Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Targeting the Epigenetic Non-Coding RNA MALAT1/Wnt Signaling Axis as a Therapeutic Approach to Suppress Stemness and Metastasis in Hepatocellular Carcinoma.

BACKGROUND: With recorded under-performance of current standard therapeutic strategies as highlighted by high rates of post-treatment (resection or local ablation) recurrence, resistance to chemotherapy, poor overall survival, and an increasing global incidence, hepatocellular carcinoma (HCC) constitutes a medical challenge. Accumulating evidence implicates the presence of HCC stem cells (HCC-SCs) in HCC development, drug-resistance, recurrence, and progression. Therefore, treatment strategies targeting both HCC-SCs and non-CSCs are essential. METHODS: Recently, there has been an increasing suggestion of MALAT1 oncogenic activity in HCC; however, its role in HCC stemness remains unexplored. Herein, we investigated the probable role of MALAT1 in the SCs-like phenotype of HCC and explored likely molecular mechanisms by which MALAT1 modulates HCC-SCs-like and metastatic phenotypes. RESULTS: We showed that relative to normal, cirrhotic, or dysplastic liver conditions, MALAT1 was aberrantly expressed in HCC, similar to its overexpression in Huh7, Mahlavu, and SK-Hep1 HCC cells lines, compared to the normal liver cell line THLE-2. We also demonstrated a positive correlation between MALAT1 expression and poor cell differentiation status in HCC using RNAscope. Interestingly, we demonstrated that shRNA-mediated silencing of MALAT1 concomitantly downregulated the expression levels of ß-catenin, Stat3, c-Myc, CK19, vimentin, and Twist1 proteins, inhibited HCC oncogenicity, and significantly suppressed the HCC-SCs-related dye-effluxing potential of HCC cells and reduced their ALDH-1 activity, partially due to inhibited MALAT1-ß-catenin interaction. Additionally, using TOP/FOP (TCL/LEF-Firefly luciferase) Flash, RT-PCR, and western blot assays, we showed that silencing MALAT1 downregulates ß-catenin expression, dysregulates the canonical Wnt signaling pathway, and consequently attenuates HCC tumorsphere formation efficiency, with concurrent reduction in CD133+ and CD90+ HCC cell population, and inhibits tumor growth in SK-Hep1-bearing mice. Conclusions: Taken together, our data indicate that MALAT1/Wnt is a targetable molecular candidate, and the therapeutic targeting of MALAT1/Wnt may constitute a novel promising anticancer strategy for HCC treatment.

Elevated PDK1 Expression Drives PI3K/AKT/MTOR Signaling Promotes Radiation-Resistant and Dedifferentiated Phenotype of Hepatocellular Carcinoma.

Resistance to radiotherapy (IR), with consequent disease recurrence, continues to limit the efficacy of contemporary anticancer treatment for patients with hepatocellular carcinoma (HCC), especially in late stage. Despite accruing evidence implicating the PI3K/AKT signaling pathway in cancer-promoting hypoxia, cancerous cell proliferation and radiotherapy-resistance, it remains unclear which molecular constituent of the pathway facilitates adaptation of aggressive HCC cells to tumoral stress signals and drives their evasion of repeated IR-toxicity. This present study investigated the role of PDK1 signaling in IR-resistance, enhanced DNA damage repair and post-IR relapse, characteristic of aggressive HCC cells, while exploring potential PDK1-targetability to improve radiosensitivity. The study employed bioinformatics analyses of gene expression profile and functional protein-protein interaction, generation of IR-resistant clones, flow cytometry-based ALDH activity and side-population (SP) characterization, siRNA-mediated loss-of-PDK1function, western-blotting, immunohistochemistry and functional assays including cell viability, migration, invasion, clonogenicity and tumorsphere formation assays. We showed that the aberrantly expressed PDK1 characterizes poorly differentiated HCC CVCL_7955, Mahlavu, SK-HEP1 and Hep3B cells, compared to the well-differentiated Huh7 or normal adult liver epithelial THLE-2 cells, and independently activates the PI3K/AKT/mTOR signaling. Molecular ablation of PDK1 function enhanced susceptibility of HCC cells to IR and was associated with deactivated PI3K/AKT/mTOR signaling. Additionally, PDK1-driven IR-resistance positively correlated with activated PI3K signaling, enhanced HCC cell motility and invasiveness, augmented EMT, upregulated stemness markers ALDH1A1, PROM1, SOX2, KLF4 and POU5F1, increased tumorsphere-formation efficiency and suppressed biomarkers of DNA damage-RAD50, MSH3, MLH3 and ERCC2. Furthermore, the acquired IR-resistant phenotype of Huh7 cells was strongly associated with significantly increased ALDH activity, SP-enrichment, and direct ALDH1-PDK1 interaction. Moreover, BX795-mediated pharmacological inhibition of PDK1 synergistically enhances the radiosensitivity of erstwhile resistant cells, increased Bax/Bcl-2 apoptotic ratio, while suppressing oncogenicity and clonogenicity. We provide preclinical evidence implicating PDK1 as an active driver of IR-resistance by activation of the PI3K/AKT/mTOR signaling, up-modulation of cancer stemness signaling and suppression of DNA damage, thus, projecting PDK1-targeting as a putative enhancer of radiosensitivity and a potential new therapeutic approach for patients with IR-resistant HCC.

Collagen 1A1 (COL1A1) Is a Reliable Biomarker and Putative Therapeutic Target for Hepatocellular Carcinogenesis and Metastasis.

Increasing evidence shows that hepatocellular carcinoma (HCC) is a principal cause of cancer-related mortality globally, especially among Asian and African populations. Collagen type I α1 (COL1A1) is the major component of type I collagen. While aberrant expression of COL1A1 and COL1A2 is implicated in numerous cancers, the differential role of COL1A1 in malignant, premalignant and normal tissues remains unclear, and its clinical significance in HCC has not been elucidated. In this study, using bioinformatics analysis of publicly-available HCC microarray data from Gene Expression Omnibus (GEO) and RNAseq data from The Cancer Genome Atlas (TCGA) database, we determined that COL1A1 is significantly upregulated in HCC tumor tissues in comparison to normal tissues. Our analysis also revealed that COL1A1 confers survival advantage and enhanced oncogenicity on HCC cells. Interestingly, the siRNA-mediated silencing of COL1A1 expression (siCOLIA1) suppressed HCC cells clonogenicity, motility, invasiveness and tumorsphere formation. Concomitantly, siCOL1A1 abrogated Slug-dependent epithelial-to-mesenchymal transition (EMT) and HCC stemness gene-signature, by attenuating expression of stemness markers SOX2, OCT4 and CD133. The present study provides some mechanistic insight into COL1A1 activity in HCC and highlights its putative role as an important diagnostic biomarker and potential therapeutic target in early development and metastasis of HCC.

Proteasome 26S Subunit, non-ATPase 3 (PSMD3) Regulates Breast Cancer by Stabilizing HER2 from Degradation.

It is well-known that human epidermal growth factor receptor 2 (HER2) is critical for breast cancer (BC) development and progression. Several studies have revealed the role of the ubiquitin/proteasome system (UPS) in cancer. In this study, we investigated the expression level of Proteasome 26S subunit, non-ATPase 3 (PSMD3) in BC using BC cell lines, human BC tissue samples, Oncomine, and TCGA databases and studied the PSMD3-HER2 protein interaction. PSMD3 was upregulated in BC, particularly in the HER2+ subtype. PSMD3 immunostaining was detected in the cytoplasm and nucleus of BC tumor tissues. Strong interaction between PSMD3 and HER2 at the protein level was observed. Knockdown of PSMD3 significantly impaired the stability of HER2, inhibited BC cell proliferation and colony formation, and induced cell apoptosis. Ubiquitination process was strongly enhanced after knockdown of PSMD3 in association with decreased HER2 level. Accumulation and Localization of LAMP-1 in the cell membrane with decreased HER2 immunostaining was observed after knockdown of PSMD3. High expression level of PSMD3 was associated with HER2 expression (p < 0.001), tumor size (p < 0.001), and clinical stage (p = 0.036). High expression level of PSMD3 predicted a short overall survival (OS), particularly for HER2+. Overall, we provide a novel function for PSMD3 in stabilizing HER2 from degradation in HER2+ BC, which suggests that PSMD3 is a novel target for HER2+ BC.

Small G protein signalling modulator 2 (SGSM2) is involved in oestrogen receptor-positive breast cancer metastasis through enhancement of migratory cell adhesion via interaction with E-cadherin.

The function of small G protein signalling modulators (SGSM1/2/3) in cancer remains unknown. Our findings demonstrated that SGSM2 is a plasma membrane protein that strongly interacted with E-cadherin/ß-catenin. SGSM2 downregulation enhanced the phosphorylation of focal adhesion kinase (FAK; Y576/577), decreased the expression of epithelial markers such as E-cadherin, ß-catenin, and Paxillin, and increased the expression of Snail and Twist-1, which reduced cell adhesion and promoted cancer cell migration. Oestrogen and fibronectin treatment was found to promote the colocalization of SGSM2 at the leading edge with phospho-FAK (Y397). The BioGRID database showed that SGSM2 potentially interacts with cytoskeleton remodelling and cell-cell junction proteins. These evidences suggest that SGSM2 plays a role in modulating cell adhesion and cytoskeleton dynamics during cancer migration.

DNA primase polypeptide 1 (PRIM1) involves in estrogen-induced breast cancer formation through activation of the G2/M cell cycle checkpoint.

The DNA primase polypeptide 1 (PRIM1) is responsible for synthesizing small RNA primers for Okazaki fragments generated during discontinuous DNA replication. PRIM1 mRNA expression levels in breast tumor samples were detected by real-time PCR analysis. Xenografted tumor model was established to study the carcinogenic role of PRIM1 and its potential therapeutic applications. The average PRIM1 mRNA (copy number × 103 /µg) expression was 4.7-fold higher in tumors than in normal tissue (*p = 0.005, n = 254). PRIM1 was detected preferentially at a higher level (>40-fold) in poorly differentiated tumor tissues (n = 46) compared with more highly differentiated tumors tissues (n = 10) (*p = 0.005). Poor overall survival rate was correlated to the estrogen receptor positive (ER+, n = 20) patients with higher PRIM1 expression when compare to the ER- (n = 10) patients (Chi Square test, p = 0.03). Stable expression of PRIM1-siRNA in the ER+ BT-474 cells-xenograft tumors significantly reduced tumor volume in SCID mice (*p = 0.005). The anti-tumoral effects of inotilone isolated from Phellinus linteus was tested and had significant effects on the inhibition of PRIM1 protein expression in ER+ breast cancer cells. In vivo study was performed by administering inotilone (10 mg/kg, twice a week for 6 weeks), which resulted in significantly reduced BT-474-xenografted tumor growth volume compared with control (n =5 per group, *p < 0.05). This study provides evidences for the prognostic effects of PRIM1 with poor overall survival rate in the ER+ patients and will be valuable to test for therapeutic purpose.

Long-term exposure to extremely low-dose of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induce non-malignant breast epithelial cell transformation through activation of the a9-nicotinic acetylcholine receptor-mediated signaling pathway.

Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in cases of second-hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non-tumorigenic breast epithelial cell line, HBL-100 used for a time-course assay, was exposed to very low levels of either Nic or NNK, or both. The time-course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL-100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48 hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL-100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9-nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells, increase Nanog expression and mammosphere-forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.

Oestrogen receptor-regulated glutathione S-transferase mu 3 expression attenuates hydrogen peroxide-induced cytotoxicity, which confers tamoxifen resistance on breast cancer cells.

PURPOSE: Glutathione S-transferase mu 3 (GSTM3) is an enzyme involving in the detoxification of electrophilic compounds by conjugation with glutathione. Higher GSTM3 mRNA levels were reported in patients with ERα-positive breast cancer who received only tamoxifen therapy after surgery. Thus, this study aimed to clarify the oncogenic characteristics of GSTM3 in breast cancer and the mechanism of tamoxifen resistance. METHODS: GSTM3 expression in human breast tumour tissues (n = 227) was analysed by RT-PCR and quantitative PCR. Western blot, promoter activity assays, and chromatin immunoprecipitation (ChIP) assays were used to investigate the mechanism of GSTM3 gene regulation. Hydrogen peroxide (H2O2)-induced cytotoxicity in breast cancer cells was detected by MTT assays and flow cytometry. The oncogenic characteristics of GSTM3 in MCF-7 cells were examined by siRNA knockdown in soft agar assays and a xenograft animal model. RESULTS: GSTM3 mRNA was highly expressed in ER- and HER2-positive breast cancers. Moreover, patients who received adjuvant Herceptin had increased GSTM3 mRNA levels in tumour tissue. Oestrogen-activated GSTM3 gene expression through ERα-mediated recruitment of SP1, EP300, and AP-1 complexes. GSTM3-silenced MCF-7 cells were more sensitive to H2O2, with significantly inhibited proliferation and colony formation abilities. Tamoxifen-resistant (Tam-R) cells lacking GSTM3 showed enhanced sensitivity to H2O2, but this result was contrary to that obtained after short-term tamoxifen exposure. The animal model suggested that GSTM3 silencing might suppress the tumourigenic ability of MCF-7 cells and increase tumour cell apoptosis. CONCLUSIONS: ROS production is one mechanism by which cancer drugs kill tumour cells, and according to our evidence, GSTM3 may play an important role in preventing breast cancer treatment-induced cellular cytotoxicity.

The impact of the effectiveness of GATA3 as a prognostic factor in breast cancer.

The transcription factor GATA3 plays a significant role in mammary gland development and differentiation. We analyzed expression of GATA3 in breast cancer (BC) cell lines and clinical specimens from BC patients in Taiwan. Semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR were carried out to determine the mRNA level of GATA3 from 241 pairs of matched tumor and adjacent normal tissues from anonymous female donors. GATA3 immunohistochemistry (IHC) staining and H-score were performed (n = 25). Inducing and silencing of GATA3 were done by exposure MCF-7 cell line to nicotine or curcumin, respectively. GATA3 expression was detected in most of the estrogen receptor-positive (ER+) tumor specimens (176/241, 73%) compared with paired normal tissues (65/241, 27%) (P < .001). The GATA3 level was highest in Luminal A, and independent t-tests revealed higher GATA3 was associated with ER+ (P = .018) and BC stages (stage II, and stage IV). Nuclear protein expression of GATA3 was detected in tumor tissues (P < .001) with higher H-score in Luminal A patients (P = .012). Kaplan-Meier survival analyses showed that ER+/progesterone receptor (PgR)+ and lower grade BC patients with relatively high GATA3 had better clinical overall survival (OS). GATA3 regulates ERα and BCL-2 as BC luminal subtype markers. Cox univariate and multivariate analyses demonstrated that the expression of GATA3 was an effective predictor of the risk of death. We demonstrated a correlation between GATA3 expression and only ER+ and suggest that a higher GATA3 expression is a good prognostic factor for OS for ER+ BC patients.

Ovatodiolide suppresses yes-associated protein 1-modulated cancer stem cell phenotypes in highly malignant hepatocellular carcinoma and sensitizes cancer cells to chemotherapy in vitro.

The cancer stem cells (CSCs) theory recently became a focus of heightened attention in cancer biology, with the proposition that CSCs may constitute an important therapeutic target for effective anticancer therapy, because of their demonstrated role in tumor initiation, chemo-, and radio-resistance. Liver CSCs are a small subpopulation of poorly- or undifferentiated liver tumor cells, implicated in tumorigenesis, metastasis, resistance to therapy and disease relapse, enriched with and associated with the functional markers corresponding to the CSCs-enriched side population (SP), high aldehyde dehydrogenase (ALDH) activity, and enhanced formation of in vitro liver CSCs models, referred to herein as hepatospheres. In this study, we found YAP1 was significantly expressed in the SP cells, as well as in generated hepatospheres compared to non-SP or parental HCC cells, at transcript and/or protein levels. In addition, downregulation of YAP1 expression levels by small molecule inhibitor and siRNA transfection, in the HCC cell lines, PLC/PRF/5 and Mahlavu, were associated with marked loss of ability to form hepatospheres and increased sensitivity to sorafenib. Consistent with the above, we demonstrated that YAP1 expression positively correlated with that of Sox2, Oct4, c-Myc and GRP78, markers of stemness and drug resistance. This is suggestive of YAP1's role as a modulator of cancer stemness, ER stress and chemoresistance. For the first time, we demonstrate that Ovatodiolide significantly attenuates YAP1 expression and subsequently suppressed YAP1-modulated CSCs phenotypes and associated disease progression, consistent with our previous finding in breast cancer. Taken together, our findings suggest that YAP1, highly expressed in malignant liver tumours, contributes to hepatocellular CSCs phenotype and is a molecular target of interest for CSCs targeted therapy in liver cancer patients.

Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade.

BACKGROUND: Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors with cholesterol-lowering properties, were recently shown to exhibit anti-cancer effects. However, the molecular mechanism underlying statin-induced cancer cell death remains to be elucidated. Elevated level of survivin is often found over-expressed in human cancers and has been implicated in the progression of tumorigenesis. Given its central role in cell division and action as an apoptosis suppressor, survivin represents a potential molecular target in cancer management. METHODS: In this study, we explored the underlying mechanisms in simvastatin-induced HCT116 colorectal cancer cell apoptosis. RESULTS: Simvastatin decreased cell viability and induced cell apoptosis in HCT116 cells. These results are associated with the modulation of p21(cip/Waf1) and survivin. Survivin knockdown using survivin siRNAs also decreased cell viability and induced cell apoptosis. Simvastatin's actions on p21(cip/Waf1), survivin and apoptosis were reduced in p53 null HCT116 cells. Simvastatin caused an increase in p53 phosphorylation and acetylation. In addition, simvastatin activated p38 mitogen-activated protein kinase (p38MAPK), whereas an inhibitor of p38MAPK signaling abrogated simvastatin's effects of increasing p53 and p21(cip/Waf1) promoter luciferase activity. Cell viability and survivin promoter luciferase activity in the presence of simvastatin were also restored by p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p53 and p63 binding to the promoter region increased after simvastatin exposure. CONCLUSIONS: Simvastatin activates the p38MAPK-p53-survivin cascade to cause HCT116 colorectal cancer cell apoptosis. GENERAL SIGNIFICANCE: This study delineates, in part, the underlying mechanisms of simvastatin in decreasing survivin and subsequent colorectal cancer cell apoptosis.