427 by alanines results in loss of Akt activity and phosphorylation induced by the epidermal growth factor (EGF), possibly because these mutations disrupt the interaction between Akt and the SH3 domain of Src. This possibility is corroborated by our observation that the Akt mutant lacking these two prolines fails to bind to Src both in vivo and in vitro. We also showed that phosphorylation of Tyr-315 in Akt induced by Src or EGF is dependent on the integrity of this proline-rich motif. Furthermore, the Akt mutant lacking this proline motif fails to block the transcription activity of Forkhead in 293 cells and poorly stimulates the proliferation of Madin-Darby canine kidney cells. Taken together, our data suggest that the interaction between the SH3 domain of Src family kinases and the proline-rich motif in the C-terminal regulatory region of Akt is required for tyrosine phosphorylation of Akt and its subsequent activation. It is noteworthy that this PXXP motif is conserved throughout several members of AGC kinase family, implying that association of this motif with the SH3 domain of an upstream regulator may represent a general mechanism applicable to these kinases as well.
Src tyrosine kinases transmit integrin-dependent signals pivotal for cell movement and proliferation. Here, we establish a mechanism for Src activation by integrins. c-Src is shown to bind constitutively and selectively to beta3 integrins through an interaction involving the c-Src SH3 domain and the carboxyl-terminal region of the beta3 cytoplasmic tail. Clustering of beta3 integrins in vivo activates c-Src and induces phosphorylation of Tyr-418 in the c-Src activation loop, a reaction essential for adhesion-dependent phosphorylation of Syk, a c-Src substrate. Unlike c-Src, Hck, Lyn, and c-Yes bind more generally to beta1A, beta2, and beta3 cytoplasmic tails. These results invoke a model whereby Src is primed for activation by direct interaction with an integrin beta tail, and integrin clustering stabilizes activated Src by inducing intermolecular autophosphorylation. The data provide a paradigm for integrin regulation of Src and a molecular basis for the similar functional defects of osteoclasts or platelets from mice lacking beta3 integrins or c-Src.
Therapies that target the EGF receptor (EGFR), such as gefitinib (IRESSA), are effective in a subset of patients with advanced non-small cell lung cancer (NSCLC). The differences in intracellular signaling networks between gefitinib-sensitive and -resistant NSCLCs remain poorly understood. In this study, we observe that gefitinib reduces phospho-Akt levels only in NSCLC cell lines in which it inhibits growth. To elucidate the mechanism underlying this observation, we compared immunoprecipitates of phosphoinositide 3-kinase (PI3K) between gefitinib-sensitive and -resistant NSCLC cell lines. We observe that PI3K associates with ErbB-3 exclusively in gefitinib-sensitive NSCLC cell lines. Gefitinib dissociates this complex, thereby linking EGFR inhibition to decreased Akt activity. In contrast, gefitinib-resistant cells do not use ErbB-3 to activate the PI3K/Akt pathway. In fact, abundant ErbB-3 expression is detected only in gefitinib-sensitive NSCLC cell lines. Two gefitinib-sensitive NSCLC cell lines with endogenous distinct activating EGFR mutations (L858R and Del747-749), frequently observed in NSCLC patients who respond to gefitinib, also use ErbB-3 to couple to PI3K. Down-regulation of ErbB-3 by means of short hairpin RNA leads to decreased phospho-Akt levels in the gefitinib-sensitive NSCLC cell lines, Calu-3 (WT EGFR) and H3255 (L858R EGFR), but has no effect on Akt activation in the gefitinib-resistant cell lines, A549 and H522. We conclude that ErbB-3 is used to couple EGFR to the PI3K/Akt pathway in gefitinib-sensitive NSCLC cell lines harboring WT and mutant EGFRs.
• Akt
• EGF receptor
Lung adenocarcinomas from patients who respond to the tyrosine kinase inhibitors gefitinib (Iressa) or erlotinib (Tarceva) usually harbor somatic gain-of-function mutations in exons encoding the kinase domain of the epidermal growth factor receptor (EGFR). Despite initial responses, patients eventually progress by unknown mechanisms of "acquired" resistance.
We show that in two of five patients with acquired resistance to gefitinib or erlotinib, progressing tumors contain, in addition to a primary drug-sensitive mutation in EGFR, a secondary mutation in exon 20, which leads to substitution of methionine for threonine at position 790 (T790M) in the kinase domain. Tumor cells from a sixth patient with a drug-sensitive EGFR mutation whose tumor progressed on adjuvant gefitinib after complete resection also contained the T790M mutation. This mutation was not detected in untreated tumor samples. Moreover, no tumors with acquired resistance had KRAS mutations, which have been associated with primary resistance to these drugs. Biochemical analyses of transfected cells and growth inhibition studies with lung cancer cell lines demonstrate that the T790M mutation confers resistance to EGFR mutants usually sensitive to either gefitinib or erlotinib. Interestingly, a mutation analogous to T790M has been observed in other kinases with acquired resistance to another kinase inhibitor, imatinib (Gleevec).
In patients with tumors bearing gefitinib- or erlotinib-sensitive EGFR mutations, resistant subclones containing an additional EGFR mutation emerge in the presence of drug. This observation should help guide the search for more effective therapy against a specific subset of lung cancers.
Evolving studies with several different targeted therapeutic agents are demonstrating that patients with genomic alterations of the target, including amplification, translocation and mutation, are more likely to respond to the therapy. Recent studies indicate that numerous components of the phosphatidylinositol-3-kinase (PI3K)/AKT pathway are targeted by amplification, mutation and translocation more frequently than any other pathway in cancer patients, with resultant activation of the pathway. This warrants exploiting the PI3K/AKT pathway for cancer drug discovery.
"Oncogene addiction" describes an unexplained dependency of cancer cells on a particular cellular pathway for survival or proliferation. We report that differential attenuation rates of prosurvival and proapoptotic signals in oncogene-dependent cells contribute to cell death following oncogene inactivation. Src-, BCR-ABL-, and EGF receptor-dependent cells exhibit a similar profile of signal attenuation following oncogene inactivation characterized by rapid diminution of phospho-ERK, -Akt, and -STAT3/5, and a delayed accumulation of the proapoptotic effector phospho-p38 MAPK. These findings implicate a transient imbalance in survival and apoptotic oncogenic outputs in the apoptotic response to oncogene inactivation. Moreover, these observations implicate a common profile of signal attenuation for multiple oncogenes and suggest that "addiction" associated with apoptosis reflects an active rather than a passive process.
The vertebrate lung consists of multiple cell types that are derived primarily from endodermal and mesodermal compartments of the early embryo. The process of pulmonary organogenesis requires the generation of precise signaling centers that are linked to transcriptional programs that, in turn, regulate cell numbers, differentiation, and behavior, as branching morphogenesis and alveolarization proceed. This review summarizes knowledge regarding the expression and proposed roles of transcription factors influencing lung formation and function with particular focus on knowledge derived from the study of the mouse. A group of transcription factors active in the endodermally derived cells of the developing lung tubules, including thyroid transcription factor-1 (TTF-1), beta-catenin, Forkhead orthologs (FOX), GATA, SOX, and ETS family members are required for normal lung morphogenesis and function. In contrast, a group of distinct proteins, including FOXF1, POD1, GLI, and HOX family members, play important roles in the developing lung mesenchyme, from which pulmonary vessels and bronchial smooth muscle develop. Lung formation is dependent on reciprocal signaling among cells of both endodermal and mesenchymal compartments that instruct transcriptional processes mediating lung formation and adaptation to breathing after birth.
Src family protein-tyrosine kinases, which play an important role in signal integration, have been implicated in tumorigenesis in multiple lineages, including breast cancer. We demonstrate, herein, that Src kinases regulate the phosphatidylinositol 3-kinase (PI3K) signaling cascade via altering the function of the PTEN tumor suppressor. Overexpression of activated Src protein-tyrosine kinases in PTEN-deficient breast cancer cells does not alter AKT phosphorylation, an indicator of signal transduction through the PI3K pathway. However, in the presence of functional PTEN, Src reverses the activity of PTEN, resulting in an increase in AKT phosphorylation. Activated Src reduces the ability of PTEN to dephosphorylate phosphatidylinositols in micelles and promotes AKT translocation to cellular plasma membranes but does not alter PTEN activity toward water-soluble phosphatidylinositols. Thus, Src may alter the capacity of the PTEN C2 domain to bind cellular membranes rather than directly interfering with PTEN enzymatic activity. Tyrosine phosphorylation of PTEN is increased in breast cancer cells treated with pervanadate, suggesting that PTEN contains sites for tyrosine phosphorylation. Src kinase inhibitors markedly decreased pervanadate-mediated tyrosine phosphorylation of PTEN. Further, expression of activated Src results in marked tyrosine phosphorylation of PTEN. SHP-1, a SH2 domain-containing protein-tyrosine phosphatase, selectively binds and dephosphorylates PTEN in Src transfected cells. Both Src inhibitors and SHP-1 overexpression reverse Src-induced loss of PTEN function. Coexpression of PTEN with activated Src reduces the stability of PTEN. Taken together, the data indicate that activated Src inhibits PTEN function leading to alterations in signaling through the PI3K/AKT pathway.
The epidermal growth factor receptor (EGFR) kinase inhibitors gefitinib and erlotinib are effective treatments for lung cancers
with EGFR activating mutations, but these tumors invariably develop drug resistance. Here, we describe a gefitinib-sensitive lung cancer
cell line that developed resistance to gefitinib as a result of focal amplification of the MET proto-oncogene. inhibition of MET signaling in these cells restored their sensitivity to gefitinib. MET amplification was detected in 4 of 18 (22%) lung cancer specimens that had developed resistance to gefitinib or erlotinib.
We find that amplification of MET causes gefitinib resistance by driving ERBB3 (HER3)–dependent activation of PI3K, a pathway thought to be specific to EGFR/ERBB
family receptors. Thus, we propose that MET amplification may promote drug resistance in other ERBB-driven cancers as well.
We used high-resolution array analysis to discover a recurrent lung cancer amplicon located at 14q13.3. Low-level gain of this region was detected in 15% of lung cancer samples, and high-level amplification was detected in an additional 4% of samples. High-level focal amplification appears to be specific to lung cancers, because it was not detected in >500 samples of other tumor types. Mapping of the commonly amplified region revealed there are three genes in the core region, all of which encode transcription factors with either established lung developmental function (TTF1/NKX2-1, NKX2-8) or potential lung developmental function (PAX9). All three genes were overexpressed to varying degrees in amplified samples, although TTF1/NKX2-1 was not expressed in the squamous cancer subtype, consistent with previous reports. Remarkably, overexpression of any pairwise combination of these genes showed pronounced synergy in promoting the proliferation of immortalized human lung epithelial cells. Analysis of human lung cancer cell lines by both RNAi and ectopic overexpression further substantiates an oncogenic role for these transcription factors. These results, taken together with previous reports of oncogenic alterations of transcription factors involved in lung development (p63, CEBPA), suggest genetic alterations that directly interfere with transcriptional networks normally regulating lung development may be a more common feature of lung cancer than previously realized.
• gene amplification
• lung development
• lung oncogene
• TTF1 NKX2-8 PAX9
• lineage addiction
Somatic alterations in cellular DNA underlie almost all human cancers. The prospect of targeted therapies and the development of high-resolution, genome-wide approaches are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection of tumours (n = 371) using dense single nucleotide polymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scale copy-number gain or loss, of which only a handful have been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in approximately 12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineage-specific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered.
In human lung adenocarcinomas harboring EGFR mutations, a second-site point mutation that substitutes methionine for threonine at position 790 (T790M) is associated with approximately half of cases of acquired resistance to the EGFR kinase inhibitors, gefitinib and erlotinib. To identify other potential mechanisms that contribute to disease progression, we used array-based comparative genomic hybridization (aCGH) to compare genomic profiles of EGFR mutant tumors from untreated patients with those from patients with acquired resistance. Among three loci demonstrating recurrent copy number alterations (CNAs) specific to the acquired resistance set, one contained the MET proto-oncogene. Collectively, analysis of tumor samples from multiple independent patient cohorts revealed that MET was amplified in tumors from 9 of 43 (21%) patients with acquired resistance but in only two tumors from 62 untreated patients (3%) (P = 0.007, Fisher's Exact test). Among 10 resistant tumors from the nine patients with MET amplification, 4 also harbored the EGFRT790M mutation. We also found that an existing EGFR mutant lung adenocarcinoma cell line, NCI-H820, harbors MET amplification in addition to a drug-sensitive EGFR mutation and the T790M change. Growth inhibition studies demonstrate that these cells are resistant to both erlotinib and an irreversible EGFR inhibitor (CL-387,785) but sensitive to a multikinase inhibitor (XL880) with potent activity against MET. Taken together, these data suggest that MET amplification occurs independently of EGFRT790M mutations and that MET may be a clinically relevant therapeutic target for some patients with acquired resistance to gefitinib or erlotinib.
• lung adenocarcinoma
• XL880
Lung cancer is a leading cause of cancer death, where the amplification of oncogenes contributes to tumorigenesis. Genomic profiling of 128 lung cancer cell lines and tumors revealed frequent focal DNA amplification at cytoband 14q13.3, a locus not amplified in other tumor types. The smallest region of recurrent amplification spanned the homeobox transcription factor TITF1 (thyroid transcription factor 1; also called NKX2-1), previously linked to normal lung development and function. When amplified, TITF1 exhibited increased expression at both the RNA and protein levels. Small interfering RNA (siRNA)-mediated knockdown of TITF1 in lung cancer cell lines with amplification led to reduced cell proliferation, manifested by both decreased cell-cycle progression and increased apoptosis. Our findings indicate that TITF1 amplification and overexpression contribute to lung cancer cell proliferation rates and survival and implicate TITF1 as a lineage-specific oncogene in lung cancer.
Gene expression profiling identified receptor tyrosine kinase ROR1, an embryonic protein involved in organogenesis, as a signature gene in B-cell chronic lymphocytic leukemia (B-CLL). To assess the suitability of ROR1 as a cell surface antigen for targeted therapy of B-CLL, we carried out a comprehensive analysis of ROR1 protein expression.
Peripheral blood mononuclear cells, sera, and other adult tissues from B-CLL patients and healthy donors were analyzed qualitatively and quantitatively for ROR1 protein expression by flow cytometry, cell surface biotinylation, Western blotting, and ELISA.
ROR1 protein is selectively expressed on the surface of B-CLL cells, whereas normal B cells, other normal blood cells, and normal adult tissues do not express cell surface ROR1. Moreover, cell surface expression of ROR1 is uniform and constitutive, i.e., independent of anatomic niches, independent of biological and clinical heterogeneity of B-CLL, independent of B-cell activation, and found at similar levels in all B-CLL samples tested. The antibody binding capacity of B-CLL cell surface ROR1 was determined to be in the range of 10(3) to 10(4) molecules per cell. A portion of B-CLL cell surface ROR1 was actively internalized upon antibody binding. Soluble ROR1 protein was detectable in sera of <25% of B-CLL patients and a similar fraction of healthy donors at concentrations below 200 ng/mL.
The restricted, uniform, and constitutive cell surface expression of ROR1 protein in B-CLL provides a strong incentive for the development of targeted therapeutics such as monoclonal antibodies.
We examined the sera of six patients before and after i.v. infusions of autologous chronic lymphocytic leukemia (CLL) cells transduced ex vivo with an adenovirus encoding CD154 (Ad-CD154). Five patients made high-titer antibodies against adenovirus and three made IgG reactive with a leukemia-associated surface antigen, which we identified as ROR1. Anti-ROR1 antibodies were not detected in the sera of untreated patients. We generated anti-ROR1 mAbs and found they reacted specifically with the CLL cells of all patients, but not with nonleukemic leukocytes, a wide variety of normal adult tissues, or blood mononuclear cells, including CD5⁺ B cells of healthy adults. ROR1 could bind Wnt5a, which induced activation of NF-κB when coexpressed with ROR1 in HEK293 cells and enhanced the survival of CLL cells in vitro, an effect that could be neutralized by posttreatment anti-ROR1 antisera. We conclude that patients with CLL can break immune tolerance to ROR1, which is an oncofetal surface antigen and survival-signaling receptor in this neoplastic disease.
• chronic lymphocytic leukemia
• neoplasia
MET amplification activates ERBB3/PI3K/AKT signaling in EGFR mutant lung cancers and causes resistance to EGFR kinase inhibitors. We demonstrate that MET activation by its ligand, HGF, also induces drug resistance, but through GAB1 signaling. Using high-throughput FISH analyses in both cell lines and in patients with lung cancer, we identify subpopulations of cells with MET amplification prior to drug exposure. Surprisingly, HGF accelerates the development of MET amplification both in vitro and in vivo. EGFR kinase inhibitor resistance, due to either MET amplification or autocrine HGF production, was cured in vivo by combined EGFR and MET inhibition. These findings highlight the potential to prospectively identify treatment naive, patients with EGFR-mutant lung cancer who will benefit from initial combination therapy.
Division of Medical Oncology and Surgical Oncology
The Ror-family receptor tyrosine kinases (RTKs) play crucial roles in the development of various organs and tissues. In mammals, Ror2, a member of the Ror-family RTKs, has been shown to act as a receptor or coreceptor for Wnt5a to mediate noncanonical Wnt signaling. Ror2- and Wnt5a-deficient mice exhibit similar abnormalities during developmental morphogenesis, reflecting their defects in convergent extension movements and planar cell polarity, characteristic features mediated by noncanonical Wnt signaling. Furthermore, mutations within the human Ror2 gene are responsible for the genetic skeletal disorders dominant brachydactyly type B and recessive Robinow syndrome. Accumulating evidence demonstrate that Ror2 mediates noncanonical Wnt5a signaling by inhibiting the beta-catenin-TCF pathway and activating the Wnt/JNK pathway that results in polarized cell migration. In this article, we review recent progress in understanding the roles of noncanonical Wnt5a/Ror2 signaling in developmental morphogenesis and in human diseases, including heritable skeletal disorders and tumor invasion.
Small cell lung cancer (SCLC) exhibits highly aggressive behavior and has a poor prognosis. While numerous investigations have been carried out, the exact mechanism of its carcinogenesis and aggressiveness is still unclear. SCLC is categorized as a neuroendocrine neoplasia and has a genetic profile characterized by universal alterations of the RB and TP53 genes. Epidemiological studies indicate the majority of SCLCs to be caused by smoking and the TP53 mutational pattern to be consistent with that evoked by smoke carcinogens; however, there is no direct evidence that such carcinogens induce alterations to RB in SCLC. While the importance of these alterations in the carcinogenesis of SCLC is strongly suggested, the exact molecular mechanism has been only little elucidated. SCLC cells almost always express mammalian achaete-scute homolog-1 (MASH1) and thyroid transcription factor-1 (TTF-1). MASH1 plays a critical role in neuroendocrine differentiation. TTF-1 is a characteristic marker of distal airway cells and pulmonary adenocarcinomas, but is also expressed in extrapulmonary neuroendocrine cancers. Thus, TTF-1 may well play a significant role in the development of neuroendocrine cancers. Recent studies indicate that the airway stem cell is committed to the neuroendocrine lineage through MASH1 and Notch signaling and that only RB-deleted neuroendocrine cells selectively proliferate in response to E2F3, eventually undergoing transformation to neuroendocrine cancer cells, probably in concert with TP53 gene aberrations. Thus, alterations of both the RB and TP53 genes are central to the carcinogenesis of SCLC, while many other factors including MASH1 and TTF-1 contribute to the development and biological behavior of SCLC.
Lung cancer remains the leading cause of cancer deaths worldwide, and advanced stage disease is largely refractory to conventional chemotherapy. Thus, there is an important need for alternative treatment strategies, and the ErbB proteins have emerged as potentially important therapeutic drug targets in this setting, apparently reflecting a state of "oncogene addiction" in some lung tumors. In this review, we discuss the recent identification of mutations that promote activation of ErbB family proteins in a subset of lung cancers, and the development of selective inhibitors of these proteins that have demonstrated clinical efficacy. We also discuss the problem of drug resistance, which severely limits the clinical utility of such agents, and has prompted intense efforts to better understand molecular mechanisms underlying drug resistance as well as strategies to overcome or prevent such resistance.
The clinical efficacy of selective kinase inhibitors suggests that some cancer cells may become dependent on a single oncogene for survival. RNAi has been increasingly used to understand such “oncogene addiction” and validate new therapeutic targets. However, RNAi approaches suffer from significant off-target effects that limit their utility. Here, we combine carefully titrated lentiviral-mediated short hairpin RNA knockdown of the epidermal growth factor receptor (EGFR) with heterologous reconstitution by EGFR mutants to rigorously analyze the structural features and signaling activities that determine addiction to the mutationally activated EGFR in human lung cancer cells. EGFR dependence is differentially rescued by distinct EGFR variants and oncogenic mutants, is critically dependent on its heterodimerization partner ErbB-3, and surprisingly, does not require autophosphorylation sites in the cytoplasmic domain. Quantitative “oncogene rescue” analysis allows mechanistic dissection of oncogene addiction, and, when broadly applied, may provide functional validation for potential therapeutic targets identified through large-scale RNAi screens.
• EGF Receptor
• RNAi
Novel human epithelial cell lines retaining characteristic features of normal peripheral airway cells were established by transfecting the SV40 large T antigen gene into primary in vitro outgrowths from normal peripheral lung specimens. These lines, designated as HPL1A to HPL1E, showed the polygonal shapes typical of epithelial cells and expressed cytokeratin in abundance. Ultrastructural examination revealed the presence of microvilli, multivesicular bodies, and multilamellar body-like structures that are characteristic of type II pneumocytes, but expression of CC1O transcripts, a highly specific marker for Clara cells, was also observed. Response to transforming growth factor beta, epidermal growth factor (EGF), and hepatocyte growth factor, all of which are thought to be important growth-regulatory molecules for cellular proliferation and developmental processes of peripheral lung, was apparent. In the HPL1A case, markedly altered cell morphology and cytoskeletal organization, potent inhibition of cell growth, and increased expression of an extracellular matrix protein were noted with transforming growth factor beta. Interestingly, both EGF and hepatocyte growth factor stimulated anchorage-dependent growth, whereas only EGF could sustain anchorage-independent proliferation. The HPL1 lines are, to our knowledge, the first series of stable epithelial lines of human peripheral lung to be described. They should be valuable for investigating various aspects of growth regulation and oncogenic processes, including the mechanisms of acquisition of anchorage independence and the interrelationships of genetic changes identified previously in lung cancers. In addition, the HPL1 lines may also prove useful for development of in vitro models for other human lung disorders as well as to elucidate the mechanisms of peripheral lung differentiation.
The non-receptor tyrosine kinase Src is important for many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first proto-oncogene to be discovered in the vertebrate genome. Src has been important, not only as an object of study in itself, but also as an entry point into the molecular genetics of cancer.
Receptor tyrosine kinases (RTKs) participate in numerous developmental decisions. Ror RTKs are a family of orphan receptors that are related to muscle specific kinase (MuSK) and Trk neurotrophin receptors. MuSK assembles acetylcholine receptors at the neuromuscular junction, and Trk receptors function in the developing nervous system (reviewed in [3-5]). Rors have been identified in nematodes, insects and mammals. Recent studies have begun to shed light on Ror function during development. In most species, Rors are expressed in many tissue types during development. Analyses of mutants that are defective in the single nematode Ror demonstrate a role in cell migration and in orienting cell polarity. Mice lacking one of the two Ror gene products display defects in bone and heart formation. Similarly, two different human bone development disorders, dominant brachydactyly B and recessive Robinow syndrome, result from mutations in one of the human Ror genes.
Tissue-specific gene expression is mediated largely by transcription factors, and a master regulatory gene is thus a potential marker of cellular lineage. Using normal fetal through adult pulmonary tissues and 64 consecutive lung adenocarcinomas, we examined the expression of thyroid transcription factor (TTF-1), which plays a crucial role in normal lung function and morphogenesis. TTF-1 was expressed consistently throughout the life stages and uniformly in the terminal respiratory unit, which is comprised of peripheral airway cells and small-sized bronchioles. Furthermore, the expression was maintained in 72% of adenocarcinomas that exhibited high correlation with surfactant apoprotein (p <0.001) and morphologic resemblance to terminal respiratory unit cells (p <0.001). The staining pattern was also uniform in the adenocarcinomas despite histologic and microenvironmental diversity in individual tumors and their metastatic foci. This consistency and uniformity, therefore, suggested that TTF-1 expression could be used as a lineage marker of terminal respiratory unit. We also identified interesting distinctions between TTF-1-positive and -negative adenocarcinomas based on their clinicopathologic features and expression of various cancer-associated genes. TTF-1-positive adenocarcinomas had statistically significant prevalence of female (p <0.01), nonsmoker (p <0.05), negative p53 staining (p <0.01), less frequent RB loss (p <0.05), and preserved expression of p27 (p <0.01). The results supported the TTF-1 lineage marker and suggested that molecular pathogenesis may in part be characterized by cellular lineage.
A series of recent studies are providing tantalizing hints of new therapeutic approaches for combating cancer. In his Perspective, [Weinstein][1] discusses new work ([ Jain et al .][2]) demonstrating that even brief inactivation of an oncogene can permanently reverse the malignant phenotype of some types of tumor.
[1]: http://www.sciencemag.org/cgi/content/full/297/5578/63
[2]: http://www.sciencemag.org/cgi/content/short/297/5578/102
The c-SRC non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been linked to the development of cancer and progression to distant metastases. These observations have led to the recent targeting of c-SRC for the development of anticancer therapeutics, which show promise as a new avenue for cancer treatment. Despite this, however, the precise functions of c-SRC in cancer remain unclear. In addition to increasing cell proliferation, a key role of c-SRC in cancer seems to be to promote invasion and motility, functions that might contribute to tumour progression.
The ErbB2-targeting antibody, trastuzumab (Herceptin), has remarkable therapeutic efficacy in certain patients with ErbB2-overexpressing tumors. The overall trastuzumab response rate, however, is limited and what determines trastuzumab response is poorly understood. Here we report that PTEN activation contributes to trastuzumab's antitumor activity. Trastuzumab treatment quickly increased PTEN membrane localization and phosphatase activity by reducing PTEN tyrosine phosphorylation via Src inhibition. Reducing PTEN in breast cancer cells by antisense oligonucleotides conferred trastuzumab resistance in vitro and in vivo. Patients with PTEN-deficient breast cancers had significantly poorer responses to trastuzumab-based therapy than those with normal PTEN. Thus, PTEN deficiency is a powerful predictor for trastuzumab resistance. Additionally, PI3K inhibitors rescued PTEN loss-induced trastuzumab resistance, suggesting that PI3K-targeting therapies could overcome this resistance.
Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in specimens from patients with non-small-cell lung cancer who have a response to anilinoquinazoline EGFR inhibitors. Despite the dramatic responses to such inhibitors, most patients ultimately have a relapse. The mechanism of the drug resistance is unknown. Here we report the case of a patient with EGFR-mutant, gefitinib-responsive, advanced non-small-cell lung cancer who had a relapse after two years of complete remission during treatment with gefitinib. The DNA sequence of the EGFR gene in his tumor biopsy specimen at relapse revealed the presence of a second point mutation, resulting in threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance.
We have previously reported that terminal-respiratory-unit (TRU) type adenocarcinoma is a distinct subset of lung adenocarcinoma in terms of molecular pathway for carcinogenesis and phenotypic profiles. This type of cancer shows TRU features, characterized by distinct cellular morphology and the expression of TTF-1 and surfactant proteins. Recently, two groups published novel mutations of the epidermal growth factor receptor (EGFR) that are closely associated with clinical response to gefitinib. The clinicopathologic features of gefitinib responders overlap with those of TRU-type adenocarcinoma, and the characteristics of TRU are likely to correspond to the bronchioloalveolar features reported as a predictor of gefitinib response. We therefore examined the characteristics of EGFR-mutated pulmonary adenocarcinomas with special reference to TRU-type adenocarcinoma. EGFR mutation was detected in 97 of 195 adenocarcinomas, 91 of 149 TRU-type adenocarcinomas and 6 of 46 tumors of other types. Conversely, 91 of 97 EGFR-mutated adenocarcinomas were categorized as TRU-type adenocarcinomas. This type-specific involvement was confirmed by logistic regression model. In addition, EGFR mutation was detected in some cases of atypical adenomatous hyperplasia, a preinvasive lesion of TRU-type adenocarcinoma. These findings further confirm that TRU-type-adenocarcinoma is a distinct adenocarcinoma subset in which a particular molecular pathway is involved.
Lung cancers with neuroendocrine features are usually aggressive, although the underlying molecular mechanisms largely remain to be determined. The basic helix-loop-helix protein, achaete-scute complex-like 1/achaete-scute homologue 1 (ASH1), is expressed in normal fetal pulmonary neuroendocrine cells and lung cancers with neuroendocrine elements and is suggested to be involved in lung carcinogenesis. In the present study, we show inhibition of ASH1 expression by plasmid-based RNA interference (RNAi) to significantly suppress growth of lung cancer cells with ASH1 expression through G2-M cell cycle arrest and accumulation of sub-G1 populations, possibly linked to cleavage of caspase-9 and caspase-7. However, lung cancer cell lines without ASH1 expression and immortalized normal BEAS2B bronchial epithelial cells were not affected. The RNAi-resistant mutant ASH1 clearly induced rescue from G2-M arrest, suggesting a target-specific effect of RNAi. An ASH1-RNAi adenovirus was also established and significantly inhibited not only in vitro cell proliferation but also in vivo xenograft growth of ASH1-positive NCI-H460 cells. Elevated levels of apoptosis were also observed in NCI-H460 xenografts with the ASH1-RNAi adenovirus. The present study therefore suggests that ASH1 plays a crucial role in lung cancer development and may be an effective therapeutic target in lung cancers with neuroendocrine features.
This study was conducted to gain insight into the relationship between expression profiles and underlying genetic changes, which are known to be important for the pathogenesis of lung cancers.
Expression profiles of 18,175 unique genes and three major targets for genetic changes, p53, epidermal growth factor receptor (EGFR), and K-ras, were investigated in 149 patients with non-small-cell lung cancer, including 90 patients with adenocarcinoma to determine their relationships with various clinicopathologic features and Gene Ontology (GO) terms.
This study successfully established a basis for expression profile-defined classification, which can classify adenocarcinomas into two major types, terminal respiratory unit (TRU) type and non-TRU type. Our GO term-based identifier of particular biologic processes, molecular functions, and cellular compartments clearly showed characteristic retention of normal peripheral lung features in TRU type, in sharp contrast to the significant association of non-TRU type with cell cycling and proliferation-related features. While significantly higher frequency of EGFR mutation was observed in TRU type, we found that the presence of EGFR mutations was a significant predictor of shorter postoperative survival for TRU type, independent of disease stage. We were also able to identify a set of genes in vivo with significant upregulation in the presence of EGFR mutations.
This study has shed light on heterogeneity in lung cancers, especially in adenocarcinomas, by establishing a molecularly, genetically, and clinically relevant, expression profile-defined classification. Future studies using independent patient cohorts are warranted to confirm the prognostic significance of EGFR mutations in TRU-type adenocarcinoma.
Although cell-lineage and differentiation models dominate tumour classification and treatment, the recognition that cancer is also a genomic disease has prompted a reconfiguration of cancer taxonomies according to molecular criteria. Recent evidence indicates that a synthesis of lineage-based and genetic paradigms might offer new insights into crucial and therapeutically pliable tumour dependencies. For example, MITF (microphthalmia-associated transcription factor), which is a master regulator of the melanocyte lineage, might become a melanoma oncogene when deregulated in certain genetic contexts. MITF and other lineage-survival genes therefore implicate lineage dependency (or lineage addiction) as a newly recognized mechanism that is affected by tumour genetic alterations.
The ErbB family of four receptor tyrosine kinases occupies a central role in a wide variety of biological processes from neuronal development to breast cancer. New information continues to expand their biologic significance and to unravel the molecular mechanisms that underlie the signaling capacity of these receptors. Here, we review several aspects of ErbB receptor physiology for which new and significant information is available. These include ligand-dependent receptor dimerization and kinase activation, which is a prerequisite for all subsequent growth factor-dependent cell responses. We also address novel roles of receptor fragments in signaling, trafficking to intracellular sites, such as the nucleus, and ErbB roles in non-cancer disease processes, including schizophrenia, chronic renal disease, hypertension, and the cellular entry of infectious pathogens.
Emerging evidence, although currently very sparse, suggests the presence of "lineage-specific dependency" in the survival mechanisms of certain cancers. TTF-1 has a decisive role as a master regulatory transcription factor in lung development and in the maintenance of the functions of terminal respiratory unit (TRU) cells. We show that a subset of lung adenocarcinoma cell lines expressing TTF-1, which presumably represent those derived from the TRU lineage, exhibit marked dependence on the persistent expression of TTF-1. The inhibition of TTF-1 by RNA interference (RNAi) significantly and specifically induced growth inhibition and apoptosis in these adenocarcinoma cell lines. Furthermore, a fraction of TTF-1-expressing tumors and cell lines displayed an increase in the gene dosage of TTF-1 in the analysis of 214 patients with non-small-cell lung cancer, including 174 adenocarcinomas, showing a tendency of higher frequency of increased gene copies at metastatic sites than at primary sites (P=0.07, by two-sided Fisher's exact test). These findings strongly suggest that in addition to the development and maintenance of TRU lineages in normal lung, sustained TTF-1 expression may be crucial for the survival of a subset of adenocarcinomas that express TTF-1, providing credence for the lineage-specific dependency model.