RhoC in association with TET2/WDR5 regulates cancer stem cells by epigenetically modifying the expression of pluripotency genes.

Emerging evidence illustrates that RhoC has divergent roles in cervical cancer progression where it controls epithelial to mesenchymal transition (EMT), migration, angiogenesis, invasion, tumor growth, and radiation response. Cancer stem cells (CSCs) are the primary cause of recurrence and metastasis and exhibit all of the above phenotypes. It, therefore, becomes imperative to understand if RhoC regulates CSCs in cervical cancer. In this study, cell lines and clinical specimen-based findings demonstrate that RhoC regulates tumor phenotypes such as clonogenicity and anoikis resistance. Accordingly, inhibition of RhoC abrogated these phenotypes. RNA-seq analysis revealed that RhoC over-expression resulted in up-regulation of 27% of the transcriptome. Further, the Infinium MethylationEPIC array showed that RhoC over-expressing cells had a demethylated genome. Studies divulged that RhoC via TET2 signaling regulated the demethylation of the genome. Further investigations comprising ChIP-seq, reporter assays, and mass spectrometry revealed that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes such as Nanog. Interestingly, clinical specimen-based investigations revealed the existence of a subset of tumor cells marked by RhoC+/Nanog+ expression. Finally, combinatorial inhibition (in vitro) of RhoC and its partners (WDR5 and TET2) resulted in increased sensitization of clinical specimen-derived cells to radiation. These findings collectively reveal a novel role for nuclear RhoC in the epigenetic regulation of Nanog and identify RhoC as a regulator of CSCs. The study nominates RhoC and associated signaling pathways as therapeutic targets.

Gene expression profiling of CD34(+) cells from patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the increased proliferation of hematopoietic stem cell precursors and mature blood cells. Mutations of Janus kinase 2 (JAK2), Calreticulin (CALR) and MPL (myeloproliferative leukemia virus) are key driver mutations in MPN. However, the molecular profile of triple negative MPN has been a subject of ambiguity over the past few years. Mutations of, methylcytosine dioxygenase TET2, polycomb group protein ASXL1 and histone-lysine N-methyltransferase EZH2 genes have accounted for certain subsets of triple negative MPNs but the driving cause for majority of cases is still unexplored. The present study performed a microarray-based transcriptomic profile analysis of bone marrow-derived CD34(+) cells from seven MPN samples. A total of 21,448 gene signatures were obtained, which were further filtered into 472 upregulated and 202 downregulated genes. Gene ontology and protein-protein interaction (PPI) network analysis highlighted an upregulation of genes involved in cell cycle and chromatin modification in JAK2V617F negative vs. positive MPN samples. Out of the upregulated genes, seven were associated with the hematopoietic stem cell signature, while forty-seven were associated with the embryonic stem cell signature. The majority of the genes identified were under the control of NANOG and E2F4 transcription factors. The PPI network indicated a strong interaction between chromatin modifiers and cell cycle genes, such as histone-lysine N-methyltransferase SUV39H1, SWI/SNF complex subunit SMARCC2, SMARCE2, chromatin remodeling complex subunit SS18, tubulin ß (TUBB) and cyclin dependent kinase CDK1. Among the upregulated epigenetic markers, there was a ~10-fold increase in MYB expression in JAK2V617F negative samples. A significant increase in total CD34 counts in JAK2V617F negative vs. positive samples (P<0.05) was also observed. Overall, the present data showed a distinct pattern of expression in JAK2V617F negative vs. positive samples with upregulated genes involved in epigenetic modification.

RhoC regulates radioresistance via crosstalk of ROCK2 with the DNA repair machinery in cervical cancer.

BACKGROUND: Radioresistance remains a challenge to the successful treatment of various tumors. Intrinsic factors like alterations in signaling pathways regulate response to radiation. RhoC, which has been shown to modulate several tumor phenotypes has been investigated in this report for its role in radioresistance. In vitro and clinical sample-based studies have been performed to understand its contribution to radiation response in cervical cancer and this is the first report to establish the role of RhoC and its effector ROCK2 in cervical cancer radiation response. METHODS: Biochemical, transcriptomic and immunological approaches including flow cytometry and immunofluorescence were used to understand the role of RhoC and ROCK2. RhoC variants, siRNA and chemical inhibitors were used to alter the function of RhoC and ROCK2. Transcriptomic profiling was performed to understand the gene expression pattern of the cells. Live sorting using an intracellular antigen has been developed to isolate the cells for transcriptomic studies. RESULTS: Enhanced expression of RhoC conferred radioprotection on the tumor cells while inhibition of RhoC resulted in sensitization of cells to radiation. The RhoC overexpressing cells had a better DNA repair machinery as observed using transcriptomic analysis. Similarly, overexpression of ROCK2, protected tumor cells against radiation while its inhibition increased radiosensitivity in vitro. Further investigations revealed that ROCK2 inhibition abolished the radioresistance phenotype, conferred by RhoC on SiHa cells, confirming that it is a downstream effector of RhoC in this context. Additionally, transcriptional analysis of the live sorted ROCK2 high and ROCK2 low expressing SiHa cells revealed an upregulation of the DNA repair pathway proteins. Consequently, inhibition of ROCK2 resulted in reduced expression of pH2Ax and MRN complex proteins, critical to repair of double strand breaks. Clinical sample-based studies also demonstrated that ROCK2 inhibition sensitizes tumor cells to irradiation. CONCLUSIONS: Our data primarily indicates that RhoC and ROCK2 signaling is important for the radioresistance phenotype in cervical cancer tumor cells and is regulated via association of ROCK2 with the proteins of DNA repair pathway involving pH2Ax, MRE11 and RAD50 proteins, partly offering insights into the mechanism of radioresistance in tumor cells. These findings highlight RhoC-ROCK2 signaling involvement in DNA repair and urge the need for development of these molecules as targets to alleviate the non-responsiveness of cervical cancer tumor cells to irradiation treatment.

Polyethylenimine-modified curcumin-loaded mesoporus silica nanoparticle (MCM-41) induces cell death in MCF-7 cell line.

Breast cancer accounts for the first highest mortality rate in India and second in world. Though current treatment strategies are effectively killing cancer cells, they also end in causing severe side effects and drug resistance. Curcumin is a nutraceutical with multipotent activity but its insolubility in water limits its therapeutic potential as an anti-cancer drug. The hydrophilicity of curcumin could be increased by nanoformulation or changing its functional groups. In this study, curcumin is loaded on mesoporous silica nanoparticle and its anti-cancer activity is elucidated with MCF-7 cell death. Structural characteristics of Mobil Composition of Matter - 41(MCM-41) as determined by high-resolution transmission electron microscopy (HR-TEM) shows that MCM-41 size ranges from 100 to 200 nm diameters with pore size 2-10 nm for drug adsorption. The authors found 80-90% of curcumin is loaded on MCM-41 and curcumin is released efficiently at pH 3.0. The 50 µM curcumin-loaded MCM-41 induced 50% mortality of MCF-7 cells. Altogether, their results suggested that increased curcumin loading and sustained release from MCM-41 effectively decreased cell survival of MCF-7 cells in vitro.