In vitro and clinical data analysis of Osteopontin as a prognostic indicator in colorectal cancer.

Osteopontin (OPN) has been shown to promote colorectal cancer (CRC) progression; however, the mechanism of OPN-induced CRC progression is largely unknown. In this study, we found that OPN overexpression led to enhanced anchorage-independent growth, cell migration and invasion in KRAS gene mutant cells but to a lesser extent in KRAS wild-type cells. OPN overexpression also induced PI3K signalling, expression of Snail and Matrix metallopeptidase 9 (MMP9), and suppressed the expression of E-cadherin in KRAS mutant cells. In human CRC specimens, a high-level expression of OPN significantly predicted poorer survival in CRC patients and OPN expression was positively correlated with MMP9 expression, and negatively correlated with E-cadherin expression. Furthermore, we have found that 15 genes were co-upregulated in OPN highly expression CRC and a list of candidate drugs that may have potential to reverse the secreted phosphoprotein 1 (SPP1) gene signature by connectivity mapping. In summary, OPN is a potential prognostic indicator and therapeutic target for colon cancer.

Combinatorial use of bone morphogenetic protein 6, noggin and SOST significantly predicts cancer progression.

Emerging evidence has indicated a role of the bone morphogenetic proteins (BMP) in the pathogenesis of certain cancers. The signaling of BMP family members is tightly regulated by their antagonists, including noggin and SOST, which are, in turn, positively regulated by BMP, thereby forming a negative feedback loop. Consequently, the expression of these antagonists should be taken into account in studies on the prognostic significance of BMP. In the present paper, we correlated protein and mRNA expression levels of BMP6, noggin and SOST, alone or in combination, with patient survival in various types of cancer. We found that BMP6 alone was not significantly correlated with esophageal squamous cell carcinoma patient survival. Instead, a high level of inhibitor of differentiation 1, a downstream factor of BMP6, was associated with shorter survival in patients whose tumors stained strongly for BMP6. Knockdown of noggin in esophageal cancer cell line EC109, which expresses BMP6 strongly and SOST weakly, enhanced the non-adherent growth of the cells. Noggin and SOST expression levels, when analyzed alone, were not significantly correlated with patient survival. However, high BMP6 activity, defined by strong BMP6 expression coupled with weak noggin or SOST expression, was significantly associated with shorter survival in esophageal squamous cell carcinoma patients. We further confirmed that BMP6 activity could be used as a prognostic indicator in prostate, bladder and colorectal cancers, using publicly available data on BMP6, noggin and SOST mRNA expression and patient survival. Our results strongly suggest that BMP6, noggin and SOST could be used in combination as a prognostic indicator in cancer progression.

The role of Pea3 group transcription factors in esophageal squamous cell carcinoma.

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P < 0.013) and between Pea3 and matrix metalloproteinase 13 in the human specimens (r = 0.462, P < 0.001). Moreover, Pea3 modulated the sensitivity of EC109 cells to doxorubicin, probably via reduced activity of the phosphatidylinositol 3-kinase-Akt-mammalian target of Rapamycin complex 1 pathway on Pea3 knockdown. In conclusion, our results suggest that Pea3 plays an important role in the progression of ESCC.

Polyomavirus enhancer activator 3 protein promotes breast cancer metastatic progression through Snail-induced epithelial-mesenchymal transition.

Polyomavirus enhancer activator 3 protein (Pea3), also known as ETV4, is a member of the Ets-transcription factor family, which promotes metastatic progression in various types of solid cancer. Pea3-driven epithelial-mesenchymal transition (EMT) has been described in lung and ovarian cancers. The mechanisms of Pea3-induced EMT, however, are largely unknown. Here we show that Pea3 overexpression promotes EMT in human breast epithelial cells through transactivation of Snail (SNAI1), an activator of EMT. Pea3 binds to the human Snail promoter through the two proximal Pea3 binding sites and enhances Snail expression. In addition, knockdown of Pea3 in invasive breast cancer cells results in down-regulation of Snail, partial reversal of EMT, and reduced invasiveness in vitro. Moreover, knockdown of Snail partially rescues the phenotype induced by Pea3 overexpression, suggesting that Snail is one of the mediators bridging Pea3 and EMT, and thereby metastatic progression of the cancer cells. In four breast cancer patient cohorts whose microarray and survival data were obtained from the Gene Expression Omnibus database, Pea3 and Snail expression are significantly correlated with each other and with overall survival of breast cancer patients. We further demonstrate that nuclear localization of Pea3 is associated with Snail expression in breast cancer cell lines and is an independent predictor of overall survival in a Chinese breast cancer patient cohort. In conclusion, our results suggest that Pea3 may be an important prognostic marker and a therapeutic target for metastatic progression of human breast cancer.

Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells.

Deregulation of nuclear factor (NF)-kappaB signalling is common in cancers and is essential for tumourigenesis. Constitutive NF-kappaB activation in extranodal natural killer (NK)-cell lymphoma, nasal type (ENKL) is known to be associated with aberrant nuclear translocation of BCL10. Here we investigated the mechanisms leading to NF-kappaB activation and BCL10 nuclear localization in ENKLs. Given that ENKLs are dependent on T-cell-derived interleukin-2 (IL2) for cytotoxicity and proliferation, we investigated whether IL2 modulates NF-kappaB activation and BCL10 subcellular localization in ENKLs. In the present study, IL2-activated NK lymphoma cells were found to induce NF-kappaB activation via the PI3K/Akt pathway, leading to an increase in the entry of G(2)/M phase and concomitant transcription of NF-kappaB-responsive genes. We also found that BCL10, a key mediator of NF-kappaB signalling, participates in the cytokine receptor-induced activation of NF-kappaB. Knockdown of BCL10 expression resulted in deficient NF-kappaB signalling, whereas Akt activation was unaffected. Our results suggest that BCL10 plays a role downstream of Akt in the IL2-triggered NF-kappaB signalling pathway. Moreover, the addition of IL2 to NK cells led to aberrant nuclear translocation of BCL10, which is a pathological feature of ENKLs. We further show that BCL10 can bind to BCL3, a transcriptional co-activator and nuclear protein. Up-regulation of BCL3 expression was observed in response to IL2. Similar to BCL10, the expression and nuclear translocation of BCL3 were induced by IL2 in an Akt-dependent manner. The nuclear translocation of BCL10 was also dependent on BCL3 because silencing BCL3 by RNA interference abrogated this translocation. We identified a critical role for BCL10 in the cytokine receptor-induced NF-kappaB signalling pathway, which is essential for NK cell activation. We also revealed the underlying mechanism that controls BCL10 nuclear translocation in NK cells. Our findings provide insight into a molecular network within the NF-kappaB signalling pathway that promotes the pathogenesis of NK cell lymphomas.

Correction: TAZ Expression as a Prognostic Indicator in Colorectal Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0054211.].

Osteopontin can act as an effector for a germline mutation of BRCA1 in malignant transformation of breast cancer-related cells.

Breast cancer-associated 1 (BRCA1) plays an important role in breast cancer initiation and progression through its functions in the cell cycle and DNA repair processes; however, its role in metastatic development in human breast cancer is still poorly understood. We have previously shown that osteopontin (OPN) expression was suppressed by wild-type BRCA1 (Wt.BRCA1) and that a natural mutant allele of BRCA1 (Mut.BRCA1) diminished the effect of Wt.BRCA1 on OPN in vitro. In this study, we show that while Wt.BRCA1 suppresses OPN-induced metastasis in a rat syngeneic system, Mut.BRCA1 enhances the development of metastasis through OPN, suggesting that OPN and BRCA1 work closely to regulate metastatic development in the rat. To test whether these findings are relevant to human breast cancer, we have investigated the relationship between BRCA1, OPN, and metastatic properties in human breast cancer-related cells. Using western blot analysis, we show that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN protein expression; and in parallel that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN-mediated in vitro properties associated with the metastatic state in both MCF-7 and MDA MB435s cells. Overall, these results suggest that Mut.BRCA1 can elicit some of the changes involved in metastatic progression in human breast cancer via the overexpression of OPN.

TWIST modulates prostate cancer cell-mediated bone cell activity and is upregulated by osteogenic induction.

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition (EMT) and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms (i) by increasing the rate of bone remodeling and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by small interfering RNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of dickkopf homolog 1 (DKK-1), a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ-confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and downregulation of TWIST through short hairpin RNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to EMT, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

MAD2 expression and its significance in mitotic checkpoint control in testicular germ cell tumour.

Chromosomal instability (CIN) is a common characteristic in testicular germ cell tumour (TGCT). A functional mitotic checkpoint control is important for accurate chromosome segregation during mitosis. Mitotic arrest deficient 2 (MAD2) is a key component of this checkpoint and inactivation of MAD2 is correlated with checkpoint impairment. The aim of this study was to investigate the function of mitotic checkpoint control in TGCT cells and to study its association with MAD2 expression using 8 TGCT cell lines as well as 23 TGCT tissue samples. We found that in response to microtubule disruption, 6 of 8 TGCT cell lines (75%) failed to arrest in mitosis demonstrated by the decreased mitotic index and aberrant expression of mitosis regulators, indicating that mitotic checkpoint defect is a common event in TGCT cells. This loss of mitotic checkpoint control was correlated with reduced MAD2 protein expression in TGCT cell lines implicating that downregulation of MAD2 may play a critical role in an impaired mitotic checkpoint control in these cells. In addition, immunohistochemistry studies on 23 seminomas and 12 normal testis tissues demonstrated that nuclear expression of MAD2 was much lower in seminomas (p<0.0001) but cytoplasmic MAD2 expression was higher in seminomas (p=0.06) than normal samples. Our results suggest that aberrant MAD2 expression may play an essential role in a defective mitotic checkpoint in TGCT cells, which may contribute to CIN commonly observed in TGCT tumours.

DJ-1 could predict worse prognosis in esophageal squamous cell carcinoma.

Recent studies have revealed an oncogenic role of DJ-1 through its ability to transform normal cells, prevent oxidative damage, and inhibit apoptosis. However, its role in esophageal squamous cell carcinoma (ESCC) is unknown. In this study, by immunohistochemistry, we analyzed the expression of DJ-1 in 81 ESCC tumors, 31 paired nonneoplastic esophageal epithelia, and 19 paired ESCC lymph node metastases. We found that cytoplasmic DJ-1 expression was significantly higher in ESCC and ESCC lymph node metastases than in nonneoplastic esophageal epithelium. ESCC specimens with high distant metastatic potential also had a significantly higher level of nuclear DJ-1 expression (P = 0.018). By Kaplan-Meier analysis, we found that a high level of nuclear DJ-1 was significantly associated with poorer patient survival in our cohort (P = 0.028). To investigate whether DJ-1 promotes ESCC progression through phosphatidylinositol 3-kinase pathway and modulation of apoptosis, we performed immunohistochemistry of pAkt and Daxx. We found that DJ-1 expression was significantly associated with pAkt, whereas nuclear DJ-1 expression was significantly correlated with nuclear expression of Daxx. These results suggest that phosphatidylinositol 3-kinase pathway and Daxx-regulated apoptosis might be important in DJ-1-mediated ESCC progression. By using multivariate Cox regression, we further showed that T(4) stage (P = 0.003) and DJ-1 (P = 0.034) are independent predictors of patient survival. In conclusion, our results suggest that DJ-1 plays a very important role in transformation and progression of ESCC and may be used as a prognostic marker in ESCC.

The prognostic significance of BMP-6 signaling in prostate cancer.

The importance of bone-morphogenetic proteins in prostate cancer is well recognized. Bone-morphogenetic protein-6 overexpression has been shown to increase the aggressiveness and invasiveness of prostate cancer cells. Recent studies on noggin and sclerostin, potent inhibitors of bone-morphogenetic protein signaling, have found that noggin also modifies the ability of prostate cancer cells to metastasize to bone. Taken together, these results suggest that bone-morphogenetic protein-6 signaling is important in prostate cancer progression. Our study investigated the expression of bone-morphogenetic protein-6, noggin and sclerostin in human prostate specimens (n=136) by immunohistochemical staining. We found that bone-morphogenetic protein-6 was increased (P<0.001), whereas sclerostin was decreased (P=0.004) in prostate cancer compared with nodular hyperplasia. In addition, significantly higher level of bone-morphogenetic protein-6 expression was observed in high-grade prostate cancer with Gleason score >or=7 (P=0.027). Bone-morphogenetic protein-6, noggin and sclerostin alone could not predict the development of distant metastasis in our patient cohort. However, high level of bone-morphogenetic protein-6 and low level of noggin, or high level of bone-morphogenetic protein-6 and low level of both noggin and sclerostin expression in primary prostate cancer significantly predicted development of distant metastasis. The predictive value was still valid when only high-grade prostate cancers were included or when patients with secondary lesion other than bone were excluded. Taken together, these results suggest that a high level of bone-morphogenetic protein-6 signaling, resulting from increased expression of bone-morphogenetic protein-6 and decreased expression of its inhibitors, might promote the development of prostate cancer metastases. Our results also imply the potential use of bone-morphogenetic protein-6, noggin and sclerostin expression together as a prognostic predictor for metastatic progression of prostate cancer.

DNA Replication Licensing Protein MCM10 Promotes Tumor Progression and Is a Novel Prognostic Biomarker and Potential Therapeutic Target in Breast Cancer.

Breast cancer is one of the most common malignancies in women worldwide. In breast cancer, the cell proliferation rate is known to influence the cancer malignancy. Recent studies have shown that DNA replication initiation/licensing factors are involved in cancer cell proliferation as well as cancer cell migration and invasion. Licensing factors have also been reported as important prognostic markers in lung, prostrate, and bladder cancers. Here, we studied the role of MCM10, a novel licensing factor, in breast cancer progression. From the public database, NCBI, we investigated six independent breast cancer patient cohorts, totaling 1283 patients. We observed a significant association between high MCM10 mRNA expression with tumor grading and patients' survival time. Most importantly, using breast cancer cohorts with available treatment information, we also demonstrated that a high level of MCM10 is associated with a better response to conventional treatment. Similarly, in in vitro studies, the expression level of MCM10 in breast cancer cell lines is significantly higher compared to paired normal breast epithelium cells. Knockdown of MCM10 expression in the cancer cell line showed significantly decreased tumorigenic properties such as cell proliferation, migration and anchorage independence. The MCF7 breast cancer cell line, after MCM10 expression knockdown, showed significantly decreased tumorigenic properties such as cell proliferation, migration, and anchorage independent growth. Mechanistically, MCM10 expression is observed to be regulated by an Estrogen Receptor (ER) signaling pathway, where its expression is suppressed by the inhibition of the ER or serum withdrawal. Our results suggest that MCM10 plays an important role in breast cancer progression and is a potential prognostic/predictive biomarker and therapeutic target for breast cancer patients.

Role of p14ARF in TWIST-mediated senescence in prostate epithelial cells.

Recently, TWIST, a basic helix-loop-helix transcription factor, is suggested to be an oncogene because of its over-expression in many types of human cancer and its positive role in promoting cell survival. The aim of this study was to investigate the role of TWIST on the growth of human epithelial cells. Using two immortalized human prostate epithelial cell lines, we demonstrated that inactivation of TWIST by small RNA technology led to the promotion of cellular senescence and growth arrest, suggesting that TWIST plays a key role in the continuous proliferation of immortalized cells. Over-expression of TWIST, in contrast, resulted in suppression of cellular senescence in response to genotoxic damage and promotion of cell proliferation with DNA damage accumulation, indicating that TWIST promotes genomic instability. In addition, we also found that the TWIST-mediated cellular senescence was regulated through its negative effect on p14(ARF) and subsequent suppression of MDM2/p53 and Chk1/2 DNA damage response pathways. Our results suggest that over-expression of TWIST results in down-regulation of p14(ARF), which leads to the impairment of DNA damage checkpoint in response to genotoxic stress. This negative effect of TWIST on DNA damage response facilitates uncontrolled cell proliferation with genomic instability and tumorigenesis in non-malignant cells.

Upregulation of Twist in oesophageal squamous cell carcinoma is associated with neoplastic transformation and distant metastasis.

BACKGROUND: The antiapoptotic and epithelial-mesenchymal transition activities of Twist have been implicated in the neoplastic transformation and the development of metastasis, respectively. Upregulation of Twist, described in several types of human cancer, also acts as a prognostic marker of poor outcome. AIM: To investigate Twist expression in oesophageal squamous cell carcinoma (SCC) and its prognostic value in a Chinese cohort of patients with oesophageal SCC. METHODS: Twist expression in primary oesophageal SCC of 87 Chinese patients was investigated by immunohistochemical staining. Twist protein level in one immortalised normal oesophageal epithelial cell line and six oesophageal SCC cell lines was measured by western blot analysis. Twist mRNA level in 30 pairs of frozen specimens of primary oesophageal SCC and non-neoplastic oesophageal epithelium from the upper resection margin of corresponding oesophagectomy specimen was also determined by semiquantitative reverse transcription-PCR. RESULTS: It was found that Twist was upregulated in oesophageal SCC cell lines, and its mRNA and protein levels were both increased in oesophageal SCC and the non-neoplastic oesophageal epithelium (p<0.001). In addition, a high level of Twist expression in oesophageal SCC was significantly associated with a greater risk for the patient of developing distant metastasis within 1 year of oesophagectomy (OR 3.462, 95% CI 1.201 to 9.978; p=0.022). CONCLUSIONS: Our results suggest that upregulation of Twist plays a role in the neoplastic transformation to oesophageal SCC and subsequent development of distant metastasis. Twist may serve as a useful prognostic marker for predicting the development of distant metastasis in oesophageal SCC.

Clinical and in vitro analysis of Osteopontin as a prognostic indicator and unveil its potential downstream targets in bladder cancer.

Osteopontin (OPN) plays an important role in cancer progression, however its prognostic significance and its downstream factors are largely elusive. In this study, we have shown that expression of OPN was significantly higher in bladder cancer specimens with higher T-stage or tumor grades. In addition, a high level of OPN was significantly associated with poorer survival in two independent bladder cancer patient cohorts totaling 389 bladder cancer patients with available survival data. We further identified Matrix metallopeptidase 9 (MMP9) and S100 calcium-binding protein A8 (S100A8) were both downstream factors for OPN in bladder cancer specimens and bladder cancer cell lines. Expression of OPN was significantly positively associated with that of MMP9 and S100A8, while overexpression of OPN resulted in upregulation of MMP9 and S100A8, and knockdown of OPN showed consistent downregulation of MMP9 and S100A8 expression levels. Importantly, expression levels of both MMP9 and S100A8 were significantly associated with higher T-stage, higher tumor grade and a shorter survival time in the bladder cancer patients. Interestingly, OPN expression only predicted survival in MMP9-high, but not MMP9-low subgroups, and in S100A8-low but not S100A8-high subgroups. Our results suggest that OPN, MMP9 and S100A8 all play a significant role in bladder cancer progression and are potential prognostic markers and therapeutic targets in bladder cancer. The mechanistic link between these three genes and bladder cancer progression warrants further investigation.

CD133 in brain tumor: the prognostic factor.

CD133 has been shown to be an important stem cell factor that promotes glioma progression. However, the mechanism for CD133-mediated glioma progression has yet to be fully elucidated. In this study, we found that CD133 mRNA expression was a prognostic marker in three independent glioma patient cohorts, corroborating a putative role for CD133 in glioma progression. Importantly, we found that CD133 expression in glioma was highly correlated with the expression of HOX gene stem cell factors (HOXA5, HOXA7, HOXA10, HOXC4 and HOXC6). The expression of these HOX genes individually was significantly associated with survival. Interestingly, the prognostic significance of CD133 was dependent on the expression level of HOX genes, and vice versa. CD133 (p = 0.021) and HOXA7 (p = 0.001) were independent prognostic markers when the three glioma patient cohorts were combined (n = 231). Our results suggest that HOX genes may play a more important role in progression of glioma when CD133 expression is low. Furthermore, we showed that low-level expression of LIM2 in CD133-high glioma was associated with poorer survival, suggesting that LIM2 could be a therapeutic target for glioma expressing a high level of CD133. Connectivity mapping identified vinblastine and vincristine as agents that could reverse the CD133/HOX genes/LIM2-signature, and we confirmed this by in vitro analysis in glioma cell lines, demonstrating that CD133 and HOX genes were co-expressed and could be downregulated by vincristine. In conclusion, our data show that CD133 and HOX genes are important prognostic markers in glioma and shed light on possible treatment strategies for glioma expressing a high level of CD133.

The prognostic significance of Cdc6 and Cdt1 in breast cancer.

DNA replication is a critical step in cell proliferation. Overexpression of MCM2-7 genes correlated with poor prognosis in breast cancer patients. However, the roles of Cdc6 and Cdt1, which work with MCMs to regulate DNA replication, in breast cancers are largely unknown. In the present study, we have shown that the expression levels of Cdc6 and Cdt1 were both significantly correlated with an increasing number of MCM2-7 genes overexpression. Both Cdc6 and Cdt1, when expressed in a high level, alone or in combination, were significantly associated with poorer survival in the breast cancer patient cohort (n = 1441). In line with this finding, the expression of Cdc6 and Cdt1 was upregulated in breast cancer cells compared to normal breast epithelial cells. Expression of Cdc6 and Cdt1 was significantly higher in ER negative breast cancer, and was suppressed when ER signalling was inhibited either by tamoxifen in vitro or letrozole in human subjects. Importantly, breast cancer patients who responded to letrozole expressed significantly lower Cdc6 than those patients who did not respond. Our results suggest that Cdc6 is a potential prognostic marker and therapeutic target in breast cancer patients.

Identification of TWIST-interacting genes in prostate cancer.

Prostate cancer is one of the most common cancers in men worldwide, and the number of diagnosed patients has dramatically increased in recent years. Currently, the clinical parameters used to diagnose prostate cancer, such as Gleason score, pathological tumor staging, and prostate-specific antigen (PSA) expression level, are considered insufficient to inform recommendation to guide clinical practice. Thus, identification of a novel biomarker is necessary. TWIST is one of the well-studied targets and is correlated with cancer invasion and metastasis in several human cancers. We have investigated two largest prostate cancer patient cohorts available in GEO database and found that TWIST expression is positive correlated with Gleason score and associated with poorer survival. By using a prostate cancer cohort and a prostate cancer cell line dataset, we have identified three potential downstream targets of TWIST, PPM1A, SRP72 and TBCB. TWIST's prognostic capacity is lost when the gene is mutated. Further investigation in the prostate cancer cohort revealed that gene expression of SERPINA, STX7, PDIA2, FMP5, GP1BB, VGLL4, KCNMA1, SHMT2, SAA4 and DIDO1 influence the prognostic significance of TWIST and vice versa. Importantly, eight out of these ten genes are prognostic indicator by itself. In conclusion, our study has further confirmed that TWIST is a prognostic marker in prostate cancer, identified its potential downstream targets and genes that could possibly give additional prognostic value to predict TWIST-mediated prostate cancer progression.

Deubiquitinating Enzyme USP9X Suppresses Tumor Growth via LATS Kinase and Core Components of the Hippo Pathway.

The core LATS kinases of the Hippo tumor suppressor pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Recent studies have identified various E3 ubiquitin ligases that negatively regulate the Hippo pathway via ubiquitination, yet few deubiquitinating enzymes (DUB) have been implicated. In this study, we report the DUB USP9X is an important regulator of the core kinases of this pathway. USP9X interacted strongly with LATS kinase and to a lesser extent with WW45, KIBRA, and Angiomotin, and LATS co-migrated exclusively with USP9X during gel filtration chromatography analysis. Knockdown of USP9X significantly downregulated and destabilized LATS and resulted in enhanced nuclear translocation of YAP and TAZ, accompanied with activation of their target genes. In the absence of USP9X, cells exhibited an epithelial-to-mesenchymal transition phenotype, acquired anchorage-independent growth in soft agar, and led to enlarged, disorganized, three-dimensional acini. YAP/TAZ target gene activation in response to USP9X knockdown was suppressed by knockdown of YAP, TAZ, and TEAD2. Deletion of USP9X in mouse embryonic fibroblasts resulted in significant downregulation of LATS. Furthermore, USP9X protein expression correlated positively with LATS but negatively with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. Overall, these results strongly indicate that USP9X potentiates LATS kinase to suppress tumor growth. Cancer Res; 77(18); 4921-33. ©2017 AACR.