PDF file - 1.3MB, Proliferation of MDA MB231 and A549 cells grown in different conditions.

PDF file - 3.5, MTT assay showing that cancer cells with K-Ras activation mutation (HCT116/Hkh-2 and DLD1/DKO3 isogenic pairs) and c-Met (HCC827 parental/GR5 pair) are more susceptible to Ran silencing-induced apoptosis. (A) In HCT116/Hkh-2 colon cancer isogenic pair, HCT116 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 5 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, Hkh-2. (B) In DLD1/DKO3 colon cancer isogenic pair, DLD1 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, DKO3. (C) In HCC827 parental/GR5 lung cancer pair, the GR5 cells, which contain c-Met amplification, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the wild-type HCC827 cells. Results are plotted as histogram showing the mean SD from three independent experiments. Key; *, ** and *** represent p < 0.05, <0.01 and <0.001, respectively. Representative blots from three independent experiments are shown.

PDF file - 1.9MB, Ran silencing induced apoptosis is concentration dependent. Effect of Ran silencing on apoptotic induction was tested using five different shRNAs (shRan1-5) targeting different regions of Ran compared to scramble shRNA (shScr) control in MDA MB231 breast cancer and A549 lung cancer cells. (A) mRNA and (B) protein levels of Ran, and (C) Flow cytometric analysis for DNA content, in MDA MB231 cells infected with different shRNAs targeting Ran. (D) Protein level and (E) Flow analysis for DNA content in A549 cells infected with different shRNAs targeting Ran. Note that the decrease in Ran mRNA level was associated with the decrease in Ran protein levels (r = 0.950, p < 0.01) in MDA MB231 cells infected with shRan1-5. In addition, the percentage of sub-G1 phase apoptotic cells induced by Ran silencing was significantly inversely associated with the levels of Ran in protein levels in MDA MB231 breast cancer cells (r = -0.880, p <0.05). Similar results were obtained in A549 cells.

PDF file - 1.8K, Percentage of sub-G1 phase apoptotic cells and apoptotic pathway induced by Ran silencing in different cell lines. (A) Cells were harvested 72 hours post-infection, fixed and stained with Propidium iodide. Significantly fewer Sub-G1 phase apoptotic cells were observed in the immortalized human embryonic kidney 293 (HEK 293) cell line compared to cancer cell lines from breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (HCT116 and DLD-1) upon Ran silencing (p < 0.01). (B) Western blots of apoptosis mediators in immortalized HEK293, breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (DLD-1 and HCT116) cancer cell lines at 72 hours post-infection with Scr or Ran shRNAs.

PDF file - 1.5MB, The importance of nucleocytoplasmic transportation and Mcl-1 expression in Ran silencing-induced apoptosis in A549 cells. Cells were grown in different conditions at 24 hours post-infection and were then harvested at 72 hours post-infection. Percentage of sub-G1 phase apoptotic cells are shown in A549 cells treated with (A) rapamycin and/or LMB, and (B) PI103 and/or LMB. Western blots of PARP, Mcl-1 and Ran, in A549 cells expressing various levels of Ran treated with (C) rapamycin and/or LMB, and (D) PI103 and/or LMB. Western blots of various transcription factors in cytoplasmic and nuclear fraction of scramble (Scr) and Ran knockdown (E) A549 cells, (F) A549 cells with or without rapamycin, (G) A549 cells with or without PD184352. (H) Quantitative PCR of Mcl-1 transcript in A549 cells infected with Scr and Ran shRNAs. (I) Western blots of Mcl-1, PARP and Ran at 72hr post-infection of Ran shRNAs in A549 cells grown in Hank's buffered salt solution (HBSS), serum-free and serum-supplemented conditions. (J) Quantification of Mcl-1 protein levels from figure (I). (K) Western blots of PARP, Mcl-1 and Bcl-2 in MDA MB231 cells upon Ran silencing in the presence of different inhibitors. (L) Quantification of Mcl-1 protein levels from figure.

PDF file - 13.3MB, High level of Ran is correlated with shorter survival time of lung cancer patients.(A) Representative images for IHC staining in lung cancer specimens. Lung cancer specimens stained with different patterns for Ran. Upper panel, 40x magnification; Lower panel, 200x magnification of the same cases. (B) Kaplan-Meier plot of the survival of lung cancer patients using combined nuclear and cytoplasmic expression of Ran; High level, high level expression of both nuclear and cytoplasmc Ran; Intermediate level, high level expression of either nuclear or cytoplasmic Ran; Low level, low level expression of both nuclear and cytoplasmic Ran. Cumulative proportion of lung cancer patients surviving with time is plotted for high, intermediate and low Ran groups. (C) Kaplan-Meier plot of survival of lung cancer patients from seven microarray datasets available in GEO database. Cumulative proportion of lung cancer patients surviving with time is plotted for high (top 25% of patients expressing a high level of Ran) and low (bottom 25% of patients expressing a low level of Ran) Ran groups.

PDF file - 4.8, Effect of chemical inhibitors on the activity of signaling pathways in MDA MB231 and A549 cells expressing various levels of Ran. Western blots of phospho- and total Akt, ERK and S6K proteins in MDA MB231 and A549 cells expressing various levels of Ran grown in different growth conditions. Representative blots from two independent experiments are shown.

PDF file - 3.6K, Cancer cells with K-Ras activation mutation (HCT116/Hkh-2 isogenic pair) are more susceptible to Ran silencing-induced apoptosis.

PDF file - 1.6MB, Percentage of sub-G1 phase apoptotic MCF10a and MDA MB231 cells grown in serum-containing medium after Ran silencing. (A-B) Cells were infected with shRan1 and shRan2. Medium was changed to serum-free medium (DMEM) 24hr post-infection. Cells were harvested at 72hr and 96hr post-infection for (A) cell cycle and (B) Western blot analyses. (A) Significantly more sub-G1 phase cells were observed in MDA MB231 breast cancer cells than in immortalized breast epithelial cells 72 and 96 hr post-infection of the shRNAs. (B) More cleaved PARP, caspase 9 and caspase 3 were observed in MDA MB231 than in MCF10A cells upon Ran silencing, suggesting a higher apoptotic response in MDA MB231 than in MCF10a cells. Levels of Ran knockdown were similar in both cell lines. Actin was used as a loading control. (C-D) Cells were infected with shRan1 and shRan2, and harvested 72hr post-infection for (C) cell cycle and (D) Western blot analyses. (C) More apoptotic cells were observed in MDA MB231 than MCF10a cells upon Ran-silencing 72hr post-infection of shRNAs, when these two cell lines were grown in their normal medium. (D) Higher levels of cleaved PARP, caspase 9 and caspase 3 were observed at 72hr post-infection of shRNAs. Results are plotted as histogram showing the mean SD from three independent experiments. Key; * represents p < 0.05 and ** represents p <0.01.

PDF file - 725K, Ran silencing-induced apoptosis depends on the growth stimulation in MDA MB231 and A549 cells using MTT assay.Percentage of cell survival in (A) MDA MB231 and (B) A549 cells expressing various levels of Ran grown in different conditions as determined by MTT assay. Results are plotted as histograms showing the mean SD from three independent experiments. Key; *, ** and *** represent p < 0.05, <0.01 and <0.001, respectively.

The Hippo pathway restricts the activity of transcriptional coactivators TAZ (WWTR1) and YAP. TAZ and YAP are reported to be overexpressed in various cancers, however, their prognostic significance in colorectal cancers remains unstudied. The expression levels of TAZ and YAP, and their downstream transcriptional targets, AXL and CTGF, were extracted from two independent colon cancer patient datasets available in the Gene Expression Omnibus database, totaling 522 patients. We found that mRNA expressions of both TAZ and YAP were positively correlated with those of AXL and CTGF (p<0.05). High level mRNA expression of TAZ, AXL or CTGF significantly correlated with shorter survival. Importantly, patients co-overexpressing all 3 genes had a significantly shorter survival time, and combinatorial expression of these 3 genes was an independent predictor for survival. The downstream target genes for TAZ-AXL-CTGF overexpression were identified by Java application MyStats. Interestingly, genes that are associated with colon cancer progression (ANTXR1, EFEMP2, SULF1, TAGLN, VCAN, ZEB1 and ZEB2) were upregulated in patients co-overexpressing TAZ-AXL-CTGF. This TAZ-AXL-CTGF gene expression signature (GES) was then applied to Connectivity Map to identify small molecules that could potentially be utilized to reverse this GES. Of the top 20 small molecules identified by connectivity map, amiloride (a potassium sparing diuretic,) and tretinoin (all-trans retinoic acid) have shown therapeutic promise in inhibition of colon cancer cell growth. Using MyStats, we found that low level expression of either ANO1 or SQLE were associated with a better prognosis in patients who co-overexpressed TAZ-AXL-CTGF, and that ANO1 was an independent predictor of survival together with TAZ-AXL-CTGF. Finally, we confirmed that TAZ regulates Axl, and plays an important role in clonogenicity and non-adherent growth in vitro and tumor formation in vivo. These data suggest that TAZ could be a therapeutic target for the treatment of colon 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.

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.

Breast cancer is a leading cause of cancer-related deaths. Anemia is common in breast cancer patients and can be treated with blood transfusions or with recombinant erythropoietin (EPO) to stimulate red blood cell production. Clinical studies have indicated decreased survival in some groups of cancer patients treated with EPO. Numerous tumor cells express the EPO receptor (EPOR), posing a risk that EPO treatment would enhance tumor growth, but the mechanisms involved in breast tumor progression are poorly understood.Here, we have examined the functional role of the EPO-EPOR axis in pre-clinical models of breast cancer. EPO induced the activation of PI3K/AKT and MAPK pathways in human breast cancer cell lines. EPOR knockdown abrogated human tumor cell growth, induced apoptosis through Bim, reduced invasiveness, and caused downregulation of MYC expression. EPO-induced MYC expression is mediated through the PI3K/AKT and MAPK pathways, and overexpression of MYC partially rescued loss of cell proliferation caused by EPOR downregulation. In a xenotransplantation model, designed to simulate recombinant EPO therapy in breast cancer patients, knockdown of EPOR markedly reduced tumor growth.Thus, our experiments in vitro and in vivo demonstrate that functional EPOR signaling is essential for the tumor-promoting effects of EPO and underline the importance of the EPO-EPOR axis in breast tumor progression.

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.

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.

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.

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.