BackgroundCurrent management of patients diagnosed with prostate cancer (PCa) is very effective; however, tumor recurrence with Castrate Resistant Prostate Cancer (CRPC) and subsequent metastasis lead to poor survival outcome, suggesting that there is a dire need for novel mechanistic understanding of tumor recurrence, which would be critical for designing novel therapies. The recurrence and the metastasis of PCa are tightly linked with the biology of prostate cancer stem cells or cancer-initiating cells that is reminiscent of the acquisition of Epithelial to Mesenchymal Transition (EMT) phenotype. Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells.Methodology/Principal FindingsIn this study, we found that PCa cells with EMT phenotype displayed stem-like cell features characterized by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1, consistent with enhanced clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice, which was associated with decreased expression of miR-200 and/or let-7 family. Reversal of EMT by re-expression of miR-200 inhibited prostasphere-forming ability of EMT-type cells and reduced the expression of Notch1 and Lin28B. Down-regulation of Lin28B increased let-7 expression, which was consistent with repressed self-renewal capability.Conclusions/SignificanceThese results suggest that miR-200 played a pivotal role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in PCa. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to Mesenchymal-Epithelial Transition (MET) phenotype using novel agents would be useful for the prevention of tumor recurrence especially by eliminating those cells that are the “Root Cause” of tumor development and recurrence.

The emergence of castrate-resistant prostate cancer (CRPC) contributes to the high mortality of patients diagnosed with prostate cancer (PCa), which in part could be attributed to the existence and the emergence of cancer stem cells (CSCs). Recent studies have shown that deregulated expression of microRNAs (miRNAs) contributes to the initiation and progression of PCa. Among several known miRNAs, let-7 family appears to play a key role in the recurrence and progression of PCa by regulating CSCs; however, the mechanism by which let-7 family contributes to PCa aggressiveness is unclear. Enhancer of Zeste homolog 2 (EZH2), a putative target of let-7 family, was demonstrated to control stem cell function. In this study, we found loss of let-7 family with corresponding over-expression of EZH2 in human PCa tissue specimens, especially in higher Gleason grade tumors. Overexpression of let-7 by transfection of let-7 precursors decreased EZH2 expression and repressed clonogenic ability and sphere-forming capacity of PCa cells, which was consistent with inhibition of EZH2 3′UTR luciferase activity. We also found that the treatment of PCa cells with BR-DIM (formulated DIM: 3,3′-diindolylmethane by Bio Response, Boulder, CO, abbreviated as BR-DIM) up-regulated let-7 and down-regulated EZH2 expression, consistent with inhibition of self-renewal and clonogenic capacity. Moreover, BR-DIM intervention in our on-going phase II clinical trial in patients prior to radical prostatectomy showed upregulation of let-7 consistent with down-regulation of EZH2 expression in PCa tissue specimens after BR-DIM intervention. These results suggest that the loss of let-7 mediated increased expression of EZH2 contributes to PCa aggressiveness, which could be attenuated by BR-DIM treatment, and thus BR-DIM is likely to have clinical impact.

Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) plays important role in glycolysis and gluconeogenesis, and is associated with invasion and metastasis of cancer cells. We have previously shown its role in the induction of Epithelial-to-Mesenchymal transition (EMT) in breast cancer cells, which led to increased aggressiveness; however, the molecular mechanism by which PGI/AMF regulates EMT is not known. Here we show, for the first time, that PGI/AMF over-expression led to an increase in the DNA-binding activity of NF-κB, which, in turn, led to increased expression of ZEB1/ZEB2. The microRNA-200s (miR-200s; miR-200a, miR-200b and miR-200c) are known to negatively regulate the expression of ZEB1/ZEB2, and we found that the expression of miR-200s was lost in PGI/AMF over-expressing MCF-10A cells as well as in highly invasive MDA-MB-231 cells, which was consistent with increased expression of ZEB1/ZEB2. Moreover, silencing of PGI/AMF expression in MDA-MB-231 cells led to over-expression of miR-200s, which was associated with reversal of EMT phenotype i.e. Mesenchymal-to-Epithelial Transition (MET), and these findings were consistent with alterations in the relative expression of epithelial (E-cadherin) and mesenchymal (vimentin, ZEB1, ZEB2) markers, and decreased aggressiveness as judged by clonogenic, motility and invasion assays. Moreover, either re-expression of miR-200 or silencing of PGI/AMF suppressed pulmonary metastases of MDA-MB-231 cells in vivo, and anti-miR-200 treatment in vivo resulted in increased metastases. Collectively, these results suggest a role of miR-200s in PGI/AMF induced EMT, and thus approaches for up-regulation of miR-200s could be a novel therapeutic strategy for the treatment of highly invasive breast cancer.

Anticancer properties of Garcinia indica–derived garcinol are just beginning to be elucidated. We have earlier reported its cancer cell–specific induction of apoptosis in breast cancer cells, which was mediated through the downregulation of NF-κB signaling pathway. To gain further mechanistic insight, here, we show for the first time that garcinol effectively reverses epithelial-to-mesenchymal transition (EMT), that is, it induces mesenchymal-to-epithelial transition (MET) in aggressive triple-negative MDA-MB-231 and BT-549 breast cancer cells. This was associated with upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers vimentin, ZEB-1, and ZEB-2. We also found that garcinol upregulates the expression of miR-200 and let-7 family microRNAs (miRNAs), which provides a molecular mechanism for the observed reversal of EMT to MET. Transfection of cells with NF-κB p65 subunit attenuated the effect of garcinol on apoptosis induction through reversal of MET to EMT. Forced transfection of p65 and anti-miR-200s could also reverse the inhibitory effect of garcinol on breast cancer cell invasion. Moreover, treatment with garcinol resulted in increased phosphorylation of β-catenin concomitant with its reduced nuclear localization. The results were also validated in vivo in a xenograft mouse model where garcinol was found to inhibit NF-κB, miRNAs, vimentin, and nuclear β-catenin. These novel findings suggest that the anticancer activity of garcinol against aggressive breast cancer cells is, in part, due to reversal of EMT phenotype, which is mechanistically linked with the deregulation of miR-200s, let-7s, NF-κB, and Wnt signaling pathways.

Background The mechanism(s) by which androgen receptor (AR) splice variants contribute to castration-resistant prostate cancer (CRPC) is still lacking. Methods Expressions of Epithelial-to-Mesenchymal Transition (EMT) and stem cell markers were molecularly tested using prostate cancer (PCa) cells transfected with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells under androgen deprivation therapy (ADT) condition. Cell migration, clonogenicity, sphere forming capacity was assessed using PCa cells under all experimental conditions and 3, 3′-diindolylmethane (DIM; BR-DIM) treatment. Human PCa samples from BR-DIM untreated or treated patients were also used for assessing the expression of AR3 and stem cell markers. Results Overexpression of AR led to the induction of EMT phenotype, while overexpression of AR3 not only induced EMT but also led to the expression of stem cell signature genes. More importantly, ADT enhanced the expression of AR and AR3 concomitant with up-regulated expression of EMT and stem cell marker genes. Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and reversed the expression of these EMT and stem cell marker genes. BR-DIM administered to PCa patients prior to radical prostatectomy inhibited the expression of cancer stem cell markers consistent with inhibition of self-renewal of PCa cells after BR-DIM treatment. Conclusion AR variants could contribute to PCa progression through induction of EMT and acquisition of stem cell characteristics, which could be attenuated by BR-DIM, suggesting that BR-DIM could become a promising agent for the prevention of CRPC and/or for the treatment of PCa

Hypoxia is known to play critical roles in cell survival, angiogenesis, tumor invasion, and metastasis. Hypoxia mediated over-expression of hypoxia-inducible factor (HIF) has been shown to be associated with therapeutic resistance, and contributes to poor prognosis of cancer patients. Emerging evidence suggest that hypoxia and HIF pathways contributes to the acquisition of epithelial-to-mesenchymal transition (EMT), maintenance of cancer stem cell (CSC) functions, and also maintains the vicious cycle of inflammation-all which lead to therapeutic resistance. However, the precise molecular mechanism(s) by which hypoxia/HIF drives these events are not fully understood. Here, we show, for the first time, that hypoxia leads to increased expression of VEGF, IL-6, and CSC signature genes Nanog, Oct4 and EZH2 consistent with increased cell migration/invasion and angiogenesis, and the formation of pancreatospheres, concomitant with increased expression of miR-21 and miR-210 in human pancreatic cancer (PC) cells. The treatment of PC cells with CDF, a novel synthetic compound inhibited the production of VEGF and IL-6, and down-regulated the expression of Nanog, Oct4, EZH2 mRNAs, as well as miR-21 and miR-210 under hypoxia. CDF also led to decreased cell migration/invasion, angiogenesis, and formation of pancreatospheres under hypoxia. Moreover, CDF decreased gene expression of miR-21, miR-210, IL-6, HIF-1α, VEGF, and CSC signatures in vivo in a mouse orthotopic model of human PC. Collectively, these results suggest that the anti-tumor activity of CDF is in part mediated through deregulation of tumor hypoxic pathways, and thus CDF could become a novel, and effective anti-tumor agent for PC therapy.

Elucidation of the role of human papillomavirus (HPV) in the etiology and prognosis of squamous carcinomas of the head and neck (HNSCC) is essential to optimize prevention and treatment strategies for this disease. We analyzed 385 HNSCC tissue blocks identified through a population-based cancer registry in Metropolitan Detroit for HPV DNA using a broad-spectrum PCR technique (SPF10-LiPA25) to correlate with patient and tumor characteristics and overall survival. Overall, HPV DNA (any type) was detected in 29.4% of all HNSCC, but it was significantly more prevalent (50.6%) in oropharyngeal sites (N=81), where 90% of HPV were type 16, than in other sites. HPV prevalence (any type) in oropharyngeal sites was highest in patients with a negative smoking indicator, Caucasians, and in regional tumor stage. Likewise, only in oropharyngeal sites did patients overall positive to HPV show significantly better survival compared with HPV-negative patients, notably among those who had been irradiated. The best and the worst survival from cancer in oropharyngeal sites were found, respectively, among HPV-positive patients with negative smoking indicator and among HPV-negative patients with positive smoking indicator. The results of the present study revealed that the presence of HPV DNA was associated with patients’ specific characteristics and better overall survival exclusively in oropharyngeal sites. To define the fraction of HNSCC preventable by HPV vaccination or amenable to less aggressive treatment, however, tobacco exposure and HPV markers other than DNA presence need to be taken into account.

To examine epigenetic events of aberrant promoter methylation as diagnostic markers in primary head and neck squamous cell carcinoma using a novel multigene approach. Promoter methylation-mediated silencing is a hallmark of several established tumor suppressor genes. Changes in DNA methylation have been reported to occur early in carcinogenesis and therefore are potentially important early indicators of existing disease. Design: A multicandidate gene probe panel interrogated DNA for aberrant methylation status in 22 cancer genes using the methylation-specific multiplex ligationdependent probe amplification (MS-MLPA) assay. Aberrant promoter hypermethylation was confirmed using methylation-specific polymerase chain reaction after bisulfite treatment. Setting: Primary care medical center. Subjects: We examined fresh-frozen primary head and neck tumor specimens from 28 patients, including 21 late-stage (19 stage IV and 2 stage III) and 7 early-stage (6 stage II and 1 stage I) tumors. Results: Promoter hypermethylation was observed in 14 of the 28 patients (50%). Genes for RARB, APC, and CHFR were most frequently hypermethylated, occurring in 11 (39%) for RARB, 7 (25%) for CHFR, and 6 (21%) for APC. Aberrant methylation of CHFR was solely a stage IV event. Methylation-specific polymerase chain reaction after bisulfite treatment with conventional and real-time polymerase chain reaction confirmed aberrant methylation for RARB and CHFR. Conclusions: Promoter methylation profiling of primary head and neck squamous cell carcinoma using multiple target genes identified RARB, APC, and CHFR as frequent epigenetic events. The clinical implications of these genes as diagnostic and treatment biomarkers are highly relevant as attractive targets for cancer therapy, given the reversible nature of epigenetic gene silencing.