TMPRSS4 promotes cancer stem–like properties in prostate cancer cells through upregulation of SOX2 by SLUG and TWIST1

Background Transmembrane serine protease 4 (TMPRSS4) is a cell surface–anchored serine protease. Elevated expression of TMPRSS4 correlates with poor prognosis in colorectal cancer, gastric cancer, prostate cancer, non–small cell lung cancer, and other cancers. Previously, we demonstrated that TMPRSS4 promotes invasion and proliferation of prostate cancer cells. Here, we investigated whether TMPRSS4 confers cancer stem–like properties to prostate cancer cells and characterized the underlying mechanisms. Methods Acquisition of cancer stem–like properties by TMPRSS4 was examined by monitoring anchorage-independent growth, tumorsphere formation, aldehyde dehydrogenase (ALDH) activation, and resistance to anoikis and drugs in vitro and in an early metastasis model in vivo. The underlying molecular mechanisms were evaluated, focusing on stemness-related factors regulated by epithelial–mesenchymal transition (EMT)-inducing transcription factors. Clinical expression and significance of TMPRSS4 and stemness-associated factors were explored by analyzing datasets from The Cancer Genome Atlas (TCGA). Results TMPRSS4 promoted anchorage-independent growth, ALDH activation, tumorsphere formation, and therapeutic resistance of prostate cancer cells. In addition, TMPRSS4 promoted resistance to anoikis, thereby increasing survival of circulating tumor cells and promoting early metastasis. These features were accompanied by upregulation of stemness-related factors such as SOX2, BMI1, and CD133. SLUG and TWIST1, master EMT-inducing transcription factors, made essential contributions to TMPRSS4-mediated cancer stem cell (CSC) features through upregulation of SOX2. SLUG stabilized SOX2 via preventing proteasomal degradation through its interaction with SOX2, while TWIST1 upregulated transcription of SOX2 by interacting with the proximal E-box element in the SOX2 promoter. Clinical data showed that TMPRSS4 expression correlated with the levels of SOX2, PROM1, SNAI2, and TWIST1. Expression of SOX2 was positively correlated with that of TWIST1, but not with other EMT-inducing transcription factors, in various cancer cell lines. Conclusions Together, these findings suggest that TMPRSS4 promotes CSC features in prostate cancer through upregulation of the SLUG- and TWIST1-induced stem cell factor SOX2 beyond EMT. Thus, TMPRSS4/SLUG–TWIST1/SOX2 axis may represent a novel mechanism involved in the control of tumor progression.

Ethanol Extract of Croton Kongensis Promotes Cell Cycle Arrest and Consequently Inhibits Anchorage-Independent Growth of Cervical Cancer Hela Cells

Extracts from Croton kongenis present anticancer activities on various cancers. However, there is no research conducted to investigate the effects of Croton kongenis extracts on cervical cancer as well as on zebrafish. In this study, we demonstrated that Croton kongenis ethanol extract expressed high toxicity to cervical cancer Hela cells with an IC50 dose of 20.4 µg/mL and to zebrafish embryos with malformations, lethality and hatching inhibition at 72-hpf at effective dose of 125 µg/mL. Interestingly, treatment with Croton kongenis ethanol extract caused cell-cycle-arrest at the G2 phase. Particularly, percentages of Croton kongenis ethanol extract-treated cells in G1, S, G2/M were 70%, 6% and 23%, while percentages of control cells in G1, S, G2/M were 65%, 15% and 18%, respectively. Consistent with cell-cycle-arrest, the expressions of CDKN1A, CDNK2A and p53 in Croton kongenis ethanol extract-treated cells were up-regulated 2.0-, 1.65- and 1.8-fold, respectively. Significantly, treatment with Croton kongenis ethanol extract inhibited anchorage-independent growth of Hela cells; the number of colonies formed in soft-agar of Croton kongenis ethanol extract-treated cells was only one-fourth of that of control cells. In conclusion, we suggest that Croton kongenis ethanol extract could be able to use as a traditional medicine for treatment of cervical cancer.

Selectively Targeting Breast Cancer Stem Cells By 8-Quinolinol and Niclosamide

Background Cancer maintenance, metastatic dissemination and drug-resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest numbers of CSCs and poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. Methods The anti-CSC efficacy of up to 17 small-drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness hallmarks were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes upon 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on the CSC proliferation and on stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSCenriched mammospheres. Finally, the efficacy of the NCS in combination with PTX was analyzed in vivo using an orthotopic mice model of MDA-MB-231 cells. Results Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The solely use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination with 8Q and NCS counteracted this pro-CSC activity of PTX whilst significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth, and limited the dissemination of the disease by reducing the circulating tumor cells and the incidence of lung metastasis. Conclusions The combination of 8Q and NCS with PTX at established ratios inhibits both, the proliferation of differentiated cancer cells and the viability of CSCs, opening a way to more efficacious TNBC treatments.

Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide

Background Cancer maintenance, metastatic dissemination and drug-resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest numbers of CSCs and poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. Methods The anti-CSC efficacy of up to 17 small-drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness hallmarks were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes upon 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on the CSC proliferation and on stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSCenriched mammospheres. Finally, the efficacy of the NCS in combination with PTX was analyzed in vivo using an orthotopic mice model of MDA-MB-231 cells. Results Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The solely use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination with 8Q and NCS counteracted this pro-CSC activity of PTX whilst significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth, and limited the dissemination of the disease by reducing the circulating tumor cells and the incidence of lung metastasis. Conclusions The combination of 8Q and NCS with PTX at established ratios inhibits both, the proliferation of differentiated cancer cells and the viability of CSCs, opening a way to more efficacious TNBC treatments.

Molecular and cellular characterization of two patient-derived ductal carcinoma in situ (DCIS) cell lines, ETCC-006 and ETCC-010

Background Currently it is unclear how in situ breast cancer progresses to invasive disease; therefore, a better understanding of the events that occur during the transition to invasive carcinoma is warranted. Here we have conducted a detailed molecular and cellular characterization of two, patient-derived, ductal carcinoma in situ (DCIS) cell lines, ETCC-006 and ETCC-010. Methods Human DCIS cell lines, ETCC-006 and ETCC-010, were compared against a panel of cell lines including the immortalized, breast epithelial cell line, MCF10A, breast cancer cell lines, MCF7 and MDA-MB-231, and another DCIS line, MCF10DCIS.com. Cell morphology, hormone and HER2/ERBB2 receptor status, cell proliferation, survival, migration, anchorage-independent growth, indicators of EMT, cell signalling pathways and cell cycle proteins were examined using immunostaining, immunoblots, and quantitative, reverse transcriptase PCR (qRT-PCR), along with clonogenic, wound-closure and soft agar assays. RNA sequencing (RNAseq) was used to provide a transcriptomic profile. Results ETCC-006 and ETCC-010 cells displayed notable differences to another DCIS cell line, MCF10DCIS.com, in terms of morphology, steroid-receptor/HER status and markers of EMT. The ETCC cell lines lack ER/PR and HER, form colonies in clonogenic assays, have migratory capacity and are capable of anchorage-independent growth. Despite being isogenic, less than 30% of differentially expressed transcripts overlapped between the two lines, with enrichment in pathways involving receptor tyrosine kinases and DNA replication/cell cycle programs and in gene sets responsible for extracellular matrix organisation and ion transport. Conclusions For the first time, we provide a molecular and cellular characterization of two, patient-derived DCIS cell lines, ETCC-006 and ETCC-010, facilitating future investigations into the molecular basis of DCIS to invasive ductal carcinoma transition.

Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage

Background Low-risk human papillomavirus (HPV), such as types 6 and 11, is considered non-oncogenic, but these types have been detected in oral cancer tissue samples, suggesting their possible involvement in oral carcinogenesis. Because double infection of high-risk HPV and Epstein-Barr virus (EBV) is known to be involved in oral carcinogenesis, we hypothesized that low-risk HPV and EBV co-infection can transform the oral cells. To verify our hypothesis, we evaluated the transformation activity of cell lines expressing both low-risk HPV E6/E7 and EBV LMP-1. Methods We transduced HPV6, 11 and 16 E6/E7 genes and EBV LMP-1 gene into primary mouse embryonic fibroblasts. The cell lines were examined for indices of transformation activity such as proliferation, induction of DNA damage, resistance to apoptosis, anchorage-independent growth, and tumor formation in nude mice. To evaluate the signaling pathways involved in transformation, NF-κB and p53 activities were analyzed. We also assessed adhesion signaling molecules associated with anchorage-independent growth such as MMP-2, paxillin and Cat-1. Results Co-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF-κB activity and reduced p53 induction. Moreover, co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escaped from apoptosis under genotoxic condition and suppression of DNA damage response (DDR). Co-expression of low-risk HPV11 E6/E7 and EBV LMP-1 demonstrated similar results. However, it led to no malignant characteristics such as anchorage-independent growth, invasiveness and tumor formation in nude mice. Compared with the cells co-expressing high-risk HPV16 E6 and EBV LMP-1 that induce transformation, co-expression of low-risk HPV6 E6 and EBV LMP-1 was associated with low MMP-2, paxillin and Cat-1 expression. Conclusions The co-expression of low-risk HPV E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to increased DNA damage and suppression of DDR. Thus, double infection of low-risk HPV and EBV could lead to precancerous lesions.

EML4-ALK induces cellular senescence in mortal normal human cells and promotes anchorage-independent growth in hTERT-transduced normal human cells

Background Chromosomal inversions involving anaplastic lymphoma kinase (ALK) and echinoderm microtubule associated protein like 4 (EML4) generate a fusion protein EML4-ALK in non-small cell lung cancer (NSCLC). The understanding of EML4-ALK function can be improved by a functional study using normal human cells. Methods Here we for the first time conduct such study to examine the effects of EML4-ALK on cell proliferation, cellular senescence, DNA damage, gene expression profiles and transformed phenotypes. Results The lentiviral expression of EML4-ALK in mortal, normal human fibroblasts caused, through its constitutive ALK kinase activity, an early induction of cellular senescence with accumulated DNA damage, upregulation of p16INK4A and p21WAF1, and senescence-associated β-galactosidase (SA-β-gal) activity. In contrast, when EML4-ALK was expressed in normal human fibroblasts transduced with telomerase reverse transcriptase (hTERT), which is activated in the vast majority of NSCLC, the cells showed accelerated proliferation and acquired anchorage-independent growth ability in soft-agar medium, without accumulated DNA damage, chromosome aberration, nor p53 mutation. EML4-ALK induced the phosphorylation of STAT3 in both mortal and hTERT-transduced cells, but RNA sequencing analysis suggested that the different signaling pathways contributed to the different phenotypic outcomes in these cells. While EML4-ALK also induced anchorage-independent growth in hTERT-immortalized human bronchial epithelial cells in vitro, the expression of EML4-ALK alone did not cause detectable in vivo tumorigenicity in immunodeficient mice. Conclusions Our data indicate that the expression of hTERT is critical for EML4-ALK to manifest its in vitro transforming activity in human cells. This study provides the isogenic pairs of human cells with and without EML4-ALK expression.

The GNAQ T96S Mutation Affects Cell Signaling and Enhances the Oncogenic Properties of Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC), the most common malignant tumor in the liver, grows and metastasizes rapidly. Despite advances in treatment modalities, the five-year survival rate of HCC remains less than 30%. We sought genetic mutations that may affect the oncogenic properties of HCC, using The Cancer Genome Atlas (TCGA) data analysis. We found that the GNAQ T96S mutation (threonine 96 to serine alteration of the Gαq protein) was present in 12 out of 373 HCC patients (3.2%). To examine the effect of the GNAQ T96S mutation on HCC, we transfected the SK-Hep-1 cell line with the wild-type or the mutant GNAQ T96S expression vector. Transfection with the wild-type GNAQ expression vector enhanced anchorage-independent growth, migration, and the MAPK pathways in the SK-Hep-1 cells compared to control vector transfection. Moreover, cell proliferation, anchorage-independent growth, migration, and the MAPK pathways were further enhanced in the SK-Hep-1 cells transfected with the GNAQ T96S expression vector compared to the wild-type GNAQ-transfected cells. In silico structural analysis shows that the substitution of the GNAQ amino acid threonine 96 with a serine may destabilize the interaction between the regulator of G protein signaling (RGS) protein and GNAQ. This may reduce the inhibitory effect of RGS on GNAQ signaling, enhancing the GNAQ signaling pathway. Single nucleotide polymorphism (SNP) genotyping analysis for Korean HCC patients shows that the GNAQ T96S mutation was found in only one of the 456 patients (0.22%). Our data suggest that the GNAQ T96S hotspot mutation may play an oncogenic role in HCC by potentiating the GNAQ signal transduction pathway.