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Aberrant Overexpression of the Cell Polarity Module Scribble in Human Cancer
Abstract
Human Scribble (Scrib) is an evolutionary-conserved cell polarity protein, but its potential role in human cancer is controversial. Herein, we show that Scrib is nearly universally overexpressed in cultured tumor cell lines and genetically disparate cancer patient series compared with matched normal tissues in vivo. Instead of a membrane association seen in normal epithelia, tumor-associated Scrib is mislocalized and found predominantly in the cytosol. Small-interfering RNA silencing of Scrib in model lung adenocarcinoma A549 cells inhibited cell migration in wound-healing assays, suppressed tumor cell invasion across Matrigel-coated inserts, and down-regulated the expression of cell motility markers and mediators of epithelial-mesenchymal transition. These data uncover a previously unrecognized exploitation of Scrib for aberrant tumor cell motility and invasion, thus potentially contributing to disease progression in humans.
... In this study, we performed proteogenomic data mining for hidden SEP-encoding genes in the human genome through the use of publicly available high-quality datasets of human cancer cell lines, which were obtained from MS-based proteomics and transcriptomic RNA sequencing (RNA-Seq). Our study successfully demonstrated the existence of a new SEP-encoding oORF on the cell polarity-determining scaffold protein gene Scribble (SCRIB), which is considered a proto-oncogene with tumor suppressor function in antitumorigenic Hippo-YAP/TAZ, Ras/ Raf/MEK/ERK (MAPK/ERK), and PI3K/Akt/mTOR and proapoptotic c-Myc-induced signaling pathways [22][23][24][25][26][27][28][29] . Together with the results from our clinical human proteomic data reanalysis and biochemical analysis, we report that the translatable oORF on SCRIB, oSCRIB, is a cis-regulatory oORF potentially providing a fail-safe mechanism to normal cells for nonexcessive downstream SCRIB expression, whereas the mechanism was dysregulated in cancer cells for their survival and proliferation. ...
... Recent studies have revealed that the downstream SCRIB is a double-agent gene as a proto-oncogene with tumor suppressor function (POTSF) in normal cells 26,27,29 . In fact, SCRIB is reported to be overexpressed at the mRNA and protein levels in multiple human cancers, including uterine and breast cancers, and the resultant mislocalization of SCRIB protein promotes tumorigenesis 23,26 . ...
... The translation product of SCRIB is known as a cell-polarity determinant and a large multi-domain scaffold POTSF protein for many key pathways such as antitumorigenic Hippo-YAP/ TAZ, Ras/Raf/MEK/ERK (MAPK/ERK), and PI3K/Akt/mTOR and proapoptotic c-Myc-induced signaling pathways [22][23][24][25][26][27][28][29] . Hence, the regulatory role of oSCRIB in SCRIB translation is likely to be associated with maintaining cellular homeostasis in normal cells. ...
Intensive gene annotation has revealed many functional and regulatory elements in the human genome. Although eukaryotic protein-coding genes are generally transcribed into monocistronic mRNAs, recent studies have discovered additional short open reading frames (sORFs) in mRNAs. Here, we performed proteogenomic data mining for hidden proteins categorized into sORF-encoded polypeptides (SEPs) in human cancers. We identified a new SEP-encoding overlapping sORF (oORF) on the cell polarity determinant Scribble (SCRIB) that is considered a proto-oncogene with tumor suppressor function in Hippo-YAP/TAZ, MAPK/ERK, and PI3K/Akt/mTOR signaling. Reanalysis of clinical human proteomic data revealed translational dysregulation of both SCRIB and its oORF, oSCRIB, during carcinogenesis. Biochemical analyses suggested that the translatable oSCRIB constitutively limits the capacity of eukaryotic ribosomes to translate the downstream SCRIB. These findings provide a new example of cis-regulatory oORFs that function as a ribosomal roadblock and potentially serve as a fail-safe mechanism to normal cells for non-excessive downstream gene expression, which is hijacked in cancer.
... Although these data suggested a model where scribble acts as an evolutionaryconserved tumour suppressor, its role in human cancer, in vivo, is more debated. In contrast to the scientific evidence reporting that scribble expression is lost during human carcinogenesis, other studies have shown that scribble levels increase and that the protein is improperly localised in certain human tumours in vivo Vaira et al. (2011). A comparative study conducted on samples of cancers of the colon, liver, prostate, uterus, thyroid, lung, bladder, breast, ovary, and stomach tissues found strong scribble immunoreactivity in all cases, except for the thyroid Vaira et al. (2011). ...
... In contrast to the scientific evidence reporting that scribble expression is lost during human carcinogenesis, other studies have shown that scribble levels increase and that the protein is improperly localised in certain human tumours in vivo Vaira et al. (2011). A comparative study conducted on samples of cancers of the colon, liver, prostate, uterus, thyroid, lung, bladder, breast, ovary, and stomach tissues found strong scribble immunoreactivity in all cases, except for the thyroid Vaira et al. (2011). For instance, scribble activity was ubiquitously high and often found in the cytosol of cells from tumour tissues representing various breast cancer types, regardless of the hormonal receptor. ...
... In Drosophila melanogaster imaginal discs, for instance, scribble works as a suppressor of Cyclin E, thus regulating entry in the S phase Humbert et al. (2008). Experimental evidence in both Drosophila melanogaster Brumby and Richardson (2003) and mammals Vaira et al. (2011) have shown that loss of function of scribble is commonly associated with uncontrolled proliferation. Conversely, overexpression of scribble was shown to repress proliferation in various mammalian in vitro models Takizawa et al. (2006). ...
Cell competition is a quality control mechanism through which tissues eliminate unfit cells. Cell competition can result from short-range biochemical signals or long-range mechanical cues. However, little is known about how cell-scale interactions give rise to population shifts in tissues, due to the lack of experimental and computational tools to efficiently characterise interactions at the single-cell level. In the work presented in this thesis, I address these challenges by combining long-term automated microscopy with deep learning image analysis to decipher how single-cell behaviour determines tissue make-up during competition. Using a novel high-throughput analysis pipeline, I show that competitive interactions between MDCK wild-type cells and cells depleted of the polarity protein scribble are governed by differential sensitivity to local density and the cell-type of each cell’s neighbours. I find that local density has a dramatic effect on the rate of division and apoptosis under competitive conditions. Strikingly, such analysis reveals that proliferation of the winner cells is up-regulated in neighbourhoods mostly populated by loser cells. These data suggest that tissue-scale population shifts are strongly affected by cellular-scale tissue organisation. I present a quantitative mathematical model that demonstrates the effect of neighbour cell-type dependence of apoptosis and division in determining the fitness of competing cell lines.
... Furthermore, Scrib deletion or mislocalization cooperates with oncogenic Ras or Myc to promote tumorigenesis (Wu et al., 2010;Zhan et al., 2008). While Scrib is not widely mutated in cancers, its mislocalization and amplification are clinically correlated with high-grade cervical carcinomas (Nakagawa and Huibregtse, 2000;Nakagawa et al., 2004), as well as malignant colon (Gardiol et al., 2006), prostate, breast cancers (Pearson et al., 2011;Zhan et al., 2008), and other epithelial cancers (Vaira et al., 2011). Several classes of viruses employ mechanisms to bypass Scrib repression. ...
... In human cancers, Scrib is predominantly mislocalized from the lateral membranes to the cytosol (Vaira et al., 2011). Since Scrib requires S-palmitoylation for membrane recruitment (Chen et al., 2016), we explored if defects in Scrib S-palmitoylation might provide a potential mechanism for mislocalization during malignancy. ...
... Tumors achieve this by uncoupling key growth suppressors from their site of action, and in certain instances by disrupting plasma membrane localization. In human cancers, Scrib is mislocalized to the cytosol, which leads to a cascade of changes that disrupt cell polarity and adhesion junctions, enhance growth signaling, and increase cell survival and proliferation (Doggett et al., 2011;Vaira et al., 2011;Yamanaka and Ohno, 2008). Based on sequence conservation, recurring annotations in numerous S-palmitoylation proteomics studies, and the correlated fluorescence imaging and biochemical S-palmitoylation studies presented here, we confirm that dynamic S-palmitoylation is critical for Scrib plasma membrane localization. ...
The multidomain scaffolding protein Scribble (Scrib) organizes key signaling complexes to specify basolateral cell polarity and suppress aberrant growth. In many human cancers, genetically normal Scrib mislocalizes from cell-cell junctions to the cytosol, correlating with enhanced growth signaling and malignancy. Here we confirm that expression of the epithelial-to-mesenchymal transcription factor (EMT-TF) Snail in benign epithelial cells leads to Scrib displacement from the plasma membrane, mimicking the mislocalization observed in aggressive cancers. Upon further examination, Snail promotes a transcriptional program that targets genes in the palmitoylation cycle, repressing many protein acyl transferases and elevating expression and activity of protein acyl thioesterase 2 (APT2). APT2 isoform-selective inhibition or knockdown rescued Scrib membrane localization and palmitoylation while attenuating MEK activation. Overall, inhibiting APT2 restores balance to the Scrib palmitoylation cycle, promoting membrane re-localization and growth attenuation. These findings emphasize the importance of S-palmitoylation as a post-translational gatekeeper of cell polarity-mediated tumor suppression.
... Immunohistochemistry. Immunohistochemical (IHC) detection of SNPH was performed on breast cancers and Cancer Universal Tissue Microarrays (CaU-TMAs) as described previously 44 . Briefly, 4 mm thick slides were incubated for 1 h with a rabbit polyclonal anti-SNPH antibody (Sigma#HPA049393 diluted 1:300). ...
... Informed consent was obtained from all subjects. Representative cores of normal mammary glands (n ¼ 114), carcinoma in situ lesions (CIS, n ¼ 79), as well as of tumour bulk (core lesion) and invasive front (IF, n ¼ 299) were arranged in tissue microarrays as previously described 44 . The patients' clinicopathological features according to the American Joint Committee on Cancer -AJCC-TNM system are summarized in Supplementary Table 2. Breast cancer metastatic burden to lymph nodes was calculated as the proportion of metastatic nonsentinel lymph nodes over the total number of analysed lymph nodes. ...
... These samples were investigated for presence of SNPH isoforms by PCR on retrotranscribed cDNA. A Cancer Universal Tissue Microarray (CaU-TMA) platform has been described 21,44 . ...
The role of mitochondria in cancer is controversial. Using a genome-wide shRNA screen, we now show that tumours reprogram a network of mitochondrial dynamics operative in neurons, including syntaphilin (SNPH), kinesin KIF5B and GTPase Miro1/2 to localize mitochondria to the cortical cytoskeleton and power the membrane machinery of cell movements. When expressed in tumours, SNPH inhibits the speed and distance travelled by individual mitochondria, suppresses organelle dynamics, and blocks chemotaxis and metastasis, in vivo. Tumour progression in humans is associated with downregulation or loss of SNPH, which correlates with shortened patient survival, increased mitochondrial trafficking to the cortical cytoskeleton, greater membrane dynamics and heightened cell invasion. Therefore, a SNPH network regulates metastatic competence and may provide a therapeutic target in cancer.
... The readout of cell polarity complexes' roles in human cancer is intricate and often context-dependent [16,17]. Preliminary data in human tumours showed SCRIB overexpression in patients with liver, colorectal and lung cancer [18][19][20]. Conversely, reports about SCRIB in breast cancer are still puzzling [21][22][23] and an increased SCRIB expression in a small series of patients with breast cancer has been reported [20,23]. Furthermore, SCRIB delocalization was responsible for acquisition of tumour-initiation capacities in breast cancer cells through suppression of Hippo signalling [21]. ...
... Preliminary data in human tumours showed SCRIB overexpression in patients with liver, colorectal and lung cancer [18][19][20]. Conversely, reports about SCRIB in breast cancer are still puzzling [21][22][23] and an increased SCRIB expression in a small series of patients with breast cancer has been reported [20,23]. Furthermore, SCRIB delocalization was responsible for acquisition of tumour-initiation capacities in breast cancer cells through suppression of Hippo signalling [21]. ...
... The positive (miR-126 and U6) and negative (scrambled miRNA probe) controls displayed homogeneous staining in the different breast tissues (Supplementary Figure S2E). SCRIB subcellular localization has been related to malignant progression in disparate tumours [20,21,26], whereas SCRIB expression in normal tissues is mainly confined to the plasma membrane and its presence in the cytoplasm, defined as mislocalization, has been related to loss of cell polarity and architecture [20,22,26]. (B) miR-296 expression was analysed in matched normal mammary glands, primary infiltrating breast cancers (IBC) and correspondent distant metastases available for 21 patients by qRT-PCR after laser-assisted microdissection of the tissues of interest ( * P < 0.05 and * * * P < 0.001; Dunn's multiple comparison test). ...
MiR-296-5p is a central regulator of signalling pathways affecting development, stem cell differentiation, and cancer. We hypothesized that miR-296-5p was involved in breast cancer onset and progression possibly through regulation of its target Scribble, a polarity protein recently implicated in the acquisition of cancer stem-cell traits and in cell motility. We found that miR-296-5p levels were consistently reduced in human breast cancer tissues compared with non-neoplastic mammary parenchyma and low expression of this miRNA predicted shorter disease-free survival independently of classic clinicopathological parameters. Further, reduced miR-296-5p levels were significantly correlated to earlier spread of cancer, in the overall series and in the subset with distant metastases. Opposite to its regulator, Scribble was overexpressed and mislocalized in primary breast cancers or locoregional or distant metastatic lesions compared to normal parenchyma. Notably, Scribble mislocalization was associated with breast cancer patients' overall survival, metastatic spread and organ tropism. Finally, direct injection of a precursor miR-296-5p into tumors of a breast cancer xenograft model significantly decreased tumor growth. Our results show that the miR-296-5p/Scribble axe plays a role in breast carcinogenesis and miR-296-5p-based therapeutic approach hampers breast cancer tumor growth in vivo. Modulation of miR-296-5p may represent a new therapeutic option for breast cancer patients.
... In transgenic mice, loss of Scribble promoted abnormal growth of mammary glands and thus, Scribble was identified as a tumor suppressor (40). Scribble has been shown to play significant roles in breast cancer development where expression is often altered and subcellular localization changes from membrane associated to cytoplasmic (41). Since AQP5 is often overexpressed in breast cancer and affects cellular features such as cell-cell adhesion (6,8), we hypothesized that AQP5 may affect Scribble and thus, cell polarity. ...
... In another study conducted on samples from 96 patients with triple-negative breast cancer, immunohistochemistry analysis showed that AQP5 expression levels were correlated with tumor size (P < 0.001), metastasis (P < 0.003), and low overall 5-yr survival rate (P < 0.024) (28). In breast cancer, counterintuitive to its tumor suppressor function as a polarity protein, Scribble was reported to be overexpressed (41). A recent genomic study that compiled data from 748 breast cancers (Cancer Genome Atlas) has shown that the Scribble gene expression varied between breast cancer subtypes and that the Scribble gene expression was significantly higher in the basal-like and luminal B tumors, as compared with luminal A, Her2-like, and normallike tumors (57). ...
Breast carcinomas originate from cells in the terminal duct-lobular unit. Carcinomas are associated with increased cell proliferation and migration, altered cellular adhesion, as well as loss of epithelial polarity. In breast cancer, aberrant and high levels of AQP5 are associated with increased metastasis, poor prognosis and cancer recurrence. AQP5 increases proliferation and migration of cancer cells, and ectopic expression of AQP5 in normal epithelial cells reduces cell-cell adhesion and increases cell detachment and dissemination from migrating cell sheets, the latter via AQP5 mediated activation of the Ras pathway. Here, we investigated if AQP5 also affects cellular polarity by examining the relationship between the essential polarity protein Scribble and AQP5. In tissue samples from invasive lobular and ductal carcinomas, the majority of cells with high AQP5 expression displayed low Scribble levels, indicating an inverse relationship. Probing for interactions via a GST pull-down experiment revealed that AQP5 and Scribble interacted. Moreover, overexpression of AQP5 in the breast cancer cell line MCF7 reduced both size and circularity of 3D spheroids and induced cell detachment and dissemination from migrating cell sheets. In addition, Scribble levels were reduced. An AQP5 mutant cell line, that cannot activate Ras (AQP5S156A), displayed unchanged spheroid size and circularity and an intermediate level of Scribble, indicating that the effect of AQP5 on Scribble is, at least in part, dependent of AQP5 mediated activation of Ras. Thus, our results suggest that high AQP5 expression negatively regulates the essential polarity protein Scribble and thus, can affect cellular polarity in breast cancer.
... In contrast to some of the previous reports, a recent study demonstrated that Scrib is overexpressed in the majority of human cancers [28], suggesting that Scrib may not only be down-regulated and mislocalized but also could be overexpressed, and possibly, mislocalized in different cancers. Moreover, contrary to the tumor suppressor function of Scrib in epithelial tissue, loss of Scrib expression delayed the onset of Eμ-myc-driven lymphoma, suggesting a potential oncogenic role of Scrib in Myc-driven lymphoma [29]. ...
... However, we found that Scrib expression is strongly induced in mouse and human HCC samples. Our observations are in line with a recent study where it was demonstrated that Scrib is overexpressed in several tumor cell lines, and in the majority of human cancers including HCC [28]. It appears that the expression and localization of Scrib is different from one type of cancer to the other. ...
Scrib is a membrane protein that is involved in the maintenance of apical-basal cell polarity of the epithelial tissues. However, Scrib has also been shown to be mislocalized to the cytoplasm in breast and prostate cancer. Here, for the first time, we report that Scrib not only translocates to the cytoplasm but also to the nucleus in hepatocellular carcinoma (HCC) cells, and in mouse and human liver tumor samples. We demonstrate that Scrib overexpression suppresses the growth of HCC cells in vitro, and Scrib deficiency enhances liver tumor growth in vivo. At the molecular level, we have identified the existence of a positive feed-back loop between Yap1 and c-Myc in HCC cells, which Scrib disrupts by simultaneously regulating the MAPK/ERK and Hippo signaling pathways. Overall, Scrib inhibits liver cancer cell proliferation by suppressing the expression of three oncogenes, Yap1, c-Myc and cyclin D1, thereby functioning as a tumor suppressor in liver cancer.
... 7 Alterations in Scrib expression levels are observed in a variety of tumor types including breast, cervical and prostate. [8][9][10][11][12][13][14] Data from cell lines and primary tissue samples suggest that altered expression and/or mislocalization of cell polarity proteins play a causal role in tumorigenesis [15][16][17] and we recently reported that mislocalization of Scrib in human prostate cancer correlates with poor survival. 14 Although the molecular mechanisms behind Scrib function are not well understood, Scrib has been implicated in numerous cellular processes including proliferation, differentiation, apoptosis, stemcell maintenance, migration and vesicle trafficking. ...
... Disruption of cell polarity is recognized as a hallmark of cancer and it was recently reported that deregulation of cell polarity regulators is associated with a number of epithelial cancers. [8][9][10][11][12][13][14] We have performed detailed assessment of public databases from the Sanger Institute (www.sanger.ac.uk), the cBioPortal for Cancer Genomics (http://www.cbioportal.org) and The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/) to specifically investigate the role of SCRIB in human cancers. ...
Lung cancer is the leading cause of cancer deaths worldwide with non small-cell lung cancer (NSCLC) accounting for 80% of all lung cancers. Although activating mutations in genes of the RAS-MAPK pathway occur in up to 30% of all NSCLC, the cooperating genetic lesions that are required for lung cancer initiation and progression remain poorly understood. Here we identify a role for the cell polarity regulator Scribble (Scrib) in NSCLC. A survey of genomic databases reveals deregulation of SCRIB in human lung cancer and we show that Scrib(+/-) mutant mice develop lung cancer by 540 days with a penetrance of 43%. To model NSCLC development in vivo, we used the extensively characterized LSL-KRas(G12D) murine model of NSCLC. We show that loss of Scrib and activated oncogenic KRas cooperate in vivo, resulting in more aggressive lung tumors, likely due to a synergistic elevation in RAS-MAPK signaling. Finally, we provide data consistent with immune infiltration having an important role in the acceleration of tumorigenesis in KRas(G12D) lung tumors following Scrib loss.Oncogene advance online publication, 25 November 2013; doi:10.1038/onc.2013.498.
... Our results support the role of Scrib as a tumor suppressor. At the same time, Scrib has also been reported to be over-expressed in cancer cells and behave as an oncogenic protein [74,75]. These findings suggest that Scrib plays a context-specific role in epithelial tissue, both as a tumor suppressor and as an oncogene [76]. ...
E-cadherin-catenin complex together with the cytoskeleton, builds the core of Adherens junctions (AJs). It has been reported that Scribble stabilizes the coupling of E-cadherin with catenins promoting epithelial cell adhesion, but the mechanism remains unknown. We show that Scribble, Lgl1, and NMII-A reside in a complex with E-cadherin-catenin complex. Depletion of either Scribble or Lgl1 disrupts the localization of E-cadherin-catenin complex to AJs. aPKCζ phosphorylation of Lgl1 regulates AJ localization of Lgl1 and E-cadherin-catenin complexes. Both Scribble and Lgl1 regulate the activation and recruitment of NMII-A at AJs. Finally, Scribble and Lgl1 are downregulated by TGFβ-induced EMT, and their re-expression during EMT impedes its progression. Our results provide insight into the mechanism regulating AJ integrity by Scribble, Lgl1, and NMII-A.
... Most human cancers, including HCC, originate from epithelial tissues, and a loss of cell polarity and tissue structure is often a hallmark of such malignancies [6,9]. Therefore, aberrant expression of SCRIB is frequently observed in several human cancers, including prostate, colorectal, and liver cancer [10][11][12][13]. ...
Objective: This study aimed to elucidate the prognostic value of the leucine rich repeat containing 1 (LRRC1) gene in hepatocellular carcinoma (HCC) and to determine the effects of high and low LRRC1 expression on mutation and immune cell infiltration.
Methods: We downloaded HCC mRNA-seq expression and clinical data from UCSC Xena. The expression of LRRC1 was compared between HCC tumor and normal samples. Tumor samples were divided according to high and low LRRC1 expression. Differentially expressed genes between the two groups were identified, and function, mutation, and immune cell infiltration were analyzed. Genes associated with immune cells were identified using weighted gene co-expression network analysis (WGCNA), and transcription factors (TFs) of these genes were predicted.
Results: The expression of LRRC1was upregulated in HCC tissues, and this indicated a poor prognosis for patients with HCC. Differentially expressed genes between tumors with high and low LRRC1 expression were significantly enriched in pathways associated with cancer, amino acid metabolism, carbohydrate metabolism, and the immune system. We identified 15 differentially infiltrated immune cells between tumors with high and low LRRC1 expression and 14 of them correlated with LRRC1gene expression. We also identified 83 genes that were associated with immune cells. Cyclic AMP-response element binding protein (CREB1) regulated ANXA5, MMP9, and LRRC1in the TF regulatory network.
Conclusion: The LRRC1 gene might serve as a potential immune-associated prognostic biomarker for HCC.
... Most human cancers, including HCC, originate from epithelial tissues, and a loss of cell polarity and tissue structure is often a hallmark of such malignancies [6,9]. Therefore, aberrant expression of SCRIB is frequently observed in several human cancers, including prostate, colorectal, and liver cancer [10][11][12][13]. ...
Objective: This study aimed to elucidate the prognostic value of the leucine rich repeat containing 1 (LRRC1) gene in hepatocellular carcinoma (HCC) and to determine the effects of high and low LRRC1 expression on mutation and immune cell infiltration. Methods: We downloaded HCC mRNA-seq expression and clinical data from UCSC Xena. The expression of LRRC1 was compared between HCC tumor and normal samples. Tumor samples were divided according to high and low LRRC1 expression. Differentially expressed genes between the two groups were identified, and function, mutation, and immune cell infiltration were analyzed. Genes associated with immune cells were identified using weighted gene co-expression network analysis (WGCNA), and transcription factors (TFs) of these genes were predicted. Results: The expression of LRRC1 was upregulated in HCC tissues, and this indicated a poor prognosis for patients with HCC. Differentially expressed genes between tumors with high and low LRRC1 expression were significantly enriched in pathways associated with cancer, amino acid metabolism, carbohydrate metabolism, and the immune system. We identified 15 differentially infiltrated immune cells between tumors with high and low LRRC1 expression and 14 of them correlated with LRRC1 gene expression. We also identified 83 genes that were associated with immune cells. Cyclic AMP-response element binding protein (CREB1) regulated ANXA5, MMP9, and LRRC1 in the TF regulatory network. Conclusion: The LRRC1 gene might serve as a potential immune-associated prognostic biomarker for HCC.
... Moreover, SCRIB has been characterized as a critical player for epithelial cell migration, in response to extracellular signals during wound healing [43]. Although it was previously reported that SCRIB was universally overexpressed in cells of various tumors including colon, liver, prostate, uterus, thyroid, lung, bladder, breast, ovary, and stomach [46], some studies have reported a down-regulation and cytoplasmic localization of SCRIB in colon, endometrial, breast, and cervical cancers [43]. Likewise, low expression of SCRIB in fibroblasts seems to be associated with the invasiveness of lung cancer cells [47]. ...
The major causative agents of head and neck squamous cell carcinomas (HNSCCs) are either environmental factors, such as tobacco and alcohol consumption, or infection with oncogenic human papillomaviruses (HPVs). An important aspect of HPV-induced oncogenesis is the targeting by the E6 oncoprotein of PDZ domain-containing substrates for proteasomal destruction. Tumor suppressors DLG1 and SCRIB are two of the principal PDZ domain-containing E6 targets. Both have been shown to play critical roles in the regulation of cell growth and polarity and in maintaining the structural integrity of the epithelia. We investigated how modifications in the cellular localization and protein expression of DLG1 and SCRIB in HPV16-positive and HPV-negative histologic oropharyngeal squamous cell carcinomas (OPSCC) might reflect disease progression. HPV presence was determined by p16 staining and HPV genotyping. Whilst DLG1 expression levels did not differ markedly between HPV-negative and HPV16-positive OPSCCs, it appeared to be relocated from cell–cell contacts to the cytoplasm in most samples, regardless of HPV16 positivity. This indicates that alterations in DLG1 distribution could contribute to malignant progression in OPSCCs. Interestingly, SCRIB was also relocated from cell–cell contacts to the cytoplasm in the tumor samples in comparison with normal tissue, regardless of HPV16 status, but in addition there was an obvious reduction in SCRIB expression in higher grade tumors. Strikingly, loss of SCRIB was even more pronounced in HPV16-positive OPSCCs. These alterations in SCRIB levels may contribute to transformation and loss of tissue architecture in the process of carcinogenesis and could potentially serve as markers in the development of OPSCCs.
... [49][50][51][52] In cancer cells, SCRIB primarily stays in cytosol. 53 Thus, maintaining the levels and functions of SCRIB appears to be essential for tissue integrity. Deregulation of SCRIB expression by increasing anti-circSCRIBs may trigger cancer development, although circSCRIBs may be needed for tissue regeneration and wound repair. ...
Circular RNAs (circRNAs) represent a large group of non-coding RNAs that are widely detected in mammalian cells. Although most circRNAs are generated in a sense orientation, there is a group of circRNAs that are synthesized in an antisense orientation. High-throughput analysis of breast cancer specimens revealed a significant enrichment of 209 antisense circRNAs. The tumor suppressor SCRIB was shown to potentially produce thirteen circRNAs, three of which are in an antisense orientation. Among these three circRNAs, circSCRIB (hsa_circ_0001831) was the most enriched in the breast cancer panel. This antisense SCRIB circRNA was shown to span one intron and two exons. We hypothesized that this circRNA could decrease pre-mRNA splicing and mRNA translation. To test this, we generated a hsa_circ_0001831 expression construct. We found that there was decreased SCRIB mRNA production but increased cancer cell proliferation, migration, and invasion. In comparison, an exonic sequence construct did not affect mRNA splicing but decreased protein translation, leading to increased E-cadherin expression and decreased expression of N-cadherin and vimentin. Thus, there was increased cell migration, invasion, proliferation, colony formation, and tumorigenesis. Our study suggests a novel modulatory role of antisense circRNAs on their parental transcripts. This may represent a promising approach for developing circRNA directed therapy.
... Originally identified in Drosophila, the cell polarity tumor suppressor Scrib has emerged as a central regulator of cell growth, invasion, and malignancy. [89][90][91] While Scrib is not widely mutated in cancers, its mislocalization and amplification are clinically correlated with high-grade cervical carcinomas, 92, 93 as well as malignant colon, 94 prostate, 95 breast cancers, 95,96 and other epithelial cancers 97 (Figure 1.4A-B). In addition, Scrib +/heterozygous male mice develop widespread epithelial hyperplasia, 95 and MMTV-driven conditional knockout of Scrib in female mice induces breast hyperplasia and widespread tumor formation. ...
Proteins are often regulated through the addition of chemical modifications that modulate localization, activity, and interactions. Protein S-palmitoylation describes the attachment of long-chain fatty acids to cysteine residues in proteins to promote membrane association, which contributes to the regulation of a number of cellular processes. Altered S-palmitoylation contributes to the pathogenesis of neurological disorders, cancer, and many other diseases. Importantly, dynamic S-palmitoylation is required to modulate the activity of proteins in response to various extracellular signaling events. This includes various membrane-associated or membrane proteins such as G proteins, small GTPases, receptors, scaffold proteins, cytoskeletal proteins, and kinases. These observations suggest that enzymes catalyzing the removal of palmitate must also play a critical role in modulating the activity of hundreds of essential proteins. Therefore, it is important to characterize how S-palmitoylation is regulated, which will provide a more complete molecular picture of how cells adjust to various signaling events. Over the last decade, potent inhibitors of depalmitoylases have been developed, yet the basic mechanisms and the cellular functions of depalmitoylases remain poorly characterized. Since depalmitoylation can modulate select cell signaling events, I hypothesized that depalmitoylase enzymes have dedicated substrates and differentially contribute to the global dynamic S-palmitoylation regulation. My dissertation addresses this hypothesis by focusing on the structure, function, and inhibition of the depalmitoylating enzymes APT1 and APT2. The first chapter of this thesis provides an overview of depalmitoylating enzymes. Second, I used isoform-selective inhibitors to understand the structure, substrate engagement and selectivity of APT1 and APT2. Third, I validated the selectivity of the APT2 inhibitor ML349 by affinity enrichment and proteomics. Finally, I explore the dynamic rate of palmitoylation regulated by depalmitoylases in cells. Overall, my research has clarified the mechanism and function of protein de-palmitoylation in cell regulation.
... Interestingly, E6 has been further shown to cause degradation of the polarity protein SCRIB (Gheit, 2019), and overexpression of SCRIB is associated with poor cervical cancer prognosis. This observation is not completely surprising as SCRIB overexpression is seen in a number of cancers, with mislocalisation usually favoring a mesenchymal phenotype (Vaira et al., 2011), and perhaps SCRIB overexpression begins in later stages of HPV associated malignant disease. ...
Human papillomavirus (HPV) infection is associated with a range of malignancies that affect anogenital and oropharyngeal sites. α-HPVs dominantly infect basal epithelial cells of mucosal tissues, where they dysregulate cell division and local immunity. The cervix is one of the mucosal sites most susceptible to HPV infections. It consists of anatomically diverse regions, and the majority of cervical intraepithelial neoplasia and cancers arise within the cervical squamo-columnar junction where undifferentiated basal progenitor cells with stem cell properties are found. The cancer stem cell theory particularly associates tumorigenesis, invasion, dissemination, and metastasis with cancer cells exhibiting stem cell properties. In this perspective, we discuss evidence of a cervical cancer stem cell niche and explore the association of stemness related genes with 5-year survival using a publicly available transcriptomic dataset of a cervical cancer cohort. We report that poor prognosis in this cohort correlates with overexpression of a subset of stemness pathway genes, a majority of which regulate the central Focal Adhesion pathway, and are also found to be enriched in the HPV infection pathway. These observations support therapeutic targeting of stemness genes overexpressed by mucosal cells infected with high-risk HPVs.
... Representative tissue blocks of tumour and non-neoplastic lung tissue derived from each patient were used to construct TMAs, as previously described [20]. Briefly, for all lung cancer samples five representative cores were selected by a pathologist whereas for non-neoplastic parenchyma one core was chosen. ...
Background:
Lung cancer is still the main cause of cancer death worldwide despite the availability of targeted therapies and immune-checkpoint inhibitors combined with chemotherapy. Cancer cell heterogeneity and primary or acquired resistance mechanisms cause the elusive behaviour of this cancer and new biomarkers and active drugs are urgently needed to overcome these limitations. p65BTK, a novel isoform of the Bruton Tyrosine Kinase may represent a new actionable target in non-small cell lung cancer (NSCLC).
Methods:
p65BTK expression was evaluated by immunohistochemistry in 382 NSCLC patients with complete clinico-pathological records including smoking habit, ALK and EGFR status, and in metastatic lymph nodes of 30 NSCLC patients. NSCLC cell lines mutated for p53 and/or a component of the RAS/MAPK pathway and primary lung cancer-derived cells from Kras/Trp53 null mice were used as a preclinical model. The effects of p65BTK inhibition by BTK Tyrosine Kinase Inhibitors (TKIs) (Ibrutinib, AVL-292, RN486) and first-generation EGFR-TKIs (Gefitinib, Erlotinib) on cell viability were evaluated by MTT. The effects of BTK-TKIs on cell growth and clonogenicity were assessed by crystal violet and colony assays, respectively. Cell toxicity assays were performed to study the effect of the combination of non-toxic concentrations of BTK-TKIs with EGFR-TKIs and standard-of-care (SOC) chemotherapy (Cisplatin, Gemcitabine, Pemetrexed).
Results:
p65BTK was significantly over-expressed in EGFR-wild type (wt) adenocarcinomas (AdC) from non-smoker patients and its expression was also preserved at the metastatic site. p65BTK was also over-expressed in cell lines mutated for KRAS or for a component of the RAS/MAPK pathway and in tumors from Kras/Trp53 null mice. BTK-TKIs were more effective than EGFR-TKIs in decreasing cancer cell viability and significantly impaired cell proliferation and clonogenicity. Moreover, non-toxic doses of BTK-TKIs re-sensitized drug-resistant NSCLC cell lines to both target- and SOC therapy, independently from EGFR/KRAS status.
Conclusions:
p65BTK results as an emerging actionable target in non-smoking EGFR-wt AdC, also at advanced stages of disease. Notably, these patients are not eligible for EGFR-TKIs-based therapy due to a lack of EGFR mutation. The combination of BTK-TKIs with EGFR-TKIs is cytotoxic for EGFR-wt/KRAS-mutant/p53-null tumors and BTK-TKIs re-sensitizes drug-resistant NSCLC to SOC chemotherapy. Therefore, our data suggest that adding BTK-TKIs to SOC chemotherapy and EGFR-targeted therapy may open new avenues for clinical trials in currently untreatable NSCLC.
... 6 By contrast, in a pathology study using human surgical material, Scribble was expressed in many types of tumors, leaving uncertain the biological role of these genes in human tumors. 7 A single transmembrane protein Crb3 was described as expressed at the apical plasma membrane of epithelial cells of diverse origins. 8,9 The Crb3 locus generates two alternativespliced isoforms, Crb3 isoform a (Crb3a) and Crb3 isoform b (Crb3b), reported to be involved in different biological processes. ...
Epithelial cell polarity regulator Crumbs3 (Crb3), a mammalian homolog within the Drosophila Crb gene family, was initially identified as an essential embryonic development factor. It is recently implicated in tumor suppression, though its specific functions are controversial. We here demonstrate that Crb3 strongly promotes tumor invasion and metastasis of human colon adenocarcinoma cells. Crb3 centrality to tumor migration was supported by strong expression at invasive front and metastatic foci of colonic adenocarcinoma of the patient tissues. Accordingly, two different Crb3‐knockout (KO) lines, Crb3‐KO (Crb3 −/−) DLD‐1 and Crb3‐KO WiDr from human colonic adenocarcinomas, were generated by the CRISPR‐Cas9 system. Crb3‐KO DLD‐1 cells exhibited loss of cellular mobility in vitro and dramatic suppression of liver metastases in vivo in contrast to the wild type of DLD‐1. Unlike DLD‐1, Crb3‐KO WiDr mobility and metastasis were unaffected, which were similar to wild‐type WiDr. Proteome analysis of Crb3‐coimmunopreciptated proteins identified different respective fibroblast growth factor receptor (FGFR) isotypes specifically bound to Crb3 isoform a through their intracellular domain. In DLD‐1, Crb3 showed membranous localization of FGFR1 leading to its functional activation, whereas Crb3 bound to cytoplasmic FGFR4 in WiDr without FGFR1 expression, leading to cellular growth. Correlative expression between Crb3 and FGFR1 was consistently detected in primary and metastatic colorectal cancer patient tissues. Taking these together, Crb3 critically accelerates cell migration, namely invasion and metastasis of human colon cancers, through specific interaction to FGFR1 on colon cancer cells.
... Polarity can be monitored by marker proteins of the apical or basolateral membranes. However, due to the need to polarize T84s on non-transparent transwell inserts were are unable to monitor the process of polarization live and fixation of these samples will not allow us to follow the same cell population over time [153][154][155] . Therefore we decided to use a less invasive way of measuring polarity: the trans-epithelial electrical resistance (TEER). ...
Cell polarity describes the asymmetric distribution of proteins, RNA, organelles and lipids. One of the best studied models of polarity are epithelial cells. They form the barriers that separate and protect the organism from the outside environment. Establishment and maintenance of polarity is ensured through a highly complex network of proteins and lipids that mediate asymmetry through trafficking processes and reorganization of the cytoskeleton. One of the key players in polarity are phosphoinositides, phosphorylated forms of the lipid phosphatidylinositol. In this thesis I investigated the role of one such phosphoinositide, PI(3)P, on cellular polarity and immunity in human intestinal epithelial cells. In my work I used established methods to evaluate the role of PI(3)P in human intestinal epithelial cells. These three methods were (1) the use of specific inhibitors targeting VPS34 (2) the development of cell lines depleted of kinases and phosphatases needed for PI(3)P biogenesis and (3) on demand depletion of PI(3)P from endosomes using a chemical dimerizer system. Each of these methods were evaluated both for their ability to modulate PI(3)P levels and for their impact on cellular polarity and immune response. While I could demonstrate that all three methods successfully modulated PI(3)P levels the chemical dimerizer (AP21967) interferes with polarity and immunity itself and we require a new dimerizer before conclusions can be made using this method. Interestingly, I determined that reduced PI(3)P levels, either by chemical inhibition or knock-down of the kinase VPS34, impaired cellular polarity in T84 cells indicating a critical role of PI(3)P in the polarity program. Additionally, knock-down of the PI(3)P metabolizing enzymes VPS34 and MTM1 showed interesting results for IFNλ production upon reovirus infection. IFNλ production in MTM1 and VPS34 knock-down cells is increased and decreased respectively. This could be due to the previously reported PI(3)P dependent TLR3 sorting adaptor WDFY1 and should be further investigated. In parallel I also created a novel viral vector system in our lab. This viral vector is based on the BacMam system which are baculovirus based vectors with a large cargo capacity. The BacMam was initially modified with an S/MAR sequence to allow for its persistence in cells. The vector was then further modified to allow expression of reporter genes. Both vectors were shown to be fully functional and will provide a valuable tool for future projects.
... The apical-basolateral polarity is a key property of epithelia to fulfill their barrier and communication roles. The scaffolding protein Scribble (Scrib), which localizes to cell-cell junctions, is involved in establishing and maintaining this polarity (Dow et al. 2003), and mislocalizes to the cytosol in many human cancers (Vaira et al. 2011). Its plasma membrane localization and tumor suppression capacity depend on the S-acylation of two conserved cysteine residues, mostly mediated by zDHHC7 (Chen et al. 2016). ...
S-Acylation (commonly referred to as S-palmitoylation) is a post-translational modification consisting in the covalent attachment of an acyl chain to a cysteine residue of the target protein. The lability of the resulting thioester bond gives S-acylation an essential characteristic: its reversibility. S-acylation dynamically regulates different aspects in the life of a protein (including stability, localization, interactome, and function) and, thus, plays critical roles in cellular physiology. For long, the reversibility of S-acylation has been neglected and thereby its potential as a regulatory mechanism for protein function undervalued. Thanks to technological advances, the field has now entered its golden era. A great diversity of interesting targets is being identified, the physio-pathological importance of the modification is starting to be revealed, structural information on the enzymes is becoming available, and the regulatory dynamics are gradually being understood. Here we will review the most recent literature in the S-acylation field, with a special focus on the molecular aspects of the modification, its regulation, and its consequences.
... As such, these proteins have been suggested to be tumour suppressors in humans, highlighting the relevance of this model to human cancer 154,158 . However, it is intriguing that the human homologues of scrib, dlg and l(2)gl are rarely deleted or mutated in cancer; rather, these genes are frequently amplified and/or overexpressed (CbioPortal) [159][160][161] . These observations indicate that the roles of SCRIB, DLG and LLGL proteins in tumour progression are poorly understood. ...
Metastasis remains the greatest challenge in the clinical management of cancer. Cell motility is a fundamental and ancient cellular behaviour that contributes to metastasis and is conserved in simple organisms. In this Review, we evaluate insights relevant to human cancer that are derived from the study of cell motility in non-mammalian model organisms. Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio permit direct observation of cells moving in complex native environments and lend themselves to large-scale genetic and pharmacological screening. We highlight insights derived from each of these organisms, including the detailed signalling network that governs chemotaxis towards chemokines; a novel mechanism of basement membrane invasion; the positive role of E-cadherin in collective direction-sensing; the identification and optimization of kinase inhibitors for metastatic thyroid cancer on the basis of work in flies; and the value of zebrafish for live imaging, especially of vascular remodelling and interactions between tumour cells and host tissues. While the motility of tumour cells and certain host cells promotes metastatic spread, the motility of tumour-reactive T cells likely increases their antitumour effects. Therefore, it is important to elucidate the mechanisms underlying all types of cell motility, with the ultimate goal of identifying combination therapies that will increase the motility of beneficial cells and block the spread of harmful cells.
... SCRIB is a scaffolding protein required for epithelial cell identity and prevents EMT progression by blocking loss of E-cadherin and ZO1 from AJ [134]. In contrast with these roles, SCRIB knockdown has been associated with impaired cell migration and downregulation of mesenchymal markers [135]. The apparent antithetical functions of SCRIB in cell migration and EMT could be partially explained by the ESRP-dependent splicing of SCRIB pre-mRNAs, where SCRIB epithelial-isoform is required for AJ stability, whereas the mesenchymal-specific variant is involved in cell motility [136]. ...
Epithelial-to-mesenchymal transition (EMT) is associated with metastasis formation as well as with generation and maintenance of cancer stem cells. In this way, EMT contributes to tumor invasion, heterogeneity and chemoresistance. Morphological and functional changes involved in these processes require robust reprogramming of gene expression, which is only partially accomplished at the transcriptional level. Alternative splicing is another essential layer of gene expression regulation that expands the cell proteome. This step in post-transcriptional regulation of gene expression tightly controls cell identity between epithelial and mesenchymal states and during stem cell differentiation. Importantly, dysregulation of splicing factor function and cancer-specific splicing isoform expression frequently occurs in human tumors, suggesting the importance of alternative splicing regulation for cancer biology.
In this review, we briefly discuss the role of EMT programs in development, stem cell differentiation and cancer progression. Next, we focus on selected examples of key factors involved in EMT and stem cell differentiation that are regulated post-transcriptionally through alternative splicing mechanisms. Lastly, we describe relevant oncogenic splice-variants that directly orchestrate cancer stem cell biology and tumor EMT, which may be envisioned as novel targets for therapeutic intervention.
... During follow-up, 9 NSCLC patients developed metastatic disease to the brain, whereas 10 patients had no evidence of metastasis (S1 Table). A Cancer Universal TMA (CaU-TMA) representative of 13 different cancer types (10 cases for each tumor type) was described previously [40]. ...
Mitochondria must buffer the risk of proteotoxic stress to preserve bioenergetics, but the role of these mechanisms in disease is poorly understood. Using a proteomics screen, we now show that the mitochondrial unfoldase-peptidase complex ClpXP associates with the oncoprotein survivin and the respiratory chain Complex II subunit succinate dehydrogenase B (SDHB) in mitochondria of tumor cells. Knockdown of ClpXP subunits ClpP or ClpX induces the accumulation of misfolded SDHB, impairing oxidative phosphorylation and ATP production while activating "stress" signals of 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and autophagy. Deregulated mitochondrial respiration induced by ClpXP targeting causes oxidative stress, which in turn reduces tumor cell proliferation, suppresses cell motility, and abolishes metastatic dissemination in vivo. ClpP is universally overexpressed in primary and metastatic human cancer, correlating with shortened patient survival. Therefore, tumors exploit ClpXP-directed proteostasis to maintain mitochondrial bioenergetics, buffer oxidative stress, and enable metastatic competence. This pathway may provide a "drugable" therapeutic target in cancer.
... The suppressive activities of wild-type SCRIB on YAP target gene expression (CTGF and CYR61) were partially reversed by loss of ZDHHC7 in MCF10A cells (Supplementary Fig. 19), confirming that ZDHHC7 has important roles in regulating SCRIB functions. SCRIB has been implicated in the regulation of cell invasion, transformation and epithelial-to-mesenchymal transition 4,8,28,35 using a previously reported Matrigel/collagen I system 8,28 . Western blot analysis showed that expression levels of HRas V12 were comparable in different MCF10A stable cell lines ( Supplementary Fig. 20). ...
Scribble (SCRIB) is a tumor-suppressor protein, playing critical roles in establishing and maintaining epithelial cell polarity. SCRIB is frequently amplified in human cancers but does not localize properly to cell-cell junctions, suggesting that mislocalization of SCRIB disrupts its tumor-suppressive activities. Using chemical reporters, here we showed that SCRIB localization was regulated by S-palmitoylation at conserved cysteine residues. Palmitoylation-deficient mutants of SCRIB were mislocalized, leading to disruption of cell polarity and loss of their tumor-suppressive activities to oncogenic YAP, MAPK and PI3K/AKT pathways. We further found that ZDHHC7 was the major palmitoyl acyltransferase regulating SCRIB. Knockout of ZDHHC7 led to SCRIB mislocalization and YAP activation, and disruption of SCRIB's suppressive activities in HRas(V12)-induced cell invasion. In summary, we demonstrated that ZDHHC7-mediated SCRIB palmitoylation is critical for SCRIB membrane targeting, cell polarity and tumor suppression, providing new mechanistic insights of how dynamic protein palmitoylation regulates cell polarity and tumorigenesis.
... It was found to be lost or mislocalized in some breast tumors (Zhan et al., 2008). It can be also overexpressed and mislocalized in breast cancers with decreased expression of miR-296 (Savi et al., 2014) and overexpressed and mislocalized in many cancer cell lines and diverse tumors (Vaira et al., 2011). Fig. 1. ...
SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. Here we report that, in breast cancer, an additional and hidden dimension of deregulations exists: an unexpected SCRIB exon usage pattern appears to mark a more malignant tumor phenotype and significantly correlates with survival. Conserved exons encoding the leucine-rich repeats tend to be overexpressed while others are underused. Mechanistic studies revealed that the underused exons encode part of the protein necessary for interaction with Vimentin and Numa1, a protein which is required for proper positioning of the mitotic spindle. Thus, the inclusion/exclusion of specific SCRIB exons is a mechanistic hallmark of breast cancer, which could potentially be exploited to develop more efficient diagnostics and therapies.
... [21] Downregulation of scribble is sufficient to induce the initiation of lung tumors. [41] Loss of the polarity proteins can directly deregulate cell adhesion processes, which is consistent with the loss of E-cadherin [42] while the situation thus will disrupt morphogenesis and promote tumorigenesis. [43] Similarly with the function of E-cadherin in ECM, the polarity proteins also involve in regulation of ECM. ...
Lung cancer is the most frequent malignant disease, since it has often metastasized to distant organs by the time of diagnosis. Epithelial-mesenchymal transition (EMT) is an important process during the progression of lung cancer. Epithelial cells lose the polarity, which contributes to uncontrolled invasion and metastasis of cancer cells. Cell polarity establishment and maintenance depends upon the three complex proteins which are par, crumbs and scribble complexes, of which are reported as tumor suppressors. The cell polarity proteins could interact with cell-cell contact and cell-extracellular matrix contact and cell-intrinsic signaling. These interactions are proved to be involved in lung cancer metastasis. However, our understanding of the mechanisms by which this occurs is poor. In this review, we will discuss the regulatory network of cell polarity in the lung cancer, especially on EMT.
... 6 In fly, loss of any of these proteins can cooperate with oncogenic Ras to induce invasive tumors; 7 whereas in mammal, homologs of these proteins show consistently aberrant expression in multiple cancers, including lung, breast, colon and prostate. [8][9][10] Despite the wellestablished connection between dysregulation of cell polarity and cancer, the factors that act downstream of the Scrib complex to mediate cancer progression remain poorly understood. ...
Both cell polarity and c-Jun N-terminal kinase (JNK) activity are essential to the maintenance of tissue homeostasis, and disruption of either is commonly seen in cancer progression. Despite the established connection between loss-of-cell polarity and JNK activation, much less is known about the molecular mechanism by which aberrant cell polarity induces JNK-mediated cell migration and tumor invasion. Here we show results from a genetic screen using an in vivo invasion model via knocking down cell polarity gene in Drosophila wing discs, and identify Rho1-Wnd signaling as an important molecular link that mediates loss-of-cell polarity-triggered JNK activation and cell invasion. We show that Wallenda (Wnd), a protein kinase of the mitogen-activated protein kinase kinase kinase family, by forming a complex with the GTPase Rho1, is both necessary and sufficient for Rho1-induced JNK-dependent cell invasion, MMP1 activation and epithelial-mesenchymal transition. Furthermore, Wnd promotes cell proliferation and tissue growth through wingless production when apoptosis is inhibited by p35. Finally, Wnd shows oncogenic cooperation with Ras(V12) to trigger tumor growth in eye discs and causes invasion into the ventral nerve cord. Together, our data not only provides a novel mechanistic insight on how cell polarity loss contributes to cell invasion, but also highlights the value of the Drosophila model system to explore human cancer biology.Oncogene advance online publication, 11 May 2015; doi:10.1038/onc.2015.137.
... PKC activation of the small G-protein Ras and of the Raf-MEK-ERK downstream pathway is also involved in tumor cell migration [72]. Le Dow et al. (2008) [73] reported that in the absence of Scrib, a protein complex involved in cell migration ability, activated Ras alters mammary epithelial cell-cell junctions, leading to increased epithelial cell migration. In fact, Ras activation prevents normal epithelial junction formation and plays a role in the alteration of polarity protein localization. ...
Alpha-fetoprotein (AFP) is a diagnostic marker for hepatocellular carcinoma (HCC). A direct relationship between poor prognosis and the concentration of serum AFP has been observed. Telomerase, an enzyme that stabilizes the telomere length, is expressed by 90% of HCC. The aim of this study was to investigate the effect of telomerase inhibition on AFP secretion and the involvement of the PI3K/Akt/mTOR signaling pathway. Proliferation and viability tests were performed using tetrazolium salt. Apoptosis was determined through the Annexin V assay using flow cytometry. The concentrations of AFP were measured using ELISA kits. The AFP mRNA expression was evaluated using RT-PCR, and cell migration was evaluated using a Boyden chamber assay. The in vivo effect of costunolide on AFP production was tested in NSG mice. Telomerase inhibition by costunolide and BIBR 1532 at 5 and 10 μM decreased AFP mRNA expression and protein secretion by HepG2/C3A cells. The same pattern was obtained with cells treated with hTERT siRNA. This treatment exhibited no apoptotic effect. The AFP mRNA expression and protein secretion by PLC/PRF/5 was decreased after treatment with BIBR1532 at 10 μM. In contrast, no effect was obtained for PLC/PRF/5 cells treated with costunolide at 5 or 10 μM. Inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP concentration. In contrast, the MAPK/ERK pathway appeared to not be involved in HepG2/C3A cells, whereas ERK inhibition decreased the AFP concentration in PLC/PRF/5 cells. Modulation of the AFP concentration was also obtained after the inhibition or activation of PKC. Costunolide (30 mg/kg) significantly decreased the AFP serum concentration of NSG mice bearing HepG2/C3A cells. Both the inhibition of telomerase and the inhibition of the PI3K/Akt/mTOR signaling pathway decreased the AFP production of HepG2/C3A and PLC/PRF/5 cells, suggesting a relationship between telomerase and AFP expression through the PI3K/Akt/mTOR pathway.
... The tumor-suppressive properties of the Scribble polarity module however are likely to be context dependent. Indeed, recent evidence suggests that upregulation of Scribble is common in many cancers including hepatocellular carcinomas, with high levels of Scribble correlated with reduced patient survival (Vaira et al. 2011;Savi et al. 2014). Importantly, studies examining large cohort of prostate or breast cancer patients have now indicated that it is the mislocalization of Scribble that is associated with poor outcome (Pearson et al. 2011;Feigin et al. 2014;Savi et al. 2014). ...
Although the Scribble polarity module has long been known as a key
regulator of apicobasal polarity, it is only recently that its broader
role in the control of near all polarity states and transitions is being
appreciated. Here we review the Scribble module in the regulation of
cell polarity and other cellular functions at the molecular and cellular
level. The more recent detailed analysis of multiple vertebrate models for
each of its component homologues, Scribble, Dlg and Lgl, has revealed
specific but also common roles for individual homologues in a variety of
developmental contexts. In addition, emerging data has also implicated
the Scribble polarity module in human developmental syndromes and the
etiology of human cancer, highlighting a need for a better understanding
of this polarity module for therapeutic purposes. Unlocking the temporal
and spatial coordination of the myriad interactions that these signaling
scaffolds regulate is a major challenge for the field and will be key
to resolve the function of Scribble, Dlg, and Lgl in the control of cell
polarity and tissue architecture.
... Gardiol et al. found increased alterations to the expression patterns of DLG and SCRIB during tumour progression in colon cancer, while down-regulation of both proteins was associated with lack of epithelial cell polarity and disorganised tissue architecture (95) . A reduction of SCRIB has been found in 81 % of lobular carcinomas (43) , whereas it was surprisingly overexpressed in tumours of the colon, bladder, ovary, prostate and uterus (96) . ...
Abstract Appropriate establishment and maintenance of cell polarity is essential for normal development and homeostasis. The vast majority of human cancers originate from epithelial tissues and tumour cell invasion and metastasis are the major cause of mortality in human cancers. Invading cells demonstrate loss of cell polarity, loss of epithelial cell-cell adhesions and tissue disorganisation. We examine the growing evidence linking loss of apicobasal polarity with tumour progression.
... Other oncogenic viruses such as human T-cell leukemia virus type 1 (HTLV-1) Tax, a causative agent for adult T-cell leukemia, are known to target Scribble (Elsum et al., 2012;Javier and Rice, 2011;Okajima et al., 2008). Additionally, mislocalization or deregulated Scribble expression has been reported in a variety of epithelial cancers (Gardiol et al., 2006;Kamei et al., 2007;Navarro et al., 2005;Ouyang et al., 2010;Pearson et al., 2011;Vaira et al., 2011;Zhan et al., 2008). The mechanism by which Scribble influences tumorigenesis is unclear, yet may lie in the interactions with oncogenic signaling cascades such as Ras, Wnt and GPCR signaling . ...
The crucial role the Crumbs and Par polarity complexes play in tight junction integrity has long been established, however very few studies have investigated the role of the Scribble polarity module. Here we use MCF10A cells, which fail to form tight junctions and express very little endogenous Crumbs3, to show that inducing expression of the polarity protein Scribble is sufficient to promote tight junction formation. We show this occurs through an epithelial to mesenchymal (EMT) pathway that involves Scribble suppressing ERK phosphorylation, leading to down regulation of the EMT inducer ZEB. Inhibition of ZEB relieves the repression on Crumbs3, resulting in increased expression of this crucial tight junction regulator. The combined effect of this Scribble mediated pathway is the upregulation of a number of junctional proteins and the formation of functional tight junctions. These data suggests a novel role for Scribble in positively regulating tight junction assembly through transcriptional regulation of an EMT signaling program.
... When siRNA or gene overexpression was conducted, HBEC3 or the indicated lung cancer cell type were seeded in Matrigel-coated inserts after 24 hours. FBS-containing medium was used as chemoattractant in the bottom chamber, and cells were allowed to migrate for 24 hours, as described previously (17,22). Cells that had invaded the lower surface of the membrane were fixed with methanol, stained with DAPI, and quantified by fluorescence microscopy (Nikon E600, Nikon Instruments Inc.). ...
Metastatic traits appear to be acquired by transformed cells with progenitor-like cancer-initiating properties, but there remains little mechanistic insight into this linkage. In this report, we show that the polarity protein Numbl, which is expressed normally in neuronal progenitors, becomes overexpressed and mislocalized in cancer cells from a variety of human tumors. Numbl overexpression relies on loss of the tumor suppressor microRNA miR-296), which actively represses translation of Numbl in normal cells. In turn, deregulated expression of Numbl mediates random tumor cell migration and invasion, blocking anoikis and promoting metastatic dissemination. In clinical specimens of non-small cell lung cancer, we found that Numbl overexpression correlated with a reduction in overall patient survival. Mechanistically, Numbl-mediated tumorigenesis involved suppression of a "stemness" transcriptional program driven by the stem cell programming transcription factor Klf4, thereby preserving a pool of progenitor-like cells in lung cancer. Our results reveal that Numbl-Klf4 signaling is critical to maintain multiple nodes of metastatic progression, including persistence of cancer-initiating cells, rationalizing its therapeutic exploitation to improve the treatment of advanced lung cancer.
... All three members of the Scribble polarity module are mislocalized in various human cancers. A recent study found Scrib mislocalized in 50% of analysed DCIS (ductal carcinoma in situ) breast cancer lesions, and Scrib and Dlg has been shown to be mislocalized in colon and cervical cancer, whereas Lgl2 is mislocalized in gastric adenocarcinomas [101,[166][167][168]. Members of the Scribble polarity module frequently show aberrant expression across a variety of tumour types, including breast, endometrial, cervical, colon, prostate and lung [101,102,[166][167][168][169][170][171][172]. Altered expression patterns of other polarity complexes, for example the Par complex, are also seen in several tumour types, including breast, oesophaegeal and ovarian carcinomas [173][174][175][176]. Furthermore, enforced expression in transformed cell lines of polarity proteins, including Scrib, Lgl and Crumbs3, results in an increase in cell adhesion and reversion to a less malignant phenotype, suggestive of a direct role for polarity regulators in tumour progression [44, [177][178][179]. ...
The Scribble, Par and Crumbs modules were originally identified in the vinegar (fruit) fly, Drosophila melanogaster, as being critical regulators of apico-basal cell polarity. In the present chapter we focus on the Scribble polarity module, composed of Scribble, discs large and lethal giant larvae. Since the discovery of the role of the Scribble polarity module in apico-basal cell polarity, these proteins have also been recognized as having important roles in other forms of polarity, as well as regulation of the actin cytoskeleton, cell signalling and vesicular trafficking. In addition to these physiological roles, an important role for polarity proteins in cancer progression has also been uncovered, with loss of polarity and tissue architecture being strongly correlated with metastatic disease.
... is a scaffolding protein associated with cell-cell junctions in epithelial cells and in epithelial MDCK cells a loss of SCRIB was associated with increased cell migration and phenotypic changes consistent with EMT [91]. In contrast, in different contexts SCRIB knockdown inhibited cell migration and invasion and induced downregulation of mesenchymal markers [92,93]. In addition it was shown that the localization of SCRIB to lateral junctions was determined by E-cadherin expression and in cells that did not express this epithelial marker, SCRIB displayed differential localization. ...
The epithelial-to-mesenchymal transition (EMT) is an important developmental process that is also implicated in disease pathophysiology, such as cancer progression and metastasis. A wealth of literature in recent years has identified important transcriptional regulators and large-scale changes in gene expression programs that drive the phenotypic changes that occur during the EMT. However, in the past couple of years it has become apparent that extensive changes in alternative splicing also play a profound role in shaping the changes in cell behavior that characterize the EMT. While long known splicing switches in FGFR2 and p120-catenin provided hints of a larger program of EMT-associated alternative splicing, the recent identification of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) began to reveal this genome-wide post-transcriptional network. Several studies have now demonstrated the truly vast extent of this alternative splicing program. The global switches in splicing associated with the EMT add an important additional layer of post-transcriptional control that works in harmony with transcriptional and epigenetic regulation to effect complex changes in cell shape, polarity, and behavior that mediate transitions between epithelial and mesenchymal cell states. Future challenges include the need to investigate the functional consequences of these splicing switches at both the individual gene as well as systems level.
This study aimed to elucidate the prognostic value of the leucine rich repeat containing 1 (LRRC1) gene in hepatocellular carcinoma (HCC) and to determine the effects of high and low LRRC1 expression on mutation and immune cell infiltration. We downloaded HCC mRNA-seq expression and clinical data from University of California Santa Cruz Xena. The expression of LRRC1 was compared between HCC tumor and normal samples. Tumor samples were divided according to high and low LRRC1 expression. Differentially expressed genes between the 2 groups were identified, and function, mutation, and immune cell infiltration were analyzed. Genes associated with immune cells were identified using weighted gene co-expression network analysis, and transcription factors of these genes were predicted. Moreover, a prognostic model was developed and its performance was evaluated. The expression of LRRC1 was upregulated in HCC tissues, and this indicated a poor prognosis for patients with HCC. Differentially expressed genes between high and low LRRC1 expression were significantly enriched in pathways associated with cancer, amino acid metabolism, carbohydrate metabolism, and the immune system. We identified 15 differentially infiltrated immune cells between tumors with high and low LRRC1 expression and 14 of them correlated with LRRC1 gene expression. Weighted gene co-expression network analysis identified 83 immune cell-related genes, 27 of which had prognostic value. Cyclic AMP-response element binding protein regulated annexin A5, matrix metallopeptidase 9, and LRRC1 in the transcription factor regulatory network. Finally, a prognostic model composed of 7 genes were generated, which could accurately predict the prognosis of HCC patients. The LRRC1 gene might serve as a potential immune-associated prognostic biomarker for HCC.
The respiratory system is composed of a multitude of cells that organize to form complex branched airways that end in alveoli, which respectively function to guide air flow and mediate gas exchange with the bloodstream. The organization of the respiratory sytem relies on distinct forms of cell polarity, which guide lung morphogenesis and patterning in development and provide homeostatic barrier protection from microbes and toxins. The stability of lung alveoli, the luminal secretion of surfactants and mucus in the airways, and the coordinated motion of multiciliated cells that generate proximal fluid flow, are all critical functions regulated by cell polarity, with defects in polarity contributing to respiratory disease etiology. Here, we summarize the current knowledge of cell polarity in lung development and homeostasis, highlighting key roles for polarity in alveolar and airway epithelial function and outlining relationships with microbial infections and diseases, such as cancer.
E-cadherin, α- and β-catenin (E-cadherin-catenin complex) together with the cytoskeleton build the core of Adherens junctions (AJs). Scribble and Lgl1 are tumor suppressors, and it has been reported that Scribble stabilizes the coupling of E-cadherin with catenins promoting epithelial cell adhesion, but the molecular mechanism remains unknown. Here, we investigated the role of Scribble, Lgl1, and myosin-IIA (NMII-A) in AJ integrity. We show that Scribble, Lgl1, and NMII-A reside in a complex with the E-cadherin-catenin complex. Depletion of either Scribble or Lgl1 disrupts the localization of E-cadherin-catenin complex to AJs. aPKCζ phosphorylation of Lgl1 regulates AJ localization of Lgl1 and E-cadherin-catenin complex. Both Scribble and Lgl1 regulate the activation and recruitment of NMII-A at AJs. Finally, Scribble and Lgl1 are downregulated by TGFβ-induced EMT, and re-expression of Scribble or Lgl1 during EMT impedes its progression. Our results provide insight into the mechanism regulating AJ integrity by Scribble, Lgl1, and NMII-A.
The multi-domain scaffolding protein Scribble (Scrib) regulates cell polarity and growth signaling at cell-cell junctions. In epithelial cancers, Scrib mislocalization and overexpression paradoxically transform Scrib from a basolateral tumor suppressor to a cytosolic driver of tumorigenicity. To address the function of Scrib (mis)localization, a Scrib-HaloTag fusion was genome engineered in polarized epithelial cells. Expression of the epithelial to mesenchymal transcription factor Snail displaced Scrib-HaloTag from cell junctions, mirroring the mislocalization observed in cancers. Interestingly, Snail expression promotes Yes-associated protein-1 (YAP1) nuclear localization independent of hippo pathway-regulated YAP-S127 phosphorylation. Furthermore, Scrib HaloPROTAC degradation attenuates YAP1-Y357 phosphorylation. Halo-ligand affinity purification mass spectrometry analysis identified the Src family kinase YES1 as a mislocalized Scrib interaction partner, preferentially recruiting the kinase active and open global conformation (αC helix in). Altogether, mislocalized Scrib enhances YAP1 phosphorylation by scaffolding active YES1.
Background
Laryngeal squamous cell carcinomas (LSCCs) have a high risk of recurrence and poor prognosis. Patient‐derived cancer cell lines remain important preclinical models for advancement of new therapeutic strategies, and comprehensive characterization of these models is vital in the precision medicine era.
Methods
We performed exome and transcriptome sequencing as well as copy number analysis of a panel of LSCC‐derived cell lines that were established at the University of Michigan and are used in laboratories worldwide.
Results
We observed a complex array of alterations consistent with those reported in The Cancer Genome Atlas head and neck squamous cell carcinoma project, including aberrations in PIK3CA, EGFR, CDKN2A, TP53, and NOTCH family and FAT1 genes. A detailed analysis of FAT family genes and associated pathways showed disruptions to these genes in most cell lines.
Conclusions
The molecular profiles we have generated indicate that as a whole, this panel recapitulates the molecular diversity observed in patients and will serve as useful guides in selecting cell lines for preclinical modeling.
Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this process is promoted by acyl protein thioesterase enzymes, which catalyze thioester hydrolysis to solubilize and displace substrate proteins from membranes. The closely related enzymes acyl protein thioesterase 1 (APT1; LYPLA1) and acyl protein thioesterase 2 (APT2; LYPLA2) were initially identified from biochemical assays as G protein depalmitoylases, yet later were shown to accept a number of S-palmitoylated protein and phospholipid substrates. Leveraging the development of isoform-selective APT inhibitors, several studies report distinct roles for APT enzymes in growth factor and hormonal signaling. Recent crystal structures of APT1 and APT2 reveal convergent acyl binding channels, suggesting additional factors beyond acyl chain recognition mediate substrate selection. In addition to APT enzymes, the ABHD17 family of hydrolases contributes to the depalmitoylation of Ras-family GTPases and synaptic proteins. Overall, enzymatic depalmitoylation ensures efficient membrane targeting by balancing the palmitoylation cycle, and may play additional roles in signaling, growth, and cell organization. In this review, we provide a perspective on the biochemical, structural, and cellular analysis of protein depalmitoylases, and outline opportunities for future studies of systems-wide analysis of protein depalmitoylation.
Since the first identification of the Scribble polarity module proteins as a new class of tumour suppressors that regulate both cell polarity and proliferation, an increasing amount of evidence has uncovered a broader role for Scribble, Dlg and Lgl in the control of fundamental cellular functions and their signalling pathways. Here, we review these findings as well as discuss more specifically the role of the Scribble module in PCP signalling.
Mechanisms regulating the transition of mammary epithelial cells (MECs) to mammary stem cells (MaSCs) and to tumor-initiating cells (TICs) have not been entirely elucidated. The p53 family member, p63, is critical for mammary gland development and contains transactivation domain isoforms, which have tumor-suppressive activities, and the ΔN isoforms, which act as oncogenes. In the clinic, p63 is often used as a diagnostic marker, and further analysis of the function of TAp63 in the mammary gland is critical for improved diagnosis and patient care. Loss of TAp63 in mice leads to the formation of aggressive metastatic mammary adenocarcinoma at 9-16 months of age. Here we show that TAp63 is crucial for the transition of mammary cancer cells to TICs. When TAp63 is lost, MECs express embryonic and MaSC signatures and activate the Hippo pathway. These data indicate a crucial role for TAp63 in mammary TICs and provide a mechanism for its role as a tumor- and metastasis-suppressor in breast cancer.Oncogene advance online publication, 21 November 2016; doi:10.1038/onc.2016.388.
Long chain acyl CoA synthetase 4 (ACSL4) is a key enzyme in fatty acid metabolism with marked preference for arachidonic acid (AA). Recent reports have implicated its crucial roles in tumorigenesis. However in gastric cancer (GC), the expression and function of ACSL4 remain unclear. In the present study, we identified ACSL4 as a potential tumor suppressor in GC. The ACSL4 expression in GC samples was evaluated by real-time PCR and immunohistochemistry. The results indicated that the mRNA and protein levels of ACSL4 were frequently downregulated in cancer tissues compared with the adjacent non-cancerous mucosa control tissues. Cell-based functional assays exhibited that ectopic expression of ACSL4 inhibits cell growth, colony formation and cell migration, whereas ACSL4 knockdown enhanced these effects. In a nude mice model, ACSL4 knockdown also promoted subcutaneous xenografts' growth in vivo. Moreover, western blot analysis revealed that ACSL4 expression had a significant effect on FAK and P21 protein level. These findings suggest that ACSL4 plays a tumor-suppressive role and could be a potential therapeutic target in GC. © 2016 IUBMB Life, 2016.
The Hippo transducers YAP and TAZ are central mediators of organ growth and tumorigenesis, regulating cell proliferation, differentiation, and epithelial stemness. In this chapter, we summarize recent findings linking the activation of YAP and TAZ to the cell's structural and architectural features, such as cell polarity, cell shape, cell adhesion, and cytoskeletal dynamics. We examine how epithelial plasticity induced by epithelial-to-mesenchymal transition (EMT) promotes Cancer Stem Cell identity and YAP/TAZ activation, and discuss the role of TAZ as molecular determinant of self-renewal and tumor-seeding potentials in cancer cells. YAP and TAZ activation can also induce EMT, generating a self-sustaining loop. We then place special emphasis on biomechanical cues as regulators of epithelial plasticity, and as dominant regulators of YAP and TAZ nuclear localization and transcriptional activities. This regulation is mediated by physical forces, such as rigidity of the extracellular matrix, compression from neighboring cells, and tension of the actomyosin cytoskeleton. These mechanical signals hold in shape individual cells and whole tissues, and are severely disturbed in cancer. In sum, we highlight new mechanisms of YAP and TAZ regulation by cell polarity and mechanical cues. This potentially adds a new dimension to our understanding of physiology and tumorigenesis, whereby the behavior of individual cells is dictated by the integration of information about tissue architecture and mechanics mediated by YAP and TAZ. © 2013 Springer Science+Business Media New York. All rights are reserved.
Apicobasal polarity is maintained by the combined action of several protein complexes – PAR, SCRIBBLE and CRUMBS – together with the structural organisation of adherent and tight junctions. Apicobasal polarity is important for the regulation of cell division, apoptosis and cell migration, and several studies show that disruption of cell polarity is involved in tumour progression. Our work focused on the biological mechanisms responsible for the altered apicobasal polarity and cell adhesion observed in breast cancers. To do so, we studied two types of breast carcinomas – invasive lobular carcinoma (ILC) and invasive micropapillary carcinoma (IMPC) – whose morphology suggests specific alterations of these cell processes. Our approach combined genomic, transcriptomic and phenotypical comparative analyses, using a group of invasive carcinomas not special type (IC-NST) as control.Lobular carcinomas are generally characterized by alterations of the E cadherin/ catenin complex and their ability to disseminate. We have shown that they also present a downregulation of PAR-3, a protein of the PAR complex, associated with deregulation of genes involved in cell adhesion (ADAM12, LOXL2), cell-extracellular matrix interactions (MMP11, COL11A1, etc…) and invasion (ACTR2, PAK1). Quantitative defects in components of the extracellular matrix were also observed. We have thus been able to establish a transcriptomic signature for this tumour entity, in agreement with the phenotypical observations.Micropapillary carcinomas show an abnormal polarity characterized by the absence of apical markers or their localization at the inverted apical pole, the loss of Golgi protein GM130 correct orientation and abnormal expression or localization of occludin (tight junctions), CDC42 and aPKC(PAR complex proteins). At the genomic level, we have identified somatic mutations in genes involved in polarity (DNAH9, FOXO3) and ciliogenesis regulation (BBS9, BBS12, SEC63), cytoskeleton organisation (HSP90B1, UBR4, ZFYVE26) and motility (FMN2). At the transcriptomic level, we observed deregulation of genes involved in cell-cell, cell-extracellular matrix adhesion and angiogenesis. IMPC also demonstrate the specific overexpression of a protein of the CRUMBS complex, LIN7A. We established an in vitro model and showed that LIN7A is an efficiently modifier of MCF10A’s polarity. Its overexpression in MCF10A cells cultured in 3-D conditions induces the formation of proliferating multi-lobar acini with no central lumen. This absence of a central lumen is due to an inhibition of apoptosis. MCF10A-LIN7A cells also show an increased ability to grow in suspension, indicating resistance to anoikis. This resistance seems to be linked to a decrease of p38protein’s phosphorylation.This detailed description of biological alterations in special types of breast carcinomas will contribute to more specific treatments provided to the patients, through the identification of new therapeutic targets or therapeutic strategies.
Satellite cells are resident skeletal muscle stem cells that supply myonuclei for homeostasis, hypertrophy, and repair in adult muscle. Scrib is one of the major cell-polarity proteins, acting as a potent tumor suppressor in epithelial cells. Here, we show that Scrib also controls satellite-cell-fate decisions in adult mice. Scrib is undetectable in quiescent cells but becomes expressed during activation. Scrib is asymmetrically distributed in dividing daughter cells, with robust accumulation in cells committed to myogenic differentiation. Low Scrib expression is associated with the proliferative state and preventing self-renewal, whereas high Scrib levels reduce satellite cell proliferation. Satellite-cell-specific knockout of Scrib in mice causes a drastic and insurmountable defect in muscle regeneration. Thus, Scrib is a regulator of tissue stem cells, controlling population expansion and self-renewal with Scrib expression dynamics directing satellite cell fate.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Aberrant localization of proteins is increasingly being suggested as a causal player in epithelial cancers. Despite this, few studies have investigated how mislocalization of a protein can alter individual biological processes that contribute to cancer progression. Using Ras as a model of transformation, we investigate how localization of the polarity protein Scribble contributes to its tumor-suppressive properties. Wild-type Scribble has been shown to modulate Ras-mitogen-activated protein kinase (MAPK) transformation both in vitro and in vivo. By utilizing a construct that carries a mutation in the LRR domain of Scribble (Scribble P305L) resulting in a cytosolic rather than the usual membrane-bound localization, we report that discrete tumor suppressive properties of Scribble are differentially sensitive to the localization of Scribble. We find that although the Scribble P305L mislocalization mutant can no longer suppress Ras-MAPK-induced invasion or epithelial to mesenchymal transition phenotypes, mislocalized Scribble can still suppress anchorage-independent cell growth. This study illustrates that the manner in which protein mislocalization contributes to cancer is likely complex and highlights the need for careful interrogation as to how cell polarity protein mislocalization, its secondary consequences, and the mutations that give rise to their mislocalization may contribute to specific aspects of cancer progression.
Loss of apico-basal polarity often results in a malignant phenotype in epithelial tissues. Aberrant expression of polarity mediator proteins is closely associated with this process. LRRC1/LANO, a putative cell polarity regulator, was previously screened from our gene expression profiling in which its expression was significantly upregulated in hepatocellular carcinoma (HCC). In the present study, we provide evidences that LRRC1 plays a potential oncogenic function in HCC. Consistent with the microarray data, quantitative real-time PCR results showed LRRC1 was aberrantly upregulated in 37/56 (66.1 %, more than twofolds) of HCC specimens compared with adjacent non-cancerous livers. Furthermore, the cellular expression of LRRC1 in all HCC cell lines examined exhibited much higher level than that in normal adult liver tissue. Functional analyses revealed that overexpression of LRRC1 promoted, while knockdown of LRRC1 by RNA interference inhibited the growth and colony formation of HCC cells. Importantly, enhanced expression of LRRC1 conferred NIH3T3 cells the ability of cell transformation. In a xenograft tumor model, we found LRRC1 overexpression increased the tumorigenicity of HCC cells. Thus, our collective findings suggest that LRRC1 contributes to HCC development, and may be a potential target for therapeutic intervention in this disease.
Adherens junctions (AD and tight junctions (TJ) play a key role in maintaining the apical-basolateral polarity and cohesive structure of epithelial cells. These epithelial junctions are maintained by the interaction of several key proteins including E-cadherin, claudins, occludin and zona-occludens. The zinc finger protein, Snail, has previously been identified as a possible regulator of several of these AJ and TJ proteins. Expression levels of ZO-1, occludin, claudins, E-cadherin, human Scribble protein and Snail were determined in HeLa and CaSki cervical carcinoma cell lines by imuunohistochemistry (IHC) and Western blotting. In tandem, tissue microarrays were utilised in the IHC-based detection of E-cadherin in 26 cases of non-cancerous cervical tissue, 15 cases of cervical intraepithelial neoplasia 1 (CIN1), 11 cases of CIN2, 12 cases of CIN3, 50 cases of squamous cell carcinoma and two cases of adenocarcinoma. This study found aberrant E-cadherin expression in CIN lesions and cases of squamous cell carcinoma compared to normal epithelium. HeLa cells were E-cadherin-negative while CaSki cells were positive, with HeLa cells showing a high level of Snail expression. Occludin and ZO-1 expression was detected in both cell lines. No expression was observed for claudin 1 or claudin 5 tight junction proteins in HeLa cells or CaSki cells. Loss of expression of claudin 1, claudin 5 and E-cadherin, with concomitant increase in Snail may be associated with loss of epithelial cell polarity and alterations in intercellular adhesion.
During development and tissue homeostasis, patterns of cellular organization, proliferation and movement are highly choreographed. Receptor tyrosine kinases (RTKs) have a crucial role in establishing these patterns. Individual cells and tissues exhibit tight spatial control of the RTKs that they express, enabling tissue morphogenesis and function, while preventing unwarranted cell division and migration that can contribute to tumorigenesis. Indeed, RTKs are deregulated in most human cancers and are a major focus of targeted therapeutics. A growing appreciation of the essential role of spatial RTK regulation during development prompts the realization that spatial deregulation of RTKs is likely to contribute broadly to cancer development and may affect the sensitivity and resistance of cancer to pharmacological RTK inhibitors.
Activating mutations in genes of the Ras-mitogen-activated protein kinase (MAPK) pathway occur in approximately 30% of all human cancers; however, mutation of Ras alone is rarely sufficient to induce tumour development. Scribble is a polarity regulator recently isolated from a Drosophila screen for events that cooperate with Ras mutation to promote tumour progression and cell invasion. In mammals, Scribble regulates directed cell migration and wound healing in vivo; however, no role has been identified for mammalian Scribble in oncogenic transformation. Here we show that in human epithelial cells expressing oncogenic Ras or Raf, loss of Scribble promotes invasion of cells through extracellular matrix in an organotypic culture system. Further, we show that the mechanism by which this occurs is in the regulation of MAPK signalling by Scribble. The suppression of MAPK signalling is a highly conserved function of Scribble as it also prevents Raf-mediated defects in Drosophila wing development. Our data identify Scribble as an important mediator of MAPK signalling and provide a molecular basis for the observation that Scribble expression is decreased in many invasive human cancers.
Human tumours exhibit a large degree of cellular and genetic heterogeneity 1. Complex cell interactions in the tumour and its microenvironment are thought to play a significant role in tumourigenesis and cancer progression 2. It is also known that cooperation between oncogenic genetic lesions is required for tumour development 3. However, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruit fly Drosophila melanogaster allow analysis of the behavior of cells with distinct mutations 4, giving this model organism a privileged position to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein RasV12
5 and loss-of-function mutations in the conserved tumour suppressor scribble (scrib)
6,7 gives rise to metastatic tumours that display many characteristics observed in human cancers 8-11. Here we show that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. We found that the RasV12 and scrib− mutations can also cause tumours when they affect different adjacent epithelial cells. We show that this interaction between RasV12 and scrib− clones involves JNK signaling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of RasV12 tumours can also be triggered by tissue damage, a stress condition that activates JNK signaling. Given the conservation of the pathways examined here, similar cooperative mechanisms could play a role in the development of human cancers.
Genomic copy number aberrations and corresponding transcriptional deregulation in the cancer genome have been suggested to have regulatory roles in cancer development and progression. However, functional evaluation of individual genes from lengthy lists of candidate genes from genomic data sets presents a significant challenge. Here, we report effective gene selection strategies to identify potential driver genes based on systematic integration of genome scale data of DNA copy numbers and gene expression profiles. Using regional pattern recognition approaches, we discovered the most probable copy number-dependent regions and 50 potential driver genes. At each step of the gene selection process, the functional relevance of the selected genes was evaluated by estimating the prognostic significance of the selected genes. Further validation using small interference RNA-mediated knockdown experiments showed proof-of-principle evidence for the potential driver roles of the genes in hepatocellular carcinoma progression (i.e., NCSTN and SCRIB). In addition, systemic prediction of drug responses implicated the association of the 50 genes with specific signaling molecules (mTOR, AMPK, and EGFR). In conclusion, the application of an unbiased and integrative analysis of multidimensional genomic data sets can effectively screen for potential driver genes and provides novel mechanistic and clinical insights into the pathobiology of hepatocellular carcinoma.
Cancer is the result of the deregulation of cell proliferation and cell migration. In advanced tumors, cells invade the surrounding tissue and eventually form metastases. This is particularly evident in carcinomas in which epithelial cells have undergone epithelial-mesenchymal transition. Increased cell migration often correlates with a weakening of intercellular interactions. Junctions between neighboring epithelial cells are required to establish and maintain baso-apical polarity, suggesting that not only loss of cell-cell adhesion but also alteration of cell polarity is involved during invasion. Accordingly, perturbation of cell polarity is an important hallmark of advanced invasive tumors. Cell polarity is also essential for cell migration. Indeed, a front-rear polarity axis has first to be generated to allow a cell to migrate. Because cells migrate during invasion, cell polarity is not completely lost. Instead, polarity is modified. From a nonmigrating baso-apically polarized epithelial phenotype, cells acquire a polarized migrating mesenchymal phenotype. The aim of this review is to highlight the molecular relationship between the control of cell polarity and the regulation of cell motility during oncogenesis.
The neoplastic tumour suppressors, Scribble, Dlg and Lgl, originally discovered in the vinegar fly Drosophila melanogaster, are currently being actively studied for their potential role in mammalian tumourigenesis. In Drosophila, these tumour suppressors function in a common genetic pathway to regulate apicobasal cell polarity and also play important roles in the control of cell proliferation, survival, differentiation and in cell migration/invasion. The precise mechanism by which Scribble, Dlg and Lgl function is not clear; however, they have been implicated in the regulation of signalling pathways, vesicle trafficking and in the Myosin II-actin cytoskeleton. We review the evidence for the involvement of Scribble, Dlg, and Lgl in cancer, and how the various functions ascribed to these tumour suppressors in Drosophila and mammalian systems may impact on the process of tumourigenesis.
Genetic studies have highlighted the key role of Scrib in the development of Metazoans. Deficiency in Scrib impairs many aspects of cell polarity and cell movement although the mechanisms involved remain unclear. In mammals, Scrib belongs to a protein complex containing betaPIX, an exchange factor for Rac/Cdc42, and GIT1, a GTPase activating protein for ARF6 implicated in receptor recycling and exocytosis. Here we show that the Scrib complex associates with PAK, a serine-threonine kinase family crucial for cell migration. PAK colocalizes with members of the Scrib complex at the leading edge of heregulin-treated T47D breast cancer cells. We demonstrate that the Scrib complex is required for epithelial cells and primary mouse embryonic fibroblasts to efficiently respond to chemoattractant cues. In Scrib-deficient cells, the pool of cortical PAK is decreased, thereby precluding its proper activation by Rac. Loss of Scrib also impairs the polarized distribution of active Rac at the leading edge and compromises the regulated activation of the GTPase in T47D cells and mouse embryonic fibroblasts. These data underscore the role of Scrib in cell migration and show the strong impact of Scrib in the function of PAK and Rac, two key molecules implicated in this process.
A simple method has been developed for detecting a broad range of genital human papillomavirus (HPV) types using the polymerase chain reaction (PCR). We utilized two consensus sequence primer pairs within the E6 and E7 open reading frames to amplify HPV DNA; malignant HPV DNA (from HPV-16, -18, -31, -33, -52b and -58) was amplified using the pU-1M/pU-2R primer pair whereas benign HPV DNA (from HPV-6 and -11) was amplified using the pU-31B/pU-2R primer pair. Identification of the amplification product was confirmed by restriction enzyme digestion. In this study, a pU-1M/pU-2R-mediated PCR was successfully applied to 39 cervical carcinoma specimens; HPV-16 was detected in 19 cases, HPV-18 in five cases, HPV-31 in two cases, HPV-33 in two cases, HPV-52b in one case, HPV-58 in three cases, and an unknown type(s) was detected in four cases. Overall, the prevalence of HPV was 84.6%. The results indicate that this detection system is useful for the detection of HPVs not only of known types but also of new types.
The high-risk human papillomavirus (HPV) E6 proteins stimulate the ubiquitination and degradation of p53, dependent on the
E6AP ubiquitin-protein ligase. Other proteins have also been shown to be targeted for degradation by E6, including hDlg, the
human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of theDrosophila Scribble (Vartul) (hScrib) tumor suppressor protein is also targeted for ubiquitination by the E6-E6AP complex in vitro and
that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that
hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope
conserved among the high-risk HPV E6 proteins. Green fluorescent protein-hScrib was localized to the periphery of MDCK cells,
where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions,
as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV
E6 proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor proteins, hDlg and hScrib, both of which are associated with cell junction complexes.
The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic
activity of high-risk HPV E6 proteins.
Members of the LAP protein family, LET-413 in Caenorhabditis elegans, Scribble in Drosophila melanogaster, and Erbin, Lano, Densin-180 and hScrib in mammals, have conserved structural features. LET-413 and Scribble are junctional proteins involved in establishing and maintaining epithelial cell polarity. scribble also behaves as a neoplastic tumor suppressor gene. We show here that, in epithelial cells, hScrib is recruited at cell-cell junctions in an E-cadherin-dependent manner as shown by calcium switch assays in MDCK cells, re-expression of E-cadherin in MDA-231 cells treated by 5-Aza-2'-deoxycytidine (5Aza), and siRNA experiments. hScrib is restricted at the basolateral membrane of epithelial cells by its LRR domain, and is enriched in Triton X-100-insoluble fractions. In breast cancers, most lobular tumors did not express hScrib and E-cadherin while ductal tumors had a less frequent downregulation of hScrib. Our data provide additional insights on the modalities of recruitment of hScrib at the cell-cell junctions, and establish a potential link between the E-cadherin and hScrib tumor suppressors.
Genomic aberrations in the form of subchromosomal DNA copy number changes are a hallmark of epithelial cancers, including breast cancer. The goal of the present study was to analyze such aberrations in breast cancer at high resolution.
We employed high-resolution array comparative genomic hybridization with 4,134 bacterial artificial chromosomes that cover the genome at 0.9 megabase resolution to analyze 47 primary breast tumors and 18 breast cancer cell lines.
Common amplicons included 8q24.3 (amplified in 79% of tumors, with 5/47 exhibiting high level amplification), 1q32.1 and 16p13.3 (amplified in 66% and 57% of tumors, respectively). Moreover, we found several positive correlations between specific amplicons from different chromosomes, suggesting the existence of cooperating genetic loci. Queried by gene, the most frequently amplified kinase was PTK2 (79% of tumors), whereas the most frequently lost kinase was PTK2B (hemizygous loss in 34% of tumors). Amplification of ERBB2 as measured by comparative genomic hybridization (CGH) correlated closely with ERBB2 DNA and RNA levels measured by quantitative PCR as well as with ERBB2 protein levels. The overall frequency of recurrent losses was lower, with no region lost in more than 50% of tumors; the most frequently lost tumor suppressor gene was RB1 (hemizygous loss in 26% of tumors). Finally, we find that specific copy number changes in cell lines closely mimicked those in primary tumors, with an overall Pearson correlation coefficient of 0.843 for gains and 0.734 for losses.
High resolution CGH analysis of breast cancer reveals several regions where DNA copy number is commonly gained or lost, that non-random correlations between specific amplicons exist, and that specific genetic alterations are maintained in breast cancer cell lines despite repeat passage in tissue culture. These observations suggest that genes within these regions are critical to the malignant phenotype and may thus serve as future therapeutic targets.
Altered expression of human Scribble is associated with invasive epithelial cancers, however, its role in tumour development remains unclear. Mutations in Drosophila Scribble result in loss of polarity, overproliferation and 3D-tumourous overgrowth of epithelial cells. Using complementation studies in Drosophila we recently demonstrated that expression of human Scribble can also regulate polarity and restrict tissue overgrowth. Here, we have undertaken a detailed study of human Scribble function in the polarized mammary cell line, MCF10A. We show that although Scribble does not seem to be required for apical-basal polarity or proliferation control in MCF10A cells, Scribble is essential for the control of polarity associated with directed epithelial cell migration. Scribble-depleted MCF10A cells show defective in vitro wound closure and chemotactic movement. The cells at the wound edge fail to polarize, show reduced lamellipodia formation and impaired recruitment of Cdc42 and Rac1 to the leading edge. Furthermore, we show that this function is relevant in vivo as Scribble mutant mice show defective epidermal wound healing. This data identifies an essential role for mammalian Scribble in the regulation of the polarity specifically involved in directed epithelial migration.
Atypical protein kinase C (aPKC) and Lethal giant larvae (Lgl) regulate apical-basal polarity in Drosophila and mammalian epithelia. At the apical domain, aPKC phosphorylates and displaces Lgl that, in turn, maintains aPKC inactive at the basolateral region. The mutual exclusion of these two proteins seems to be crucial for the correct epithelial structure and function. Here we show that a cortical aPKC loading induces Lgl cytoplasmic release and massive overgrowth in Drosophila imaginal epithelia, whereas a cytoplasmic expression does not alter proliferation and epithelial overall structure. As two aPKC isoforms (iota and zeta) exist in humans and we previously showed that Drosophila Lgl is the functional homologue of the Human giant larvae-1 (Hugl-1) protein, we argued if the same mechanism of mutual exclusion could be impaired in human epithelial disorders and investigated aPKCiota, aPKCzeta and Hugl-1 localization in cancers deriving from ovarian surface epithelium. Both in mucinous and serous histotypes, aPKCzeta showed an apical-to-cortical redistribution and Hugl-1 showed a membrane-to-cytoplasm release, perfectly recapitulating the Drosophila model. Although several recent works support a causative role for aPKCiota overexpression in human carcinomas, our results suggest a key role for aPKCzeta in apical-basal polarity loosening, a mechanism that seems to be driven by changes in protein localization rather than in protein abundance.
We performed a technical and cost-effectiveness analysis of quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) for the assessment of HER2 in breast cancer. We evaluated 44 frozen and 55 formalin-fixed paraffin-embedded (FFPE) breast carcinoma specimens by Q-RT-PCR, immunohistochemical analysis, and fluorescent in situ hybridization (FISH). Immunohistochemical and FISH analyses were performed on individual slides and on tissue microarray. Costs of techniques were calculated to study 1 case and 10 or 40 cases. Q-RT-PCR provided reliable data in frozen and FFPE specimens, which were significantly correlated. HER2 messenger RNA levels were significantly stratified in agreement with immunohistochemical data (P < .05). There was complete concordance between Q-RT-PCR and immunohistochemical results for negative and strongly positive (3+) cases. The intermediate immunohistochemical group (2+), including FISH+ and FISH- cancers, could also be stratified by Q-RT-PCR. Cost analysis documented the advantage of Q-RT-PCR in all US Food and Drug Administration-approved assays. Our data support the use of Q-RT-PCR for testing breast cancer specimens to select patients for HER2 inhibitory therapy.
Cell-cycle defects are responsible for cancer onset and growth. We studied the expression profile of 60 genes involved in cell cycle in a series of malignant mesotheliomas (MMs), normal pleural tissues, and MM cell cultures using a quantitative polymerase chain reaction-based, low-density array. Nine genes were significantly deregulated in MMs compared with normal controls. Seven genes were overexpressed in MMs, including the following: CDKN2C, cdc6, cyclin H, cyclin B1, CDC2, FoxM1, and Chk1, whereas Ube1L and cyclin D2 were underexpressed. Chk1 is a principal mediator of cell-cycle checkpoints in response to genotoxic stress. We confirmed the overexpression of Chk1 in an independent set of 87 MMs by immunohistochemistry using tissue microarrays. To determine whether Chk1 down-regulation would affect cell-cycle control and cell survival, we transfected either control or Chk1 siRNA into two mesothelioma cell lines and a nontumorigenic (Met5a) cell line. Results showed that Chk1 knockdown increased the apoptotic fraction of MM cells and induced an S phase block in Met5a cells. Furthermore, Chk1 silencing sensitized p53-null MM cells to both an S phase block and apoptosis in the presence of doxorubicin. Our results indicate that cell-cycle gene expression analysis by quantitative polymerase chain reaction can identify potential targets for novel therapies. Chk1 knockdown could provide a novel therapeutic approach to arrest cell-cycle progression in MM cells, thus increasing the rate of cell death.
Loss of cell polarity proteins such as Scribble induces neoplasia in Drosophila by promoting uncontrolled proliferation. In mammals, the role that polarity proteins play during tumorigenesis is not well understood. Here, we demonstrate that depletion of Scribble in mammary epithelia disrupts cell polarity, blocks three-dimensional morphogenesis, inhibits apoptosis, and induces dysplasia in vivo that progress to tumors after long latency. Loss of Scribble cooperates with oncogenes such as c-myc to transform epithelial cells and induce tumors in vivo by blocking activation of an apoptosis pathway. Like depletion, mislocalization of Scribble from cell-cell junction was sufficient to promote cell transformation. Interestingly, spontaneous mammary tumors in mice and humans possess both downregulated and mislocalized Scribble. Thus, we demonstrate that scribble inhibits breast cancer formation and that deregulation of polarity pathways promotes dysplastic and neoplastic growth in mammals by disrupting morphogenesis and inhibiting cell death.
In the past 20 years, the discovery and characterization of the molecular machinery that controls cellular polarization have enabled us to achieve a better understanding of many biological processes. Spatial asymmetry or establishment of cell polarity during embryogenesis, epithelial morphogenesis, neuronal differentiation, and migration of fibroblasts and T cells are thought to rely on a small number of evolutionarily conserved proteins and pathways. Correct polarization is crucial for normal cell physiology and tissue homeostasis, and is lost in cancer. Thus, cell polarity signaling is likely to have an important function in tumor progression. Recent findings have identified a regulator of cell polarity, the Par complex, as an important signaling node in tumorigenesis. In normal cell types, the Par complex is part of the molecular machinery that regulates cell polarity and maintains normal cell homeostasis. As such, the polarity regulators are proposed to have a tumor suppressor function, consistent with the loss of polarity genes associated with hyperproliferation in Drosophila melanogaster. However, recent studies showing that some members of this complex also display pro-oncogenic activities suggest a more complex regulation of the polarity machinery during cellular transformation. Here, we examine the existing data about the different functions of the Par complex. We discuss how spatial restriction, binding partners and substrate specificity determine the signaling properties of Par complex proteins. A better understanding of these processes will very likely shed some light on how the Par complex can switch from a normal polarity regulation function to promotion of transformation downstream of oncogenes.
The polarity protein complex Par6/atypical protein kinase (aPKC)/Cdc42 regulates polarization processes during epithelial morphogenesis, astrocyte migration, and axon specification. We, as well as others, have shown that this complex is also required for disruption of apical-basal polarity during the oncogene ErbB2-induced transformation and transforming growth factor beta-induced epithelial-mesenchymal transition of mammary epithelial cells. Here, we report that expression of Par6 by itself in mammary epithelial cells induces epidermal growth factor-independent cell proliferation and development of hyperplastic three-dimensional acini without affecting apical-basal polarity. This is dependent on the ability of Par6 to interact with aPKC and Cdc42, but not Lgl and Par3, and its ability to promote sustained activation of MEK/ERK signaling. Down-regulation of Cdc42 or aPKC expression suppresses the ability of Par6 to induce proliferation, demonstrating that Par6 promotes cell proliferation by interacting with aPKC and Cdc42. We also show that Par6 is overexpressed in breast cancer-derived cell lines and in both precancerous breast lesions and advanced primary human breast cancers, suggesting that Par6 overexpression regulates tumor initiation and progression. Thus, in addition to regulating cell polarization processes, Par6 is an inducer of cell proliferation in breast epithelial cells.
Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. We
provide evidence from Drosophila that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation.
Scribble (Scrib) is a cell junction–localized protein required for polarization of embryonic and, as demonstrated here, imaginal
disc and follicular epithelia. We show that the tumor suppressors lethal giant larvae(lgl) and discs-large (dlg) have identical effects on all three epithelia, and that scribalso acts as a tumor suppressor. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is
required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Our data indicate that the three tumor suppressors act together in a common pathway to
regulate cell polarity and growth control.
Loss of cell polarity is one of the hallmarks of malignant carcinomas. Most of the understanding about the link between cell polarity and proliferation control comes from studies on the Drosophila tumor suppressors discs large (Dlg), scribble (Scrib) and lethal giant larvae (lgl). Mammalian homologues of these proteins have been described and are conserved in sequence and function. Human Dlg (hDlg) and Scrib were independently shown to be down-regulated during malignant progression. This, and other lines of evidence, points toward the participation of both hDlg and hScrib in a common pathway involved in polarity control and tumor suppression. We investigated the correlation between the expression of both proteins in tissues and their relative contributions to the maintenance of tissue architecture during colon cancer development. We analyzed the levels and distribution of hDlg and hScrib by immunohistochemistry, using serial sections of the same sample. We used normal and neoplastic colon mucosa, since it offers a good model for analyzing these features in progressive dysplastic stages. The results demonstrate that both proteins localize at the same regions in polarized colon epithelia, and that in normal samples the proteins' distribution varies as cells differentiate at the surface mucosa. In neoplasia, alterations in the expression pattern of hDlg and of hScrib increase during tumor progression; down-regulation of both proteins being associated with lack of epithelial cell polarity and disorganized tissue architecture. The results, therefore, demonstrate that there is an inverse relationship between the levels of hDlg and hScrib expression and the loss of cell polarity and tissue architecture in the colon.
Metastatic disease is the primary cause of death for most cancer patients. Complex and redundant pathways involving the tumor cell and the microenvironment mediate tumor invasion at the primary site, survival and arrest in the bloodstream, and progressive outgrowth at a distant site. Understanding these pathways and their dynamic interactions will help identify promising molecular targets for cancer therapy and key obstacles to their clinical development.
The mechanism of drug resistance in cancer is multifactorial, and the accumulation of multiple genetic changes may lead to drug-resistant phenotypes. This study sought to determine characteristic genetic changes in chemoresistant serous ovarian carcinomas using high-resolution array comparative genomic hybridization (aCGH), and identified genomic aberrations that could be used as predictive markers of chemoresistant disease. Seventeen primary ovarian tumors from optimally debulked stage IIIc serous ovarian carcinoma patients were analyzed using aCGH. Ten patients had chemoresistant disease (progression within 12 months of initial chemotherapy), whereas seven patients had chemosensitive disease (no recurrence for more than 36 months). Receiver operating characteristics curve analysis was used to select chromosomal aberrations that could help distinguish chemoresistant disease from chemosensitive disease. In 17 tumors, frequent increases in DNA copy number were seen on 1p36.33, 3q26.2, 8q24.3, 10q26.3, 12p11.21, 20q13.33, and 21q22.3, and frequent losses were observed on 4p12, 5q13.2, 7q11.21, 8p23.1, 14q32.33, Xq13.3, and Xq21.31. The gains on 5p15.33 and 14q11.2, and losses on 4q34.2, 4q35.2, 5q15, 8p21.1, 8p21.2, 11p15.5, 13q14.13, 13q14.2, 13q32.1, 13q34, 16q22.2, 17p11.2, 17p12, and 22q12.3 were more frequent in chemoresistant disease. The losses on 13q32.1 and 8p21.1 had the largest areas under the curve (AUC 0.90 and 0.85, respectively). The most reliable combination of chromosomal aberrations for detecting chemoresistant disease was the loss on 13q32.1 and 8p21.1 (AUC 0.950). Our findings suggest that these chromosomal aberrations are potential predictive markers of chemoresistant disease in patients with serous ovarian carcinomas.
Mammalian Scribble (Scrib) plays a conserved role in polarization of epithelial and neuronal cells. Polarization is essential for migration of a variety of cell types; however, the function of Scrib in this context remains unclear. Scrib has been shown to interact with betaPIX, a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42. Cdc42 controls cell polarity from yeast to mammals during asymmetric cell division and epithelial cell polarization, as well as during cell migration. Cdc42 is, in particular, required for polarization and orientation of astrocytes in a scratch-induced polarized migration assay. Using this assay, we characterized Scrib function during polarized cell migration.
Depletion of Scrib by siRNA or expression of dominant-negative constructs inhibits astrocyte polarization. Like Cdc42, Scrib controls protrusion formation, cytoskeleton polarization, and centrosome and Golgi reorientation. Scrib interacts and colocalizes with betaPIX at the front edge of polarizing astrocytes. Perturbation of Scrib localization or of Scrib-betaPIX interaction inhibits betaPIX polarized recruitment. We further show that betaPIX is required for astrocyte polarization and that both the Scrib-binding motif and the GEF activity of betaPIX are essential for its function. Scrib and betaPIX control Cdc42 activation and localization during astrocyte polarization. Thereby, Scrib regulates Cdc42-dependent APC and Dlg1 recruitment to the leading edge to promote cell orientation.
We conclude that Scrib plays a key role in the establishment of cell polarity during migration. By interacting with betaPIX, Scrib controls localization and activation of the small GTPase Cdc42 and regulates Cdc42-dependent polarization pathways.
The loss of epithelial polarity and tissue architecture is a diagnostic feature of malignant tumors. In Drosophila, genetic studies identified 3 neoplastic tumor suppressor genes (nTSGs), and a loss of nTSGs has been shown to result in a disruption of apical-basal polarity and neoplastic growth in epithelial cells. Scribble is one type of the Drosophila nTSGs, which encodes a membrane-associated cytoplasmic protein containing the multi-PDZ domain. In contrast to Drosophila scribble, the oncogenic roles of its mammalian homologues have not yet been established. We herein immunohistochemically examined the distributions of hScrib protein in human colorectal neoplasia using affinity-purified antibody. In 50 cases of colorectal adenomas and adenocarcinomas, the accumulation of hScrib protein was commonly observed in comparison with the adjacent normal epithelia. Furthermore, the overexpression and distribution of hScrib was observed to extensively overlap with the cytoplasmic accumulation of beta-catenin. Like beta-catenin, the intense immunoreactivity of hScrib was often observed in small adenomas, thus, suggesting that hScrib could be involved in an early step of colon carcinogenesis. Five corresponding liver metastases showed a comparable immunoreactivity for anti-hScrib in comparison with their primary sites. In an immunofluorescence analysis on cultured cell lines, the loss of membranous staining of hScrib was observed according to the cytoplasmic translocation of beta-catenin. We herein demonstrate that the accumulation of hScrib protein might therefore be involved in colon carcinogenesis while also providing a possible link between hScrib and beta-catenin.
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