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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 .

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.

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.

RhoC is an important regulator of metastasis and tumour progression across various tumour models. Since RhoC has been found to have no major contribution towards normal embryogenic development, it has emerged as a suitable therapeutic target for effective cancer treatment. Recent evidence has shown that Rho-based peptide vaccines have favourable outcomes in prostate cancer patients, by bringing about activation of CD4+ T-cells. Antigen presentation on the surface of cells is brought about by the MHC-I/MHC-II complex. This work provides conclusive evidence to show that the seemingly cytosolic protein, RhoC, is in fact, present on the surface of tumour cells. This report goes on to prove that the presentation of RhoC peptides is brought about in association with MHC-II, becoming the first piece of scientific evidence to report this phenomenon. Competing Interest Statement The authors have declared no competing interest.

In their quest for autonomy, tumor cells are known to reroute metabolic networks to aid their proliferation and survival. These metabolic alterations are governed by the tumor sub-population, thereby contributing towards an additional layer of complexity within the already heterogeneous tumor. For instance, bulk proliferative tumor cells rely on completely different pathways for their metabolic requirements as opposed to the stem-like metastatic cells. However, the molecular switch that drives these metabolic changes remains unknown. RhoC is a well-established contributor towards multiple aspects of tumor development including proliferation, EMT, migration, invasion and metastasis. A transcriptomics-based approach on a RhoC over-expressing cervical cancer cell line unveiled distinct metabolic signatures existent in these cells. Oxidative phosphorylation, TCA cycle, nucleic acid metabolism and fatty acid elongation were some of the specific pathways that emerged as up-regulated. This study therefore provides insight into the intricate metabolic circuitry functional in aggressive RhoC-high cells and thus proposes a pivotal role for RhoC in oncometabolism.

The domestic ferret ( Mustela putorius furo ) has proven to be a useful species for modeling human genetic and infectious diseases of the lung and brain. However, biomedical research in ferrets has been hindered by the lack of rapid and cost-effective methods for genome engineering. Here, we utilized CRISPR/Cas9-mediated, homology-independent insertion at the ROSA26 “safe harbor” locus in ferret zygotes and created transgenic animals expressing a dual-fluorescent Cre-reporter system flanked by PhiC31 and Bxb1 integrase att P sites. Out of 151 zygotes injected with circular transgene-containing plasmid and Cas9 protein loaded with the ROSA26 intron-1 sgRNA, there were 23 births of which 5 had targeted integration events (22% efficiency). The encoded tdTomato transgene was highly expressed in all tissues evaluated. Targeted integration was verified by PCR analyses, Southern blot, and germ-line transmission. Function of the ROSA26 -CAG- LoxP tdTomato StopLoxP EGFP ( ROSA -TG) Cre-reporter was confirmed in primary cells following Cre expression. The Phi31 and Bxb1 integrase att P sites flanking the transgene will also enable rapid directional insertion of any transgene without a size limitation at the ROSA26 locus. These methods and the model generated will greatly enhance biomedical research involving lineage tracing, the evaluation of stem cell therapy, and transgenesis in ferret models of human disease.

Two anti-transferrin receptor (TfR) nanobodies, V H H123 specific for mouse TfR and V H H188 specific for human TfR, were used to track transplants non-invasively by PET/CT in mouse models, without the need for genetic modification of the transferred cells. We provide a comparison of the specificity and kinetics of the PET signals acquired when using nanobodies radiolabeled with 89 Zr, 64 Cu, and 18 F, and find that the chelation of the 89 Zr and 64 Cu radioisotopes to anti-TfR nanobodies results in radioisotope release upon endocytosis of the radiolabeled nanobodies. We used a knock-in mouse that expresses a TfR with a human ectodomain (Tfrc hu/hu ) as a source of bone marrow for transplants into C57BL/6 recipients and show that V H H188 detects such transplants by PET/CT. Conversely, C57BL/6 bone marrow and B16.F10 melanoma cell line transplanted into Tfrc hu/hu recipients can be imaged with V H H123. In C57BL/6 mice impregnated by Tfrc hu/hu males, we saw an intense V H H188 signal in the placenta, showing that TfR-specific V H Hs accumulate at the placental barrier but do not enter the fetal tissue. We were unable to observe accumulation of the anti-TfR radiotracers in the central nervous system (CNS) by PET/CT but showed evidence of CNS accumulation by radiospectrometry. The model presented here can be used to track many transplanted cell types by PET/CT, provided cells express TfR, as is typically the case for proliferating cells such as tumor lines.

Two anti-transferrin receptor (TfR) nanobodies, V H H123 specific for mouse TfR and V H H188 specific for human TfR, were used to track transplants non-invasively by PET/CT in mouse models, without the need for genetic modification of the transferred cells. We provide a comparison of the specificity and kinetics of the PET signals acquired when using nanobodies radiolabeled with 89 Zr, 64 Cu, and 18 F, and find that the chelation of the 89 Zr and 64 Cu radioisotopes to anti-TfR nanobodies results in radioisotope release upon endocytosis of the radiolabeled nanobodies. We used a knock-in mouse that expresses a TfR with a human ectodomain (Tfrc hu/hu ) as a source of bone marrow for transplants into C57BL/6 recipients and show that V H H188 detects such transplants by PET/CT. Conversely, C57BL/6 bone marrow and B16.F10 melanoma cell line transplanted into Tfrc hu/hu recipients can be imaged with V H H123. In C57BL/6 mice impregnated by Tfrc hu/hu males, we saw an intense V H H188 signal in the placenta, showing that TfR-specific V H Hs accumulate at the placental barrier but do not enter the fetal tissue. We were unable to observe accumulation of the anti-TfR radiotracers in the central nervous system (CNS) by PET/CT but showed evidence of CNS accumulation by radiospectrometry. The model presented here can be used to track many transplanted cell types by PET/CT, provided cells express TfR, as is typically the case for proliferating cells such as tumor lines.

According to this traditional model, the selective advantage is conferred by a small set of driver mutations, but as the subclones that bear them successively expand, they also accumulate passenger mutations, which can be detected in sequencing experiments1. Genomes of individual tumors contain hundreds to many thousands of these genetic variants at a wide range of frequencies5,6. Because genetic drift can drive novel variants to high frequencies, it is of great interest to discern the relative importance of selection and drift in shaping the frequency distribution of variants in any given tumor. [...] the deterministic model of tumor growth described by Williams et al. relies on strong biological assumptions, including synchronous cell divisions, constant cell death, and constant mutation and division rates. [...]discrimination of neutral and non-neutral simulated tumors by using a linear fit is almost arbitrary, with 53.5% false-positive neutral calls in nonneutral tumors (Fig. 1b) and an area under the receiver operating characteristic curve of 0.42 for the classification of 1,919 neutral and 1,919 non-neutral tumors (Fig. 1c).

Two anti-transferrin receptor (TfR) nanobodies, VHH123 specific for mouse TfR and VHH188 specific for human TfR (huTfR) were used to track transplants non-invasively by PET/CT in mouse models, without the need for genetic modification of the transferred cells. We provide a comparison of the specificity and kinetics of the PET signals acquired when using nanobodies radiolabeled with 89Zr, 64Cu and 18F. We used a knock-in mouse that expresses a TfR with a human ectodomain (huTfR +/+) as a source of transplants into C57BL/6 recipients and show that VHH188 detects such transplants by PET/CT. Conversely, C57BL/6 transplants into huTfR +/+ recipients can be imaged with VHH123. In C57B/6 mice impregnated by huTfR+/+ males we saw an intense VHH188 signal in the placenta showing that TfR-specific VHHs accumulate at the placental barrier but do not enter the fetal tissue. The model presented here can be used to track many transplanted cell types by PET/CT, provided cells express TfR, as is typically the case for proliferating cells such as tumor lines.Competing Interest StatementThe authors have declared no competing interest.