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.

A simple high-volume culture technique-Good substitute for polymerase chain reaction for the detection of Aspergillus species in bronchoalveolar lavage samples.

BACKGROUND: Aspergillus species is the most common agent of invasive pulmonary fungal disease. Culture-based diagnosis considered as gold standard is limited by the fungal load in samples. Detection of Aspergillus by polymerase chain reaction (PCR) has been included as a diagnostic criterion by European Organisation for Research and Treatment of Cancer (EORTC). Most routine laboratories lack facilities for molecular diagnosis. Better yield using high-volume culture (HVC) technique has been reported. Studies have not compared HVC and PCR for detection of Aspergillus species in respiratory samples from patients with suspected invasive pulmonary Aspergillosis (IPA) not on antifungal therapy. OBJECTIVE: This pilot study compared HVC and PCR for the detection of Aspergillus species in respiratory samples from treatment naïve patients. METHODS: Bronchoalveolar lavage (BAL) samples from 30 patients with clinical suspicion of IPA were evaluated. Direct microscopy, culture both conventional (CC) and HVC and qualitative Pan Aspergillus PCR were performed. Latent class model was used for statistical analysis. RESULTS: Sensitivity of HVC (100%) was better compared with CC (60%) and comparable to that of PCR (100%). Specificities of CC, HVC and PCR were 100%, 100% and 25%, respectively. CONCLUSION: High-volume culture is a simple cost-effective technique with a high sensitivity and specificity. It can be easily introduced in routine microbiology laboratories. In centres with the availability of infrastructure for molecular analysis, Aspergillus PCR with other mycological techniques can be used for better diagnosis and management of patients with IPA.

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.

RhoC: a fascinating journey from a cytoskeletal organizer to a Cancer stem cell therapeutic target.

Tumor heterogeneity results in differential response to therapy due to the existence of plastic tumor cells, called cancer stem cells (CSCs), which exhibit the property of resistance to therapy, invasion and metastasis. These cells have a distinct, signaling network active at every stage of progression. It is difficult to envisage that the CSCs will have a unique set of signaling pathways regulating every stage of disease progression. Rather, it would be easier to believe that a single pivotal pathway having significant contribution at every stage, which can further turn on a battery of signaling mechanisms specific to that stage, would be instrumental in regulating the signaling network, enabling easy transition from one state to another. In this context, we discuss the role of RhoC which has contributed to several phenotypes during tumor progression.RhoC (Ras homolog gene family member C) has been widely reported to regulate actin organization. It has been shown to impact the motility of cancer cells, resultantly affecting invasion and metastasis, and has contributed to carcinoma progression of the breast, pancreas, lung, ovaries and cervix, among several others. The most interesting finding has been its indispensable role in metastasis. Also, it has the ability to modulate various other phenotypes like angiogenesis, motility, invasion, metastasis, and anoikis resistance. These observations suggest that RhoC imparts the plasticity required by tumor cells to exhibit such diverse functions based on microenvironmental cues. This was further confirmed by recent reports which show that it regulates cancer stem cells in breast, ovary and head and neck cancers. Studies also suggest that the inhibition of RhoC results in abolition of advanced tumor phenotypes.Our review throws light on how RhoC, which is capable of modulating various phenotypes may be the apt core signaling candidate regulating disease progression. Additionally, mice studies show that RhoC is not essential for embryogenesis, giving scope for its development as a possible therapeutic target. This review thus stresses on the need to understand the protein and its functioning in greater detail to enable its development as a stem cell marker and a possible therapeutic target.