Oncogene

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Online ISSN: 0950-9232
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Article
  • Xiaosong HuXiaosong Hu
  • Ruochen LiuRuochen Liu
  • Jianbing HouJianbing Hou
  • [...]
  • Hongjuan CuiHongjuan Cui
SMARCE1 gene, encoding a core subunit of SWI/SNF chromatin remodeling complex, is situated on chromosome 17q21-ter region that is frequently gained in neuroblastoma. However, its role in the tumorigenesis remains unknown. Here, we showed that high expression of SMARCE1 was associated with poor prognosis of patients with neuroblastoma, especially those with MYCN amplification. Knockdown of SMARCE1 reduced proliferation, colony formation, and tumorigenicity of neuroblastoma cells. Mechanistically, SMARCE1 directly interacted with MYCN, which was necessary for MYCN-mediated transcriptional activation of downstream target genes including PLK1, ODC1, and E2F2. Overexpression of PLK1, ODC1 or E2F2 significantly reversed the inhibiting effect of SMARCE1 knockdown on the proliferation, colony formation, and tumorigenicity of MYCN-amplified neuroblastoma cells. Moreover, we revealed that MYCN directly regulated SMARCE1 transcription through binding to a non-canonical E-box of SMARCE1 promoter, thus enhancing SMARCE1-MYCN cooperativity. These findings establish SMARCE1 is a critical oncogenic factor in neuroblastoma and provide a new potential target for treatment of neuroblastoma with 17q21-ter gain and MYCN amplification.
 
Article
  • Luyao WeiLuyao Wei
  • Wantao WangWantao Wang
  • Junxia YaoJunxia Yao
  • [...]
  • Zun-Ji KeZun-Ji Ke
Most basal-like breast cancers (BLBCs) are triple-negative breast cancers (TNBCs), which is associated with high malignancy, high rate of recurrence and distant metastasis, and poor prognosis among all types of breast cancer. However, there are currently no effective therapies for BLBC. Furthermore, chemoresistance limits the therapeutic options for BLBC treatment. In this study, we screen out protein activator of the interferon-induced protein kinase (PACT) as an essential gene in BLBC metastasis. We find that high PACT expression level was associated with poor prognosis among BLBC patients. In vivo and in vitro investigations indicated that PACT could regulate BLBC metastasis by interacting with SUMO-conjugating enzyme Ubc9 to stimulate the SUMOylation and thus consequently the activation of Rac1. BLBC patients receiving chemotherapy presents poorer prognosis with PACT high expression, and PACT disruption sensitizes experimental mammary tumor metastases to chemotherapy, thus providing insights to consider PACT as a potential therapeutic target to overcome acquired chemoresistance in BLBC.
 
Article
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.
 
Article
Transcriptomic analyses of pancreatic ductal adenocarcinoma (PDAC) have identified two major epithelial subtypes with distinct biology and clinical behaviours. Here, we aimed to clarify the role of FGFR1 and FGFR4 in the definition of aggressive PDAC phenotypes. We found that the expression of FGFR4 is exclusively detected in epithelial cells, significantly elevated in the classical PDAC subtype, and associates with better outcomes. In highly aggressive basal-like/squamous PDAC, reduced FGFR4 expression aligns with hypermethylation of the gene and lower levels of histone marks associated with active transcription in its regulatory regions. Conversely, FGFR1 has more promiscuous expression in both normal and malignant pancreatic tissues and is strongly associated with the EMT phenotype but not with the basal-like cell lineage. Regardless of the genetic background, the increased proliferation of FGFR4-depleted PDAC cells correlates with hyperactivation of the mTORC1 pathway both in vitro and in vivo. Downregulation of FGFR4 in classical cell lines invariably leads to the enrichment of basal-like/squamous gene programs and is associated with either partial or full switch of phenotype. In sum, we show that endogenous levels of FGFR4 limit the malignant phenotype of PDAC cells. Finally, we propose FGFR4 as a valuable marker for the stratification of PDAC patients.
 
PrPC and Caveolin-1 relationship across cancer
A Heatmap showing the top genes most positively and negatively correlated with the expression on PRNP in the CCLE. B qRT-PCR analysis of the relative expression of PRNP and CAV1 in PC3 prostate cancer cells versus PNT2 normal prostate cells (left panel); relative expression of PrPC and CAV1 mRNA (middle panel) or protein (right panel) in PRNP-silenced versus control PC3 prostate cancer cells. C qRT-PCR analysis of the relative expression of CAV1 PrPC-overexpressing versus control LoVo colon cancer cells (day 3 post-transfection) (left panel); relative expression of PrPC and CAV1 mRNA (middle panel) or protein (right panel) in PRNP-silenced versus control MDST8 colon cancer cells. Results are expressed as means of n = 2 independent triplicates of cell preparations ± s.e.m. *p < 0.05, ***p < 0.001 vs. control (Mann-Whitney test). Quantification for western blots is provided in Supplementary Fig. S2, with protein levels normalized to α-tubulin (α-tub). D Proximity ligation assay showing co-localization of PrPC and CAV1 in MDST8 cells (orange spots, right panel). Cells incubated with anti-PrPC antibody alone (left panel) or anti-Caveolin-1 antibody alone (middle panel) were used as controls. Nuclei were stained with DAPI.
PrPC controls Aβ levels in prostate and colon cancer cells
A qRT-PCR analysis of the relative expression of APP and BACE1 in PC3 prostate cancer cells versus PNT2 normal prostate cells (left panel). Extracellular Aβ40 and Aβ42 levels were measured in the supernatants of PNT2 and PC3 cells (right panels). BAPP and BACE1 mRNA levels, APP protein levels and extracellular Aβ40 and Aβ42 levels were measured in PRNP-silenced versus control PC3 prostate cancer cell extracts and supernatants. CAPP and BACE1 mRNA levels, APP protein levels and extracellular Aβ40 and Aβ42 levels were measured in PRNP-silenced versus control MDST8 colon cancer cell extracts and supernatants. DAPP and BACE1 mRNA levels and extracellular Aβ40 and Aβ42 levels were measured in PrPC-overexpressing versus control LoVo colon cancer cell extracts (day 3 post-transfection) and supernatants (days 3, 4 and 5 post-transfection). In B and C, protein levels were normalized to α-tubulin (α-tub). Results are expressed as means of n = 2 independent triplicates of cell preparations ± s.e.m. *p < 0.05, **p < 0.01 vs. control (Mann-Whitney test except for D middle and right panels Kruskal-Wallis and posthoc Wilcoxon rank-sum test with Holm’s correction). Quantification for western blots is provided in Supplementary Fig. S2C, D.
Identification of potential Aβ-PrPC targets and impact of antibody-mediated blockade of the Aβ-PrPC interaction
A qRT-PCR analysis of the relative expression of DKK1 and PDGFC in PC3 prostate cancer cells versus PNT2 normal prostate cells (left panels) as well as in PRNP-silenced versus control PC3 cells (middle panel) and qRT-PCR analysis of the relative expression of DKK1, DKK3 and PDGFC in PRNP-silenced versus control MDST8 cells (right panel). BDKK1 and PDGFC mRNA levels and extracellular Aβ40, Aβ42 and TGFβ levels were measured in cell extracts and supernatants of PC3 prostate cancer cells exposed to 6D11 antibodies versus control isotype antibodies. CDKK3 and PDGFC mRNA levels and extracellular Aβ40, Aβ42 and TGFβ levels were measured in cell extracts and supernatants of MDST8 colon cancer cells exposed to 6D11 antibodies versus control isotype antibodies. Results are expressed as means of n = 2 independent triplicates of cell preparations (except for A and Bn = 2 independent duplicates of cell preparations) ± s.e.m. *p < 0.05, ***p < 0.001 vs. control (Mann-Whitney test). D GSEA analysis showing the downregulation of the TGFβ signalling, regulation of actin cytoskeleton and focal adhesion signatures in 6D11-treated versus control MDST8 cells. NES normalized enrichment score.
The Aβ-PrPC axis can be targeted in vivo and has clinical relevance in basal prostate cancer and mesenchymal colon cancer
A Top panels. Tumour growth, survival curves and number of metastases in mice bearing PC3 xenografts and treated with 5, 7.5 or 10 mg per kg of 6D11 antibody (Ab-5, Ab-7.5, Ab-10, respectively) versus control mice (Sham). Bottom panels. Tumour size (measured at autopsy for the sham and Aβ groups, and at day 40 for the Ab-10 and the Aβ + Ab-10 groups), survival curves and number of metastases in mice bearing PC3 xenografts and treated with recombinant Aβ, 6D11 antibody (at 10 mg per kg, Ab-10) or both Aβ and 6D11 antibody (Aβ + Ab-10) versus control mice (Sham). Data for tumour size and number of metastases are expressed as means ± s.e.m. of n = 5 values (*p < 0.05 and **p < 0.01 versus Sham, §p < 0.05 and §§p < 0.01 versus Aβ alone, Kruskal-Wallis and posthoc Wilcoxon rank-sum test with Holm’s correction for tumour size and number of metastases, log-rank test for survival curves. ns not significant). B Volcano plot showing the enrichment of CAV1, DKK1, DKK3 and PRNP transcripts in basal versus luminal benign prostate cancers from the Zhang study [26] (left panel); Enrichment of APP, BACE1, CAV1, DKK1, DKK3, PDGFC and PRNP transcripts in CD49fHi versus CD49fLo prostate tissue from the Smith study [27] (middle panel) (*p < 0.05 and ***p < 0.001 versus CD49fLo, two-tailed t-test); Volcano plot showing the upregulation of CAV1, DKK1, DKK3, PDGFC and PRNP transcripts after ADT in the Rajan study [32] (right panel). C Heatmaps showing the distribution of PRNP-associated genes in prostate cancer patients (E-MATB6128 data set) according to the PAM50 classification by Zhao [29] (left panel) or in colon cancer patients (GSE39582 dataset) according to the CMS classification by Guinney [31] (right panel). LumA: Luminal A. Lum B: Luminal B. NT: non tumour. See Supplementary Fig. S5 and S7 for statistics. D Kaplan-Meier overall survival (OS) (left panel) and relapse free survival (RFS) (right panel) according to high and low BACE1 gene expression was determined in colon cancer patients of the GSE39582 dataset. Hazard ratios were adjusted for TNM stage, MMR status and adjuvant chemotherapy.
Article
The cellular prion protein PrPC partners with caveolin-1 (CAV1) in neurodegenerative diseases but whether this interplay occurs in cancer has never been investigated. By leveraging patient and cell line datasets, we uncover a molecular link between PrPC and CAV1 across cancer. Using cell-based assays, we show that PrPC regulates the expression of and interacts with CAV1. PrPC additionally controls the expression of the amyloid precursor protein APP and of the Aβ generating enzyme BACE1, and regulates the levels of Aβ, whose accumulation is a central event in Alzheimer’s disease. We further identify DKK1 and DKK3, involved in both Alzheimer’s disease and cancer progression, as targets of the PrPC-dependent axis. Finally, we establish that antibody-mediated blocking of the Aβ-PrPC interaction delays the growth of prostate cancer cell line-derived xenografts and prevents the development of metastases. Our data additionally support an enrichment of the Aβ-PrPC-dependent pathway in the basal subtype of prostate cancer, associated with anti-hormonal therapy resistance, and in mesenchymal colon cancer, associated with poor prognosis. Thus, based on a parallel with neurodegenerative diseases, our results bring to light an Aβ-PrPC axis and support the potential of targeting this pathway in patients with selected subtypes of prostate and colon cancer.
 
Article
LKB1 loss of function is one key oncogenic event in lung cancer. Clinical data suggest that LKB1 loss of function is associated with patients’ smoking status. The responsible ingredients and molecular mechanisms in tobacco for LKB1 loss of function, however, are not defined. In this study, we reported that NNAL, a major metabolite of a tobacco-specific carcinogen NNK, induces LKB1 phosphorylation and its loss of function via the β-AR/PKA signaling pathway in an isomer-dependent manner in human lung cancer cells. NNAL exposure also resulted in enhanced lung cancer cell migration and chemoresistance in an LKB1-dependent manner. A 120-day NNAL exposure in lung cancer cells, mimicking its chronic exposure among smokers, resulted in more prominent LKB1 phosphorylation, cell migration, and chemoresistance even in the absence of NNAL, indicating the long-lasting LKB1 loss of function although such an effect eventually disappeared after NNAL was removed for two months. These observations were confirmed in a lung cancer xenograft model. More importantly, human lung cancer tissues revealed elevated LKB1 phosphorylation in comparison to the paired normal lung tissues. These results suggest that LKB1 loss of function in human lung cancer could be extended to its phosphorylation, which may be mediated by NNAL from tobacco smoke in an isomer-dependent manner via the β-AR/PKA signaling pathway.
 
Article
Uncontrolled proliferation of intestinal epithelial cells caused by mutations in genes of the WNT/β-catenin pathway is associated with development of intestinal cancers. We previously reported that intestinal stromal cell-derived angiopoietin-like protein 2 (ANGPTL2) controls epithelial regeneration and intestinal immune responses. However, the role of tumor cell-derived ANGPTL2 in intestinal tumorigenesis remained unclear. Here, we show that tumor cell-derived ANGPTL2 promotes β-catenin-driven intestinal tumorigenesis. ANGPTL2 deficiency suppressed intestinal tumor development in an experimental mouse model of sporadic colon cancer. We also found that increased ANGPTL2 expression in colorectal cancer (CRC) cells augments β-catenin pathway signaling and promotes tumor cell proliferation. Relevant to mechanism, our findings suggest that tumor cell-derived ANGPTL2 upregulates expression of OB-cadherin, which then interacts with β-catenin, blocking destruction complex-independent proteasomal degradation of β-catenin proteins. Moreover, our observations support a model whereby ANGPTL2-induced OB-cadherin expression in CRC cells is accompanied by decreased cell surface integrin α5β1 expression. These findings overall provide novel insight into mechanisms of β-catenin-driven intestinal tumorigenesis.
 
Article
Heme oxygenase-1 (HO-1) is an inducible heme degradation enzyme that plays a cytoprotective role against various oxidative and inflammatory stresses. However, it has also been shown to exert an important role in cancer progression through a variety of mechanisms. Although transcription factors such as Nrf2 are involved in HO-1 regulation, the posttranslational modifications of HO-1 after oxidative insults and the underlying mechanisms remain unexplored. Here, we screened and identified that the deubiquitinase USP7 plays a key role in the control of redox homeostasis through promoting HO-1 deubiquitination and stabilization in hepatocytes. We used low-dose arsenic as a stress model which does not affect the transcriptional level of HO-1, and found that the interaction between USP7 and HO-1 is increased after arsenic exposure, leading to enhanced HO-1 expression and attenuated oxidative damages. Furthermore, HO-1 protein is ubiquitinated at K243 and subjected to degradation under resting conditions; whereas when after arsenic exposure, USP7 itself can be ubiquitinated at K476, thereafter promoting the binding between USP7 and HO-1, finally leading to enhanced HO-1 deubiquitination and protein accumulation. Moreover, depletion of USP7 and HO-1 inhibit liver tumor growth in vivo, and USP7 positively correlates with HO-1 protein level in clinical human hepatocellular carcinoma (HCC) specimens. In summary, our findings reveal a critical role of USP7 as a HO-1 deubiquitinating enzyme in the regulation of oxidative stresses, and suggest that USP7 inhibitor might be a potential therapeutic agent for treating HO-1 overexpressed liver cancers.
 
Article
A plethora of studies have shown that both DNMT1 and EZH2 have great effects on the progression of a variety of cancers. However, it remains unclear whether the expression profiles of these two epigenetic enzymes are molecularly intertwined in prostate cancer (PC), especially in castration-resistant prostate cancer (CRPC). Here, we found that DNMT1 is highly expressed and facilitates PC cell proliferation and migration. Importantly, we demonstrate that the abrogation of DNMT1 expression can induce the decreased expression of EZH2, resulting in the less aggressive capacity of PC cells. Mechanistically, we discovered that DNMT1 promotes PC tumorigenesis and metastasis by inhibiting TRAF6 transcriptional expression and subsequent TRAF6-mediated EZH2 ubiquitination. Finally, we confirmed that there is a negative correlation between DNMT1 and TRAF6 expression and a positive correlation between DNMT1 and EZH2 expression in PC patients. In this study, we first disclose that there is a direct crosstalk between DNA methyltransferase DNMT1 expression and histone methyltransferase EZH2 expression in tumorigenesis and cancer metastasis in vitro and in vivo. Our results also show that targeting DNMT1 with its inhibitor decitabine (an FDA-approved drug) is an appealing treatment strategy for CRPC patients through epigenetic suppression of both DNMT1-mediated DNA methylation and EZH2-modulated histone methylation.
 
Article
PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.
 
Article
Circular RNAs (circRNAs) play critical roles in clear cell renal cell carcinoma (ccRCC). However, their involvement in sunitinib resistance remains largely unknown. Herein, we identified a novel circRNA, named circME1, which contributes to sunitinib resistance development in ccRCC. CircME1 also promoted proliferation, migration, and invasion of ccRCC cells. Further mechanism analysis showed that circME1 interacted with U1 snRNP at the promoter of its parental gene ME1, thereby upregulating the expression of ME1, enhancing aerobic glycolysis of ccRCC, and promoting its malignant phenotype. Furthermore, ME1 specific inhibitor could effectively repress the oncogenic functions of circME1. Taken together, our study demonstrates that the circME1/ME1 pathway is involved in ccRCC progression and sunitinib resistance development, which may be exploited for anticancer therapy.
 
Article
The acquisition of novel detrimental cellular properties following exposure to cytotoxic drugs leads to aggressive and metastatic tumors that often translates into an incurable disease. While the bulk of the primary tumor is eliminated upon exposure to chemotherapeutic treatment, residual cancer cells and non-transformed cells within the host can engage a stable cell cycle exit program named senescence. Senescent cells secrete a distinct set of pro-inflammatory factors, collectively termed the senescence-associated secretory phenotype (SASP). Upon exposure to the SASP, cancer cells undergo cellular plasticity resulting in increased proliferation, migration and epithelial-to-mesenchymal transition. The molecular mechanisms by which the SASP regulates these pro-tumorigenic features are poorly understood. Here, we report that breast cancer cells exposed to the SASP strongly upregulate Lipocalin-2 (LCN2). Furthermore, we demonstrate that LCN2 is critical for SASP-induced increased migration in breast cancer cells, and its inactivation potentiates the response to chemotherapeutic treatment in mouse models of breast cancer. Finally, we show that neoadjuvant chemotherapy treatment leads to LCN2 upregulation in residual human breast tumors, and correlates with worse overall survival. These findings provide the foundation for targeting LCN2 as an adjuvant therapeutic approach to prevent the emergence of aggressive tumors following chemotherapy.
 
Article
Response to cancer immunotherapy in primary versus metastatic disease has not been well-studied. We found primary pancreatic ductal adenocarcinoma (PDA) is responsive to diverse immunotherapies whereas liver metastases are resistant. We discovered divergent immune landscapes in each compartment. Compared to primary tumor, liver metastases in both mice and humans are infiltrated by highly anergic T cells and MHCIIloIL10+ macrophages that are unable to present tumor-antigen. Moreover, a distinctive population of CD24+CD44−CD40− B cells dominate liver metastases. These B cells are recruited to the metastatic milieu by Muc1hiIL18hi tumor cells, which are enriched >10-fold in liver metastases. Recruited B cells drive macrophage-mediated adaptive immune-tolerance via CD200 and BTLA. Depleting B cells or targeting CD200/BTLA enhanced macrophage and T-cell immunogenicity and enabled immunotherapeutic efficacy of liver metastases. Our data detail the mechanistic underpinnings for compartment-specific immunotherapy-responsiveness and suggest that primary PDA models are poor surrogates for evaluating immunity in advanced disease.
 
Article
Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies worldwide. Recently, our group identified purine-rich element binding protein alpha (PURα), a single-stranded DNA/RNA-binding protein, to be significantly associated with the progression of ESCC. Additional immunofluorescence staining demonstrated that PURα forms cytoplasmic stress granules to suppress mRNA translation initiation. The expression level of cytoplasmic PURα in ESCC tumor tissues was significantly higher than that in adjacent epithelia and correlated with a worse patient survival rate by immunohistochemistry. Functionally, PURα strongly preferred to bind to UG-/U-rich motifs and mRNA 3´UTR by CLIP-seq analysis. Moreover, PURα knockout significantly increased the protein level of insulin-like growth factor binding protein 3 (IGFBP3). In addition, it was further demonstrated that PURα-interacting proteins are remarkably associated with translation initiation factors and ribosome-related proteins and that PURα regulates protein expression by interacting with translation initiation factors, such as PABPC1, eIF3B and eIF3F, in an RNA-independent manner, while the interaction with ribosome-related proteins is significantly dependent on RNA. Specifically, PURα was shown to interact with the mRNA 3´UTR of IGFBP3 and inhibit its expression by suppressing mRNA translation initiation. Together, this study identifies cytoplasmic PURα as a modulator of IGFBP3, which could be a promising therapeutic target for ESCC treatment.
 
Article
DNA repair gene mutations are frequent in castration-resistant prostate cancer (CRPC), suggesting eligibility for poly(ADP-ribose) polymerase inhibitor (PARPi) treatment. However, therapy resistance is a major clinical challenge and genes contributing to PARPi resistance are poorly understood. Using a genome-wide CRISPR-Cas9 knockout screen, this study aimed at identifying genes involved in PARPi resistance in CRPC. Based on the screen, we identified PARP1, and six novel candidates associated with olaparib resistance upon knockout. For validation, we generated multiple knockout populations/clones per gene in C4 and/or LNCaP CRPC cells, which confirmed that loss of PARP1, ARH3, YWHAE, or UBR5 caused olaparib resistance. PARP1 or ARH3 knockout caused cross-resistance to other PARPis (veliparib and niraparib). Furthermore, PARP1 or ARH3 knockout led to reduced autophagy, while pharmacological induction of autophagy partially reverted their PARPi resistant phenotype. Tumor RNA sequencing of 126 prostate cancer patients identified low ARH3 expression as an independent predictor of recurrence. Our results advance the understanding of PARPi response by identifying four novel genes that contribute to PARPi sensitivity in CRPC and suggest a new model of PARPi resistance through decreased autophagy.
 
FOXA1 down-regulation in PCa cells induces hypoxia programs
A GO of FOXA1-repressed genes with adjusted p < = 0.001 and log2 fold change > = 2. RNA-seq of LNCaP cells with control and FOXA1 KD was performed in triplicate experiments. Cells were treated with 100 µM CoCl2 for 24 h to mimic tumor hypoxia. B GSAA revealed that hypoxia hallmark gene signature is significantly enriched for up-regulation upon FOXA1 KD (FDR q = 0.00). Heatmap depicts the leading edge genes according to GSAA. C Heatmaps of hypoxia-induced (vs. normoxia) genes in LNCaP (left) and PC3 cells (right), derived from the published dataset (GSE106305), in our RNA-seq data of control and FOXA1 KD (shFOXA1) LNCaP cells. D FOXA1 expression is negatively correlated with hypoxia marker CA9 in TCGA PRAD tumors (n = 498). (The x/y values are Z-scores.). E IHC staining (20X) of CA9 (dilution 1:500, top) and FOXA1 (dilution 1:200, bottom) in several LuCaP PDX tumors.
FOXA1 directly inhibits HIF1A transcription through an intragenic enhancer
FOXA1 KD enhances HIF1A expression in LNCaP, C4-2B, and 22Rv1 cells. PCa cells were infected with shCtrl, shFOXA1 lentivirus, followed by 1ug/ml puromycin selection. Then cells were treated with 100 µM CoCl2 for 24 h to mimic hypoxia before being harvested. Cells were analyzed by A qRT-PCR (results are shown as mean ± s.e.m. of three technical replicates from one representative experiment, *p < 0.05) and B WB analyses. C FOXA1 overexpression represses HIF1A expression in PC-3M cells. PC-3M cells were infected with empty vector control or FOXA1-HA-Flag lentivirus. At 24 h before harvesting, cells were treated with 100 µM CoCl2 to induce hypoxia. Cells were analyzed by western blot. D Genome browser view showing FOXA1 binding at a HIF1A intragenic enhancer. FOXA1 ChIP-seq was performed in shCtrl and shFOXA1 LNCaP cells. A zoomed-in view of the FOXA1 binding region shows several FOXA1 motifs and identifies the sgRNAs targeting the intragenic enhancer for deletion (corresponding to Fig. 2G). E ChIP-qPCR confirms FOXA1 binding at the HIF1A enhancer in LNCaP cells. FOXA1 and IgG ChIP were performed in LNCaP cells with or without FOXA1 KD. Results are shown as mean ± s.e.m. of three technical replicates from one representative experiment, *p < 0.05. F RNA Pol II (phosphorylated at Ser5) occupancy at the HIF1A promoter is increased upon FOXA1 KD. ChIP was performed in shCtrl or shFOXA1 LNCaP cells using antibodies against phospho-Pol II Ser5. Results are shown as mean ± s.e.m. of 3 technical replicates from one representative experiment, *p < 0.05. G qRT-PCR analysis of HIF1A gene expression (relative to GAPDH) in LNCaP cells treated with CRISPR/Cas9 sgRNAs targeting the HIF1A intragenic enhancer region (depicted in Fig. 2D). Results are shown as mean ± s.e.m. of 3 technical replicates from one representative experiment, *p < 0.05.
FOXA1 inhibits hypoxia programs and CCL2 gene expression through HIF1A
A RNA-seq analyses of shCtrl, shFOXA1, shHIF1A, and shFOXA1 + shHIF1A LNCaP cells treated with 100 µM CoCl2 for 24 h to mimic hypoxia. FOXA1-repressed genes depicted in the heatmap were identified using an adjusted p-value < = 0.01 and fold change > = 2 as a cutoff. FOXA1-repressed genes formed two clusters: Cluster I and II. Cluster I consists of genes upregulated upon FOXA1 KD, in a HIF1A-dependent manner (n = 300). Cluster II consists of genes upregulated upon FOXA1 KD, independent of HIF1A (n = 222). B GO analysis for Hallmark gene sets was performed on Clusters I and II FOXA1-repressed genes to identify Hallmark gene sets significantly enriched in each cluster. C HIF1A KD reverses FOXA1 loss-induced CCL2 expression. LNCaP, C4-2B, and 22Rv1 cells were infected with shCtrl, shFOXA1, or shFOXA1 + shHIF1A lentivirus, followed by 1 ug/ml puromycin selection. The cells were then treated with 100 µM CoCl2 for 24 h to induce hypoxia before being harvested, and CCL2 mRNA expression (relative to GAPDH) was analyzed by qRT-PCR. Results are shown as mean ± s.e.m. of 3 technical replicates from one representative experiment, *p < 0.05. D ELISA of the conditioned medium of LNCaP and C4-2B cells infected with shCtrl, shFOXA1, or shFOXA1 + shHIF1A lentivirus, under hypoxic condition (+100 µM CoCl2), was performed to measure CCL2 secretion. Results are shown as mean ± S.D. of 2 technical replicates from one experiment, *p < 0.05. E CCL2 and FOXA1 expression are negatively correlated in human PCa samples from public datasets GSE21034 [37] and GSE3933 [38]. (The x/y values are Z-scores.). F CCL2 and HIF1A expression are positively correlated in human PCa samples from public datasets. (The x/y values are Z-scores).
FOXA1 loss-induced HIF1A promotes macrophage infiltration and cell invasion
FOXA1 KD promotes macrophage infiltration. A, B U937M cells were seeded in the upper chamber of transwell plates, with control medium, shCtrl, and shFOXA1 LNCaP or C4-2B cells (treated with 100 µM CoCl2) in the bottom chamber. Migrated macrophages were counted from several random fields within each transwell after 24 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one representative experiment, *p < 0.05. C FOXA1 KD promotes macrophage infiltration, while concomitant HIF1A KD reverses FOXA1 loss-induced macrophage infiltration. U937M cells were cultured in the upper chamber of the transwell system, with shCtrl, shFOXA1, or shFOXA1 + shHIF1A LNCaP cells (treated with 100 µM CoCl2) in the bottom chamber. Migrated macrophages were counted from several random fields within each transwell after 24 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one representative experiment, *p < 0.05. D FOXA1 KD in LNCaP cells induces cell invasion, which can be blocked by shHIF1A. Ctrl, shFOXA1, and shFOXA1 + shHIF1A LNCaP cells (treated with 100 µM CoCl2) were cultured in the top chamber of Matrigel-coated transwells, with the medium in the bottom chamber for a transwell invasion assay. Invaded PCa cells were counted from several random fields within each transwell after 72 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one experiment, *p < 0.05. E FOXA1 KD in PC-3M cells induces macrophage infiltration, which can be blocked by shHIF1A, while FOXA1 OE reduces macrophage infiltration. U937M cells were cultured in the upper chamber of the transwell co-culture system, with Ctrl, shFOXA1, shFOXA1 + shHIF1A, and FOXA1 OE PC-3M cells (treated with 100 µM CoCl2) in the bottom chamber. Migrated macrophages were counted from several random fields within each transwell after 36 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one representative experiment, *p < 0.05. F FOXA1 KD in PC-3M cells induces cell invasion, which can be blocked by shHIF1A, while FOXA1 OE reduces cell invasion. Ctrl, shFOXA1, shFOXA1 + shHIF1A, and FOXA1 OE PC-3M cells (treated with 100 µM CoCl2) were cultured in the top chamber of Matrigel-coated transwells, with the medium in the bottom chamber. Invaded PCa cells were counted from several random fields within each transwell after 72 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one representative experiment, *p < 0.05.
Targeting HIF1A/CCL2 axis abolishes FOXA1 loss-induced hypoxia programs, macrophage infiltration, and cell invasion
A LNCaP, C4-2B, and 22Rv1 cells were infected with shCtrl or shFOXA1 lentivirus and treated with 40 µM KC7F2 for 24 h. Cells were also treated with 100 µM CoCl2 for 24 h to mimic hypoxia, and HIF1A protein expression was measured by western blot. B RNA-seq analysis of shCtrl and shFOXA1 LNCaP cells with or without 40 µM HIF1A inhibitor KC7F2 treatment, under hypoxic conditions (+100 µM CoCl2). The heatmap shows a normalized expression of shFOXA1-induced genes across all samples. C GSAA of Hallmark hypoxia gene set in shFOXA1 vs. shFOXA1 + KC7F2 LNCaP RNA-seq data. D GSEA of Hallmark EMT genes in shFOXA1 vs. shFOXA1 + KC7F2 LNCaP RNA-seq data. E KC7F2 suppressed FOXA1 loss-induced CCL2 mRNA expression. LNCaP cells infected with either shCtrl or shFOXA1 lentivirus were treated with 40 µM KC7F2 for 24 h under hypoxic conditions (+100 µM CoCl2). Then HIF1A and CCL2 gene expression (relative to GAPDH) were analyzed by qRT-PCR. Results are shown as mean ± s.e.m. of 3 technical replicates from one representative experiment, using the same shCtrl and shFOXA1 C4-2B/22Rv1 samples as Fig. 3C, *p < 0.05. F KC7F2 reversed FOXA1 loss-induced macrophage infiltration. U937M cells were cultured in the upper chamber of the transwell system, and shCtrl or shFOXA1 LNCaP cells treated with or without 40 µM KC7F2 were in the bottom chamber (in RPMI media + 100 µM CoCl2). Migrated macrophages were counted from several random fields within each transwell after 24 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one representative experiment, using the same shCtrl and shFOXA1 samples as Fig. 4C, *p < 0.05. G KC7F2 treatment blocks FOXA1 KD-induced cell invasion in LNCaP cells. Ctrl, shFOXA1, and shFOXA1 + 40 µM KC7F2 LNCaP cells (+100 µM CoCl2) were cultured in the top chamber of Matrigel-coated transwells, with the medium in the bottom chamber. Invaded PCa cells were counted from several random fields within each transwell after 72 h. Results are expressed as the average number of cells per field (mean ± S.D.) from one experiment, *p < 0.05. H A model depicting FOXA1 regulation of hypoxia programs and PCa progression through HIF1A. FOXA1 loss in PCa cells induces HIF1A expression, which upregulates hypoxia response genes and CCL2 expression, recruiting tumor-promoting M2-like macrophages and promoting PCa EMT and cell invasion, which can be blocked using HIF1A-CCL2 inhibitors.
Article
Intratumoral hypoxia is associated with castration-resistant prostate cancer (CRPC), a lethal disease. FOXA1 is an epithelial transcription factor that is down-regulated in CRPC. We have previously reported that FOXA1 loss induces epithelial-mesenchymal transition (EMT) and cell motility through elevated TGFβ signaling. However, whether FOXA1 directly regulates hypoxia pathways of CRPC tumors has not been previously studied. Here we report that FOXA1 down-regulation induces hypoxia transcriptional programs, and FOXA1 level is negatively correlated with hypoxia markers in clinical prostate cancer (PCa) samples. Mechanistically, FOXA1 directly binds to an intragenic enhancer of HIF1A to inhibit its expression, and HIF1A, in turn, is critical in mediating FOXA1 loss-induced hypoxia gene expression. Further, we identify CCL2, a chemokine ligand that modulates tumor microenvironment and promotes cancer progression, as a crucial target of the FOXA1-HIF1A axis. We found that FOXA1 loss leads to immunosuppressive macrophage infiltration and increased cell invasion, dependent on HIF1A expression. Critically, therapeutic targeting of HIF1A-CCL2 using pharmacological inhibitors abolishes FOXA1 loss-induced macrophage infiltration and PCa cell invasion. In summary, our study reveals an essential role of FOXA1 in controlling the hypoxic tumor microenvironment and establishes the HIF1A-CCL2 axis as one mechanism of FOXA1 loss-induced CRPC progression.
 
Article
Accumulating evidence identifies non-genetic mechanisms substantially contributing to drug resistance in cancer patients. Preclinical and clinical data implicate the transcriptional co-activators YAP1 and its paralog TAZ in resistance to multiple targeted therapies, highlighting the strong need for therapeutic strategies overcoming YAP1/TAZ-mediated resistance across tumor entities. Here, we show particularly high YAP1/TAZ activity in MITFlow/AXLhigh melanomas characterized by resistance to MAPK pathway inhibition and broad receptor tyrosine kinase activity. To uncover genetic dependencies of melanoma cells with high YAP1/TAZ activity, we used a genome-wide CRISPR/Cas9 functional screen and identified SLC35B2, the 3′-phosphoadenosine-5′-phosphosulfate transporter of the Golgi apparatus, as an essential gene for YAP1/TAZ-driven drug resistance. SLC35B2 expression correlates with tumor progression, and its loss decreases heparan sulfate expression, reduces receptor tyrosine kinase activity, and sensitizes resistant melanoma cells to BRAF inhibition in vitro and in vivo. Thus, targeting heparan sulfation via SLC35B2 represents a novel approach for breaking receptor tyrosine kinase-mediated resistance to MAPK pathway inhibitors.
 
Article
Metastasis accounts for the major cause of cancer-related mortality. How disseminated tumor cells survive under suspension conditions and avoid anoikis is largely unknown. Here, using a metabolic enzyme-centered CRISPR-Cas9 genetic screen, we identified methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1 (MTHFD1) as a novel suppressor of anoikis. MTHFD1 depletion obviously restrained the capacity of cellular antioxidant defense and inhibited tumor distant metastasis. Mechanistically, MTHFD1 was found to bind the protein arginine methyltransferase 5 (PRMT5) and then undergo symmetric dimethylation on R173 by PRMT5. Under suspension conditions, the interaction between MTHFD1 and PRMT5 was strengthened, which increased the symmetric dimethylation of MTHFD1. The elevated methylation of MTHFD1 largely augmented its metabolic activity to generate NADPH, therefore leading to anoikis resistance and distant organ metastasis. Therapeutically, genetic depletion or pharmacological inhibition of PRMT5 declined tumor distant metastasis. And R173 symmetric dimethylation status was associated with metastasis and prognosis of ESCC patients. In conclusion, our study uncovered a novel regulatory role and therapeutic implications of PRMT5/MTHFD1 axis in facilitating anoikis resistance and cancer metastasis.
 
Article
Despite its clinical efficacy in HER2-positive cancers, resistance to trastuzumab inevitably occurs. The DNA damage response (DDR) pathway is essential for maintaining genomic stability and cell survival. However, the role of the DDR pathway in HER2-positive tumors and trastuzumab resistance remains elusive. In this study, we verified that increased PARP1 expression in trastuzumab-resistant (TR) cells, owing to its augmented stability by escape from proteasomal degradation, confers tolerability to trastuzumab-induced DNA damage. Interruption of PARP1 in TR cells restrains its cellular growth, while simultaneously activating ATM to retain its genome stability. Dual inhibition of PARP and ATM induces synthetic lethality in TR cells by favoring the toxic NHEJ pathway instead of the HRR pathway. Our results highlight the potential of clinical development of DDR-targeting strategies for trastuzumab-resistant HER2-positive cancer patients.
 
Article
WTAP, an essential component of the RNA N-6-methyladenosine (m6A) modification complex, guides METLL3-METLL14 heteroduplexes to target RNAs in the nuclear speckles of mammalian cells. Here, we show that TTC22 is widely coexpressed with WTAP and FTO in many human tissues by mining Genotype-Tissue Expression (GTEx) datasets. Our results indicate that the direct interaction of TTC22 with 60S ribosomal protein L4 (RPL4) promotes the binding of WTAP mRNA to RPL4, enhances the stability and translation efficiency of WTAP mRNA, and consequently increases the level of WTAP protein. Also, WTAP mRNA itself is an m6A target and YTHDF1 is characterized as an essential m6A binding protein interacting with m6A-modified WTAP mRNA. TTC22 triggers a positive feedback loop between WTAP expression and WTAP mRNA m6A modification, leading to an increased m6A level in total RNA. The knockdown of RPL4, WTAP, or YTHDF1 expression diminishes the TTC22-induced increase in the m6A level of total RNA. Thus, TTC22 caused dramatic expression changes in genes related to metabolic pathways, ribosomal biogenesis, the RNA spliceosome, and microorganism infections. Importantly, TTC22 upregulates the expression of SNAI1 by increasing m6A level and thus promotes lung metastases of colon cancer cells in mice. In conclusion, our study showed that TTC22 upregulates WTAP and SNAI1 expression, which contributes to TTC22-induced colon cancer metastasis.
 
Article
The transcription factor, forkhead box M1 (FOXM1), has been implicated in the natural history and outcome of newly diagnosed high-risk myeloma (HRMM) and relapsed/refractory myeloma (RRMM), but the mechanism with which FOXM1 promotes the growth of neoplastic plasma cells is poorly understood. Here we show that FOXM1 is a positive regulator of myeloma metabolism that greatly impacts the bioenergetic pathways of glycolysis and oxidative phosphorylation (OxPhos). Using FOXM1-deficient myeloma cells as principal experimental model system, we find that FOXM1 increases glucose uptake, lactate output, and oxygen consumption in myeloma. We demonstrate that the novel 1,1-diarylethylene small-compound FOXM1 inhibitor, NB73, suppresses myeloma in cell culture and human-in-mouse xenografts using a mechanism that includes enhanced proteasomal FOXM1 degradation. Consistent with the FOXM1-stabilizing chaperone function of heat shock protein 90 (HSP90), the HSP90 inhibitor, geldanamycin, collaborates with NB73 in slowing down myeloma. These findings define FOXM1 as a key driver of myeloma metabolism and underscore the feasibility of targeting FOXM1 for new approaches to myeloma therapy and prevention.
 
DDR-focused RNAi screen identifies HORMAD1-driven genetic dependencies
A Schematic diagram describing workflow for parallel siRNA screens in parental SUM159, and clonally-derived HORMAD1-inducible SUM159. Cells were reverse-transfected into siRNA-containing 384-well plates, and doxycycline added 24 h post-transfection. Cell viability was measured 5 days post-transfection using CellTiter-Glo. CellTiter-Glo readings were converted into Z scores, and doxycycline-inducible effects were identified using drug effect (DE) Z-scores. Candidate genetic dependencies were selected using the following criteria: 1) DE Z-score < -3 in HORMAD1-inducible clone 1, 2) DE Z-score > -2 in SUM159 parental clone and 3) Z-score > -3 in DMSO-treated arms. B Scatter plot displaying the distribution of DE-Z scores in HORMAD1-inducible SUM159 clone 1. Negative DE Z-scores are indicative of HORMAD1-driven dependencies. A numerical threshold of DE Z-score < -3 was used for candidate selection. Fourteen candidate DDR genetic dependencies were interrogated in secondary deconvolution experiments, of which 5 were validated as HORMAD1-induced genetic dependencies (marked in red). C–G Bar plots displaying increased normalised percentage inhibition (NPI) of clonally-derived HORMAD1-inducible SUM159 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA pool or four individual siRNAs targeting ATR, BRIP1, POLH, TDP1 and XRCC1 and exposed to HORMAD1 expression for 4 days. Non-targeting (siALLSTAR) and targeting (siPLK1) siRNAs were used as normalisation controls. Error bars indicate SD from mean effects (n = 3), p values represent multiple Student t-tests (***p = < 0.0001, **p = < 0.001, *p = < 0.05).
HORMAD1 drives ATR, BRIP1, POLH, TDP1 and XRCC1 dependencies in multiple cellular models
A Bar plot displaying reduced colony counts of MCF10A cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA pool targeting ATR, BRIP1, POLH, TDP1 and XRCC1 exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) siRNA was used as normalisation control. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests. B Representative colony images from experiment A. C Bar plot displaying the percentage of ATR, BRIP1, POLH, TDP1 and XRCC1 mRNA expression following siRNA-mediated gene knockdown for experiments described in A, measured by RT-qPCR and normalised to ACTB. D Bar plot displaying reduced colony counts of RPE1 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA pool targeting ATR, BRIP1, POLH, TDP1 and XRCC1 and exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) siRNA was used as normalisation control. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests. E Representative colony images from experiment D. F Bar plot displaying the percentage of ATR, BRIP1, POLH, TDP1, and XRCC1 mRNA expression following siRNA-mediated gene knockdown for experiments described in D, measured by RT-qPCR and normalised to ACTB.
Additional validation of HORMAD1-driven POLH dependency
A Bar plot displaying reduced surviving fractions of clonally-derived HORMAD1-inducible SUM159 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA pool or 4 individual siRNAs targeting POLH and exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) and targeting (siPLK1) siRNAs were used as transfection controls and surviving fractions calculated from mock-transfected cells. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t tests (***p = < 0.0001, **p = < 0.001, *p = < 0.05). B Bar plot displaying the percentage of POLH mRNA expression following siRNA-mediated depletion of POLH described in A, measured by RT-qPCR and normalised to ACTB. C Bar plot displaying reduced surviving fractions of clonally-derived HORMAD1-inducible SUM159 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) expressing constitutive Cas9-mCherry, transfected with 5 Edit-R crRNAs targeting POLH, and exposed to HORMAD1 expression for 14 days (in total). Surviving fractions were calculated relative to Cas9-expressing mock-transfected controls. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests (***p = < 0.0001, **p = < 0.001, *p = < 0.05). D Western blot analysis of POLH protein knockout from experiment C. E, FLeft, growth curves displaying reduced cellular growth of HORMAD1-expressing breast cancer cell lines E HCC38 and F BT549 expressing constitutive Cas9-mCherry and bulk-transfected with 3 POLH-targeting Edit-R crRNAs. Cell number was normalised relative to T0 counts. Error bars indicate SD from mean effects (n = 3). p-values represent two-way repeated-measures ANOVA. Right, western blot analysis of HORMAD1 expression and POLH protein knockout from experiments described in left panel.
HORMAD1 drives broad genetic dependency on TLS polymerases
A Bar plot displaying reduced colony counts of SUM159 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA pool targeting REV3L and REV7 and exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) siRNA was used as normalisation control. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests. B Representative colony images from experiment A. C Bar plot displaying the percentage of REV3L and REV7 mRNA expression following siRNA-mediated gene knockdown for experiments described in A, measured by RT-qPCR and normalised to ACTB. D Bar plot displaying reduced colony counts of MCF10A cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA SMARTpool targeting POLK, REV1 and REV7 and exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) siRNA was used as normalisation control. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests. E Representative colony images from experiment D. F Bar plot displaying the percentage of POLK, REV1 and REV7 mRNA expression following siRNA-mediated gene knockdown for experiments described in D, measured by RT-qPCR and normalised to ACTB. G Bar plot displaying reduced colony counts of RPE1 cells (+DOX/ + HORMAD1 vs. -DOX/-HORMAD1) transfected with an siRNA SMARTpool targeting POLK, REV3L and REV7 and exposed to HORMAD1 expression for 14 days (in total). Non-targeting (siALLSTAR) siRNA was used as normalisation control. Error bars indicate SD from mean effects (n = 3), p-values represent multiple Student t-tests. H Representative colony images from experiment G. I Bar plot displaying the percentage of POLK, REV3L, and REV7 mRNA expression following siRNA-mediated gene knockdown for experiments described in G, measured by RT-qPCR and normalised to ACTB.
Article
HORMAD1 expression is usually restricted to germline cells, but it becomes mis-expressed in epithelial cells in ~60% of triple-negative breast cancers (TNBCs), where it is associated with elevated genomic instability (1). HORMAD1 expression in TNBC is bimodal with HORMAD1-positive TNBC representing a biologically distinct disease group. Identification of HORMAD1-driven genetic dependencies may uncover novel therapies for this disease group. To study HORMAD1-driven genetic dependencies, we generated a SUM159 cell line model with doxycycline-inducible HORMAD1 that replicated genomic instability phenotypes seen in HORMAD1-positive TNBC (1). Using small interfering RNA screens, we identified candidate genes whose depletion selectively inhibited the cellular growth of HORMAD1-expressing cells. We validated five genes (ATR, BRIP1, POLH, TDP1 and XRCC1), depletion of which led to reduced cellular growth or clonogenic survival in cells expressing HORMAD1. In addition to the translesion synthesis (TLS) polymerase POLH, we identified a HORMAD1-driven dependency upon additional TLS polymerases, namely POLK, REV1, REV3L and REV7. Our data confirms that out-of-context somatic expression of HORMAD1 can lead to genomic instability and reveals that HORMAD1 expression induces dependencies upon replication stress tolerance pathways, such as translesion synthesis. Our data also suggest that HORMAD1 expression could be a patient selection biomarker for agents targeting replication stress.
 
Article
Altered expression of Urea Cycle (UC) enzymes occurs in many tumors, resulting a metabolic hallmark termed as UC dysregulation. Polyamines are synthesized from ornithine, and polyamine synthetic genes are elevated in various tumors. However, the underlying deregulations of UC/ polyamine synthesis in cancer remain elusive. Here, we characterized a hypoxia-induced lncRNA LVBU (lncRNA regulation via BCL6/urea cycle) that is highly expressed in colorectal cancer (CRC) and correlates with poor cancer prognosis. Increased LVBU expression promoted CRC cells proliferation, foci formation and tumorigenesis. Further, LVBU regulates urea cycle and polyamine synthesis through BCL6, a negative regulator of p53. Mechanistically, overexpression of LVBU competitively bound miR-10a/miR-34c to protect BCL6 from miR-10a/34c-mediated degradation, which in turn allows BCL6 to block p53-mediated suppression of genes (arginase1 ARG1, ornithine transcarbamylase OTC, ornithine decarboxylase 1 ODC1) involved in UC/polyamine synthesis. Significantly, ODC1 inhibitor attenuated the growth of patient derived xenografts (PDX) that sustain high LVBU levels. Taken together, elevated LVBU can regulate BCL6-p53 signaling axis for systemic UC/polyamine synthesis reprogramming and confers a predilection toward CRC development. Our data demonstrates that further drug development and clinical evaluation of inhibiting UC/polyamine synthesis are warranted for CRC patients with high expression of LVBU.
 
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Lung cancer is the most common malignancy and the leading cause of cancer death worldwide, and lung adenocarcinoma (LUAD) is the most prevalent subtype. Considering the emergence of resistance to therapies, it is urgent to develop more effective therapies to improve the prognosis. Here we reported that pancreatic progenitor cell differentiation and proliferation factor (PPDPF) deficiency inhibited LUAD development both in vitro and in vivo. Mechanistically, PPDPF induces hyperactive STAT3 by interfering STAT3-PTPN1 interaction. Activated STAT3 promoted BMPR2 transcription, which further inhibited apoptosis. Moreover, PPDPF reduced NK cell infiltration and activation to develop an immunosuppressive microenvironment, which was also mediated by STAT3. Furthermore, we identified that the expression of PPDPF was positively correlated with the malignant features of LUAD, as well as BMPR2 and p-STAT3 level in clinical samples. Therefore, our study suggests that PPDPF positively regulates BMPR2 expression and facilitates immune escape via regulating STAT3 activity, providing a potential therapy target for LUAD.
 
Article
We previously found that lactic acidosis in the tumor environment was permissive to cancer cell surviving under glucose deprivation and demonstrated that neutralizing lactic acidosis restored cancer cell susceptibility to glucose deprivation. We then reported that alternate infusion of bicarbonate and anticancer agent into tumors via tumor feeding artery markedly enhanced the efficacy of transarterial chemoembolization (TACE) in the local control of hepatocellular carcinoma (HCC). Here we sought to further investigate the mechanism by which bicarbonate enhances the anticancer activity of TACE. We propose that interfering cellular pH by bicarbonate could induce a cascade of molecular events leading to cancer cell death. Alkalizing cellular pH by bicarbonate decreased pH gradient (ΔpH), membrane potential (ΔΨm), and proton motive force (Δp) across the inner membrane of mitochondria; disruption of oxidative phosphorylation (OXPHOS) due to collapsed Δp led to a significant increase in adenosine monophosphate (AMP), which activated the classical AMPK-mediated autophagy. Meanwhile, the autophagic flux was ultimately blocked by increased cellular pH, reduced OXPHOS, and inhibition of lysosomal proton pump in alkalized lysosome. Bicarbonate also induced persistent mitochondrial permeability (MPT) and damaged mitochondria. Collectively, this study reveals that interfering cellular pH may provide a valuable approach to treat cancer.
 
Article
Glioblastoma is the most common malignant brain cancer with dismal survival and prognosis. Temozolomide (TMZ) is a first-line chemotherapeutic agent for glioblastoma, but the emergence of drug resistance limits its anti-tumor activity. We previously discovered that the interferon inducible guanylate binding protein 3 (GBP3) is highly elevated and promotes tumorigenicity of glioblastoma. Here, we show that TMZ treatment significantly upregulates the expression of GBP3 and stimulator of interferon genes (STING), both of which increase TMZ-induced DNA damage repair and reduce cell apoptosis of glioblastoma cells. Mechanistically, relying on its N-terminal GTPase domain, GBP3 physically interacts with STING to stabilize STING protein levels, which in turn induces expression of p62 (Sequestosome 1), nuclear factor erythroid 2 like 2 (NFE2L2, NRF2), and O6-methlyguanine-DNA-methyltransferase (MGMT), leading to the resistance to TMZ treatment. Reducing GBP3 levels by RNA interference in glioblastoma cells markedly increases the sensitivity to TMZ treatment in vitro and in murine glioblastoma models. Clinically, GBP3 expression is high and positively correlated with STING, NRF2, p62, and MGMT expression in human glioblastoma tumors, and is associated with poor outcomes. These findings provide novel insight into TMZ resistance and suggest that GBP3 may represent a novel potential target for the treatment of glioblastoma.
 
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Despite paclitaxel’s wide use in cancer treatment, patient response rate is still low and drug resistance is a major clinical obstacle. Through a Phos-tag-based kinome-wide screen, we identified MARK2 as a critical regulator for paclitaxel chemosensitivity in PDAC. We show that MARK2 is phosphorylated by CDK1 in response to antitubulin chemotherapeutics and in unperturbed mitosis. Phosphorylation is essential for MARK2 in regulating mitotic progression and paclitaxel cytotoxicity in PDAC cells. Mechanistically, our findings also suggest that MARK2 controls paclitaxel chemosensitivity by regulating class IIa HDACs. MARK2 directly phosphorylates HDAC4 specifically during antitubulin treatment. Phosphorylated HDAC4 promotes YAP activation and controls expression of YAP target genes induced by paclitaxel. Importantly, combination of HDAC inhibition and paclitaxel overcomes chemoresistance in organoid culture and preclinical PDAC animal models. The expression levels of MARK2, HDACs, and YAP are upregulated and positively correlated in PDAC patients. Inhibition of MARK2 or class IIa HDACs potentiates paclitaxel cytotoxicity by inducing mitotic abnormalities in PDAC cells. Together, our findings identify the MARK2-HDAC axis as a druggable target for overcoming chemoresistance in PDAC.
 
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Aldo-keto reductase family 1 member C3 (AKR1C3) serves as a contributor to numerous kinds of tumors, and its expression is elevated in patients with hepatocellular carcinoma (HCC). However, the biological function of AKR1C3 in HCC remains unclear. Here we investigated the role of AKR1C3 in liver carcinogenesis using in vitro and in vivo models. We determined that AKR1C3 is frequently increased in HCC tissues with poor prognosis. Genetically manipulated cells with AKR1C3 construction were examined to highlight the pro-tumoral growth of both wild-type AKR1C3 and mutant in vitro and in vivo. We observed promising treatment effects of AKR1C3 shRNA by intratumoral injection in mice. Mechanically, we demonstrated that the transcription factor heterodimer NRF2/MAFG was able to bind directly to AKR1C3 promoter to activate its transcription. Further, AKR1C3 stabilized PARP1 by decreasing its ubiquitination, which resulted in HCC cell proliferation and low sensitivity of Cisplatin. Moreover, we discovered that the tumorigenic role of AKR1C3 was non-catalytic dependent and the NRF2/MAFG-AKR1C3-PARP1 axis might be one of the important proliferation pathways in HCC. In conclusion, blockage of AKR1C3 expression provides potential therapeutic benefits against HCC.
 
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Yes-associated protein 1 (YAP1), a central component of the Hippo pathway, plays an important role in tumor metastasis; however, the underlying mechanism remains to be elucidated. Invadopodia are actin-rich protrusions containing multiple proteases and have been widely reported to promote cell invasiveness by degrading the extracellular matrix. In the present study, we report that YAP1 induces invadopodia formation and promotes tumor metastasis in breast cancer cells. We also identify TIAM1, a guanine nucleotide exchange factor, as a target of the YAP1–TEAD4 complex. Our results demonstrate that YAP1 could promote TEAD4 binding to the enhancer region of TIAM1, which activates TIAM1 expression, subsequently increasing RAC1 activity and inducing invadopodia formation. These findings reveal the functional role of Hippo signaling in the regulation of invadopodia and provide potential molecular targets for preventing tumor metastasis in breast cancer.
 
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Large-scale fecal shotgun metagenomic sequencing revealed the high abundance of Parvimonas micra in colorectal cancer (CRC) patients. We investigated the role and clinical significance of P. micra in colorectal tumorigenesis. The abundance of P. micra was examined in 309 fecal samples and 165 colon biopsy tissues of CRC patients and healthy subjects. P. micra was significantly enriched in fecal samples from 128 CRC patients compared to 181 healthy subjects (P < 0.0001); and in colon tissue biopsies from 52 CRC patients compared to 61 healthy subjects (P < 0.0001). Multivariate analysis showed that P. micra is an independent risk factor of poor survival in CRC patients (Hazard Ratio: 1.93). P. micra strain was isolated from feces of a CRC patient. Apcmin/+ mice gavaged with P. micra showed significantly higher tumor burden and tumor load (both P < 0.01). Consistently, gavage of P. micra significantly promoted colonocyte proliferation in conventional mice, which was further confirmed by germ-free mice. P. micra colonization up-regulated genes involved in cell proliferation, stemness, angiogenesis and invasiveness/metastasis; and enhanced Th17 cells infiltration and expression of Th17 cells-secreted cytokines (Il-17, Il-22, and Il-23) in the colon of Apcmin/+, conventional and germ-free mice. P. micra-conditioned medium significantly promoted the differentiation of CD4⁺ T cells to Th17 cells (IL-17⁺CD4⁺ phenotype) and enhanced the oncogenic Wnt signaling pathway. In conclusion, P. micra promoted colorectal tumorigenesis in mice by inducing colonocyte proliferation and altering Th17 immune response. P. micra may act as a prognostic biomarker for poor survival of CRC patients.
 
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Aberrations in nuclear size and shape are commonly used to identify cancerous tissue. However, it remains unclear whether the disturbed nuclear structure directly contributes to the cancer pathology or is merely a consequence of other events occurring during tumorigenesis. Here, we show that highly invasive and proliferative breast cancer cells frequently exhibit Akt-driven lower expression of the nuclear envelope proteins lamin A/C, leading to increased nuclear deformability that permits enhanced cell migration through confined environments that mimic interstitial spaces encountered during metastasis. Importantly, increasing lamin A/C expression in highly invasive breast cancer cells reflected gene expression changes characteristic of human breast tumors with higher LMNA expression, and specifically affected pathways related to cell-ECM interactions, cell metabolism, and PI3K/Akt signaling. Further supporting an important role of lamins in breast cancer metastasis, analysis of lamin levels in human breast tumors revealed a significant association between lower lamin A levels, Akt signaling, and decreased disease-free survival. These findings suggest that downregulation of lamin A/C in breast cancer cells may influence both cellular physical properties and biochemical signaling to promote metastatic progression.
 
Article
Plasma membrane Na ⁺ /Ca ²⁺ exchanger 1 (NCX1) is a bidirectional ion transporter to operate in Ca ²⁺ entry or exit modes, and TRPC1 is Ca ²⁺ -permeable channel. Both NCX1 and TRPC1 play critical roles in maintaining cytosolic free Ca ²⁺ ([Ca ²⁺ ] cyt ) homeostasis in mammalian cells. Although either TRPC1 channel or Ca ²⁺ entry mode of NCX1 is implicated in some tumorigenesis, it has not been explored if a coordination of NCX1 and TRPC1 involves in the pathogenesis of H. pylori- associated human gastric cancer (GC). Here we found the protein expression of NCX1 was significantly enhanced in human GC specimens, which correlated with tumor progression and poor survival in GC patients. TRPC1 and NCX1 were parallelly enhanced, co-localized and bound in human GC cells. By a functional coupling, TRPC1 drives NCX1 to the Ca ²⁺ entry mode, raising [Ca ²⁺ ] cyt in GC cells. Moreover, CaCl 2 , H. pylori and their virulence factors all enhanced expressions and activities of NCX1 and TRPC1, and evoked aberrant Ca ²⁺ entry to promote proliferation, migration, and invasion of GC cells through AKT/β-catenin pathway. Tumor growth and metastasis also depended on the enhanced expression of NCX1 in subcutaneously xenografted GC mouse model. Overall, our findings indicate that TRPC1/NCX1 coupling may promote H. pylori- associated GC through the Ca ²⁺ /AKT/β-catenin pathway. Since the Ca ²⁺ exit mode and the Ca ²⁺ entry mode of NCX1 play different roles under mostly physiological and pathological conditions respectively, targeting TRPC1/NCX1 coupling could be a novel strategy for selectively blocking Ca ²⁺ entry mode to potentially treat digestive cancer with less side effect.
 
Article
HFE (Hemochromatosis) is a conventional iron level regulator and its loss of function due to gene mutations increases the risk of cancers including hepatocellular carcinoma (HCC). Likewise, studies focusing on HFE overexpression in cancers are all limited to linking up these events as a consequence of iron level deregulation. No study has explored any iron unrelated role of HFE in cancers. Here, we first reported HFE as an oncogene in HCC and its undescribed function on promoting abscission in cytokinesis during mitotic cell division, independent of its iron-regulating ability. Clinical analyses revealed HFE upregulation in tumors linking to large tumor size and poor prognosis. Functionally and mechanistically, HFE promoted cytokinetic abscission via facilitating ESCRT abscission machinery recruitment to the abscission site through signaling a novel HFE/ALK3/Smads/LIF/Hippo/YAP/YY1/KIF13A axis. Pharmacological blockage of HFE signaling axis impeded tumor phenotypes in vitro and in vivo. Our data on HFE-driven HCC unveiled a new mechanism utilized by cancer cells to propel rapid cell division. This study also laid the groundwork for tumor intolerable therapeutics development given the high cytokinetic dependency of cancer cells and their vulnerability to cytokinetic blockage.
 
Incidence of GNAS mutations in pan-cancer
A Frequency of GNAS mutations across various pan-cancers obtained from the cBioportal database and (B), estimated number of GNAS mutant cases in 2021 using USA cancer statistics. C Mutation map of GNAS protein. Lollipops along the domain structure of the protein represent the total frequencies of all mutated alleles of GNAS in a population. Diagram circles represent different GNAS variants in 1050 pan-cancer samples (green = Missense mutations, black = Truncating mutations (Nonsense, Frameshift insertion/deletion), purple = Other types of mutations). The x-axis and y-axis represent the amino acid number and the frequency of the mutation, respectively. Mutation maps were generated using the web-based tool MutationMapper for plotting lollipop plot available in cBioPortal. D Co-mutation network of all GNASR201 variants with other mutant genes in colorectal cancer samples from the institutional molecular database of The University of Texas MD Ander Cancer Center. Edge weights in the network were scaled with respect to the magnitude of odds ratio of the Chi-square test performed between the pair of mutated genes.
Evaluation of GNAS knockout and overexpressing CRC cell lines
AGNAS was knocked out from KM12, SNU175, and SKCO1 cell lines using a CRISPR-mediated approach, and its effect on clonogenic capacity was assessed by evaluating growth in standard culture conditions for 12–15 days. Colonies were fixed in 1% paraformaldehyde and stained with 1% crystal violet prior to counting (mean ± SD, n = 5, 4, 4 respectively). BGNAS was overexpressed in the LS174T cell line using a doxycycline inducible promoter, and the resulting effect on clonogenic capacity was assessed as previously described (mean ± SD, n = 3 in each group). C Growth of GNASKO SNU175 cells (number of colonies, total colony area) evaluated with a 3D organoid assay. D A single clone (GNAS352delG) was selected from a pool of GNASKO cells, and growth (number of colonies, total area) was evaluated with a 3D organoid assay. E Growth of GNASR201 overexpressing LS174T cell lines evaluated with a 3D organoid assay (mean ± SD, n = 3 in each group).
GNAS promotes tumor formation from KM12 and LS174T cells in the peritoneum of NSG mice
A NSG mice were intraperitoneally injected with 10⁶ parental (GNASR201H) or GNAS-knockout (GNASKO-pool) KM12 cells. At 1–6 weeks post-injection, D-luciferin (150 mg/kg) was injected via IP and the bioluminescence signal was obtained using an in vivo imaging system (IVIS) with small binning and 1 s exposures at 10 min after administration of luciferin. Three representative merged images are shown. B Data expressed as mean ± SEM, n = 6 in each group. C Survival curve of NSG mice injected with KM12 cells (GNASR201H or GNASKO-pool). D Tissue sections of mouse tumors were stained with H&E. 1× bar, 3 mm (left) and 4× bar, 600 μm (right). E NSG mice were intraperitoneally injected with 2.5 × 10⁴ parental, GNASWT, GNASR201C, and GNASR201H overexpressing LS174T cells. Bioluminescent signal was obtained 1–6 weeks post-injection as described in (A), and four representative images are shown. F Data are expressed as mean ± SEM, n = 3 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.
GNAS signals through the cAMP-PKA axis
A KM12 GNASR201H and GNAS352delG cells were treated with forskolin (0–250 μM) for 15 min. Following the treatment, cells were lysed, and cAMP levels were measured as described in the methods. B LS174T GNASR201H cells were treated with and without doxycycline, and cAMP levels measured after induction with forskolin (0–2.5 μM) for 15 min. C KM12 GNASR201H and GNAS352delG organoids were grown in 3D-matrigel domes and treated with the PKA inhibitor H-89 (60 μM) for 10 days.
β-catenin is a downstream mediator of mutant GNAS signaling
A Top 10 hallmark genesets from the KM12 GNASKO-pool and LS174T GNAS overexpression datasets were selected and overlapping genesets were plotted. B Ratio of pT41/S45 phosphoβ-catenin/β-catenin based on the RPPA analysis results. C Immunohistochemistry (IHC) staining of β-catenin in LS174T dox (−) and dox (+) tissues from intraperitoneal mouse tumors. D Quantification of IHC tumors showing % of cells staining positive of β-catenin. E Quantification of the cytoplasmic and nuclear distribution of β-catenin based on IHC staining. F Effect of LF3 (β-catenin inhibitor) on the growth of KM12 (GNASR201H and GNAS352delG) organoids. DMSO (0.2%) was used as control treatment and organoids were cultured for 10 days.
Article
The GNASR201 gain-of-function mutation is the single most frequent cancer-causing mutation across all heterotrimeric G proteins, driving oncogenesis in various low-grade/benign gastrointestinal and pancreatic tumors. In this study, we investigated the role of GNAS and its product Gαs in tumor progression using peritoneal models of colorectal cancer (CRC). GNAS was knocked out in multiple CRC cell lines harboring GNASR201C/H mutations (KM12, SNU175, SKCO1), leading to decreased cell-growth in 2D and 3D organoid models. Nude mice were peritoneally injected with GNAS-knockout KM12 cells, leading to a decrease in tumor growth and drastically improved survival at 7 weeks. Supporting these findings, GNAS overexpression in LS174T cells led to increased cell-growth in 2D and 3D organoid models, and increased tumor growth in PDX mouse models. GNAS knockout decreased levels of cyclic AMP in KM12 cells, and molecular profiling identified phosphorylation of β-catenin and activation of its targets as critical downstream effects of mutant GNAS signaling. Supporting these findings, chemical inhibition of both PKA and β-catenin reduced growth of GNAS mutant organoids. Our findings demonstrate oncogene addiction to GNAS in peritoneal models of GNASR201C/H tumors, which signal through the cAMP/PKA and Wnt/β-catenin pathways. Thus, GNAS and its downstream mediators are promising therapeutic targets for GNAS mutant tumors.
 
TRPC3 behaves as an oncogene and acts as a downstream target of PLAA. A RT-qPCR analysis of TRPC3 expression in 56 tissue samples of ovarian cancer and 12 tissue samples of ovarian benign tumor. B RT-qPCR analysis of TRPC3 expression in ovarian cancer tissues of late stages and early stages. C Kaplan-Meier analysis of progression-free survival of patients from TCGA data with different TRPC3 expression. A2780-M cells were transfected with two TRPC3 siRNAs or negative control. TRPC3 expression was determined by RT-qPCR (D) or immunoblot analysis (E). F A2780-M cells were transfected with two TPRC3 siRNAs or negative control. Cellular migration and invasion were detected by transwell assay. Scale bar, 100 μm. G Cellular migration and invasion of A2780-M cells treated with Pry3 or DMSO were detected by transwell assay. Scale bar, 100 μm. H, I A2780-M cells were transfected with PLAA plasmid, PLAA plasmid plus TRPC3 plasmid, and negative control, respectively (H). A2780 cells were transfected with PLAA siRNA, PLAA siRNA plus TRPC3 siRNA, PLAA siRNA plus Pyr3 treatment (2 μM, 24 h), and negative control, respectively (I). Cellular migration and invasion were detected by transwell assay. Scale bar, 100 μm. J Ovarian cancer cells were orthotopically transplanted into SCID mice, and mice were intravenously treated with DMSO or Pyr3 (5 mg/kg) for twice a week from the first week after implantation. Representative bioluminescence images of dissected primary and metastatic ovarian cancer at the time of execution. The photon count indicated the tumor burden. Data are representative of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns no significant.
Clinical characteristics of 79 ovarian cancer patients depending on PLAA protein level.
Article
Wide metastasis contributes to a high death rate in ovarian cancer, and understanding of the molecular mechanism helps to find effective targets for metastatic ovarian cancer therapy. It has been found that phospholipase A2-activating protein (PLAA) is inactivated in some cancers, but its role in cancer metastasis remains unknown. Here, we found that PLAA was significantly downregulated in ovarian cancer highly metastatic cell lines and patients, and the low expression of PLAA was associated with poorer prognosis and high-risk clinicopathological features of patients. PLAA inhibited the migration and invasion of ovarian cancer cells and metastasis of transplanted tumor in the orthotopic xenograft mouse model. Meanwhile, PLAA inhibited metastasis of ovarian cancer by inhibiting transient receptor potential channel canonical 3 (TRPC3)-mediated the intracellular Ca2+ level. Mechanistically, PLAA inhibited methyltransferase-like 3 (METTL3) expression through the ubiquitin-mediated degradation, and METTL3 stabilized TRPC3 mRNA expression via N6-methyladenosine (m6A) modification. Our study verified the function and mechanism of the PLAA-METTL3-TRPC3 axis involved in ovarian cancer metastasis, with a view to providing a potential therapeutic approach for ovarian cancer.
 
Article
Loss of α6β4-dependent hemidesmosomal adhesions has been observed during prostate cancer progression. However, the significance and underlying mechanisms by which aberrant hemidesmosome assembly may modulate tumorigenesis remain elusive. Using an extensive CRISPR/Cas9-mediated genetic engineering approaches in different prostate cancer cell lines combined with in vivo tumorigenesis studies in mice, bone marrow-on-chip assays and bioinformatics, as well as histological analysis of prostate cancer patient cohorts, we demonstrated that simultaneous loss of PTEN and hemidesmosomal adhesions induced several tumorigenic properties including proliferation, migration, resistance to anoikis, apoptosis, and drug treatment in vitro, and increased metastatic capacity in vivo. These effects were plectin-depended and plectin was associated with actin-rich adhesions upon hemidesmosome disruption in PTEN-negative prostate cancer cells leading to activation of EGFR/PI3K/Akt- and FAK/Src-pathways. These results suggest that analysis of PTEN and hemidesmosomal proteins may have diagnostic value helping to stratify prostate cancer patients with high risk for development of aggressive disease and highlight actin-associated plectin as a potential therapeutic target specifically in PTEN/hemidesmosome dual-negative prostate cancer.
 
Article
Glioblastoma is a lethal primary brain tumor with abundant immune-suppressive glioblastoma-associated macrophage (GAM) infiltration. Skewing immune suppressive GAMs towards an immune-activating phenotype represents a promising immunotherapeutic strategy against glioblastoma. Herein, we reported that genetic deletion of miRNA-processing enzyme Dicer in macrophages inhibited the growth of GL261 murine glioblastoma xenografts and prolonged survival of tumor-bearing mice. Single cell RNA sequencing (scRNA-seq) of the tumor-infiltrating immune cells revealed that Dicer deletion in macrophages reduced the proportion of cell-cycling GAM cluster and reprogramed the remaining GAMs towards a proinflammatory activation state (enhanced phagocytotic and IFN-producing signature). Dicer-deficient GAMs showed reduced level of cyclin-dependent kinases (CDK1 and CDK2) and increased expression of CDK inhibitor p27 Kip1, thus manifesting impaired proliferation. Dicer knockout enhanced phagocytotic activity of GAMs to eliminate GL261 tumor cells. Increased proinflammatory GAM clusters in macrophage Dicer-deficient mice actively interacted with tumor-infiltrating T cells and NK cells through TNF paracrine signaling to create a pro-inflammatory immune microenvironment for tumor cell elimination. Our work identifies the role of Dicer deletion in macrophages in generating an immune-activating microenvironment, which could be further developed as a potential immunotherapeutic strategy against glioblastoma.
 
Article
Human papillomavirus (HPV) infection is very common in sexually active women, but cervical cancer only develops in a small fraction of HPV-infected women, suggesting that unknown intrinsic factors associated with the unique genetic/genomic background of the high-risk population play a critical role in cervical carcinogenesis. Although our previous studies have identified the hyperactivated YAP1 oncogene as a critical contributor to cervical cancer, the molecular mechanism by which YAP1 drives cervical cancer is unknown. In the present study, we found that although the hyperactivated YAP1 caused a malignant transformation of immortalized cervical epithelial cells, it induced cellular senescence in cultures of primary human cervical epithelial cells (HCvECs). However, the hyperactivated YAP1 induced malignant transformation of HCvECs in the presence of high-risk HPV E6/E7 proteins, suggesting that the hyperactivated YAP1 synergizes with HPV to initiate cervical cancer development. Our mechanistic studies demonstrate that YAP1, via up-regulating LATS2, formed a YAP1-LATS2 negative feedback loop in cervical epithelial cells to maintain homeostasis of cervical tissue. Intriguingly, we found that high-risk HPV targets LATS2 to disrupt the feedback loop leading to the malignant transformation of cervical epithelial cells. Finally, we report that mitomycin C, an FDA-approved drug that could upregulate LATS2 and drive cellular senescence in vitro and in vivo, induced a regression of cervical cancer in a pre-clinial animal model. Thus, high-risk HPV targeting the YAP1-LATS2 feedback loop represents a new mechanism of cervical cancer development.
 
Article
Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.
 
Article
Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The retinoblastoma protein (RB1), a regulator of cell proliferation, is functionally inactivated in HCC by CYCLIN D/E-mediated phosphorylation. However, the mechanism of RB1-inactivation is unclear because only small percentages of HCCs exhibit amplification of CYCLIN D/E or mutations in the CDK-inhibitory genes. We show that FOXM1, which is overexpressed and critical for HCC, plays essential roles in inactivating RB1 and suppressing RB1-induced senescence of the HCC cells. Mechanistically, FOXM1 binds RB1 and DNMT3B to repress the expression of FOXO1, leading to a decrease in the levels of the CDK-inhibitors, creating an environment for phosphorylation and inactivation of RB1. Consistent with that, inhibition of FOXM1 causes increased expression of FOXO1 with consequent activation of RB1, leading to senescence of the HCC cells, in vitro and in vivo. Also, repression-deficient mutants of FOXM1 induce senescence that is blocked by depletion of RB1 or FOXO1. We provide evidence that human HCCs rely upon this FOXM1–FOXO1 axis for phosphorylation and inactivation of RB1. The observations demonstrate the existence of a new autoregulatory loop of RB1-inactivation in HCC involving a FOXM1–FOXO1 axis that is required for phosphorylation of RB1 and for aggressive progression of HCC.
 
Article
The dynamics of mitochondrial biogenesis regulation is critical in maintaining cellular homeostasis for immune regulation and tumor prevention. Here, we report that mitochondrial biogenesis disruption through TFAM reduction significantly impairs mitochondrial function, induces autophagy, and promotes esophageal squamous cell carcinoma (ESCC) growth. We found that TFAM protein reduction promotes mitochondrial DNA (mtDNA) release into the cytosol, induces cytosolic mtDNA stress, subsequently activates the cGAS-STING signaling pathway, thereby stimulating autophagy and ESCC growth. STING depletion or mtDNA degradation by DNase I abrogates mtDNA stress response, attenuates autophagy, and decreases the growth of TFAM depleted cells. In addition, autophagy inhibitor also ameliorates mitochondrial dysfunction-induced activation of the cGAS-STING signaling pathway and ESCC growth. In conclusion, our results indicate that mtDNA stress induced by mitochondria biogenesis perturbation activates the cGAS-STING pathway and autophagy to promote ESCC growth, revealing an underappreciated therapeutic strategy for ESCC. Schematic depicting the TFAM downregulation promotes autophagy and ESCC survival through mtDNA stress-mediated STING pathway