Emerging role of caldesmon in cancer:A potential biomarker for colorectal cancer and other cancers

Colorectal cancer(CRC) is a devastating disease, mainly because of metastasis. As a result, there is a need to better understand the molecular basis of invasion and metastasis and to identify new biomarkers and therapeutic targets to aid in managing these tumors. The actin cytoskeleton and actin-binding proteins are known to play an important role in the process of cancer metastasis because they control and execute essential steps in cell motility and contractility as well as cell division. Caldesmon(CaD) is an actin-binding protein encoded by the CALD1 gene as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight CaD, expressed in smooth muscle, and low-molecular weight CaD(l-CaD), expressed in nonsmooth muscle cells. According to our comprehensive review of the literature, CaD, particularly l-CaD, plays a key role in the development, metastasis, and resistance to chemoradiotherapy in colorectal, breast, and urinary bladder cancers and gliomas, among other malignancies. CaD is involved in many aspects of the carcinogenic hallmarks, including epithelial mesenchymal transition via transforming growth factor-beta signaling, angiogenesis, resistance to hormonal therapy, and immune evasion. Recent data show that CaD is expressed in tumor cells as well as in stromal cells, such as cancerassociated fibroblasts, where it modulates the tumor microenvironment to favor the tumor. Interestingly, CaD undergoes selective tumor-specific splicing, and the resulting isoforms are generally not expressed in normal tissues, making these transcripts ideal targets for drug design. In this review, we will analyze these features of CaD with a focus on CRC and show how the currently available data qualify CaD as a potential candidate for targeted therapy in addition to its role in the diagnosis and prognosis of cancer.

Role of AXL in invasion and drug resistance of colon and breast cancer cells and its association with p53 alterations

AIM To characterize AXL receptor tyrosine kinase(AXL)expression in relationship to tumor protein P53(TP53gene,p53 protein)and its role in tumor invasion and response to therapy.METHODS We used 14 cell lines,including 3 isogenic pairs carrying mutant/knockout p53,to gain insight into the relationship between AXL and TP53.These included HCT116,HCT116.p53 mutant,RKO,and RKO.p53-/-lines(all from colon cancers)as well as breast cancer cell lines MCF7 and 1001(MCF7-p53 mutant clone).He La cell line was used as a positive control for epithelial to mesenchymal transition(EMT).AXL expression was determined by Western blotting using rabbit monoclonal antibody clone C89E7.AXL si RNA silencing was performed and followed by collagen invasion assay.Cell viability analysis using the sulforhodamine B assay and the invasion assay were performed after exposure to chemotherapeutic agents(doxorubicin for breast cancer cells;5FU or irinotecan for colon cancer cells).RESULTS We showed that the introduction of p53 mutations or knockout increased expression levels of AXL in isogenic cells compared to the matching p53 wild-type parental cells.Overall,we found a trend for correlation between the potential EMT candidate AXL,p53 alterations,and EMT markers in colorectal and breast cancers.The expression of AXL in RKO cells,a rare colon cancer cell line with inactive Wnt signaling,suggests that the AXL oncogene might provide an alternative genetic pathway for colorectal carcinogenesis in the absence of Wnt signaling activation and TP53 mutation.AXL silencing in the TP53 mutant isogenic cell lines 1001,HCT116.p53 mutant and RKO.P53-/-was>95%efficient and the silenced cells were less invasive compared to the parental TP53 wild-type cells.AXL silencing showed a subtle trend to restore colon cancer cell sensitivity to5FU or irinotecan.Importantly,AXL expressing cells developed more invasive potential after exposure to chemotherapy compared to the AXL-silenced cells.CONCLUSION AXL is influenced by p53 status and could cause the emergence of aggressive clones after exposure to chemotherapy.These findings could have applications in cancer management.

Calponin 3 promotes invasion and drug resistance of colon cancer cells

BACKGROUND Calponin 3(CNN3)is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells.It is required for cytoskeletal rearrangement and wound healing.AIM To dissect the role of CNN3 in carcinogenesis with a focus on colon cancer.METHODS A total of 20 cancer cell lines(8 breast,11 colon,and HeLa cervical cancer cell as a positive control for mesenchymal phenotype)and 57 formalin-fixed,paraffinembedded sections from archived sporadic colorectal carcinomas were included in this study.CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses.The CNN3 gene was silenced by specific small interfering RNA(commonly known as siRNA),followed by confirmation of the silencing efficiency by western blotting.Then,the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers,invasion,and response to 5-fluoruracil treatment.We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins.RESULTS CNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells.Interestingly,the colorectal adenocarcinoma line SW480 was negative,while the cell line developed from its matching lymph node metastasis(SW620)was positive for CNN3.CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others.We selected SW620 for subsequent functional analyses.CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers.CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil.Phosphokinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase,β-Catenin,mutant p53,c-Jun,and heat shock protein 60 activities but increased that of checkpoint kinase 2.CNN3 was expressed in 20/57(35%)colon cancer cases as shown by immunohistochemistry.CNN3 was associated with a decrease in overall survival in colon cancer in silico.CONCLUSION These results show the involvement of CNN3 in lymph node metastasis and resistance to chemotherapy in colon cancer and suggest that significant oncogenic pathways are involved in these CNN3-related actions.

Insight into molecular mechanisms underlying hepatic dysfunction in severe COVID-19 patients using systems biology

BACKGROUND The coronavirus disease 2019(COVID-19),a pandemic contributing to more than 105 million cases and more than 2.3 million deaths worldwide,was described to be frequently accompanied by extrapulmonary manifestations,including liver dysfunction.Liver dysfunction and elevated liver enzymes were observed in about 53%of COVID-19 patients.AIM To gain insight into transcriptional abnormalities in liver tissue of severe COVID-19 patients that may result in liver dysfunction.METHODS The transcriptome of liver autopsy samples from severe COVID-19 patients against those of non-COVID donors was analyzed.Differentially expressed genes were identified from normalized RNA-seq data and analyzed for the enrichment of functional clusters and pathways.The differentially expressed genes were then compared against the genetic signatures of liver diseases including cirrhosis,fibrosis,non-alcoholic fatty liver disease(NAFLD),and hepatitis A/B/C.Gene expression of some differentially expressed genes was assessed in the blood samples of severe COVID-19 patients with liver dysfunction using qRT-PCR.RESULTS Analysis of the differential transcriptome of the liver tissue of severe COVID-19 patients revealed a significant upregulation of transcripts implicated in tissue remodeling including G-coupled protein receptors family genes,DNAJB1,IGF2,EGFR,and HDGF.Concordantly,the differential transcriptome of severe COVID-19 liver tissues substantially overlapped with the disease signature of liver diseases characterized with pathological tissue remodeling(liver cirrhosis,Fibrosis,NAFLD,and hepatitis A/B/C).Moreover,we observed a significant suppression of transcripts implicated in metabolic pathways as well as mitochondrial function,including cytochrome P450 family members,ACAD11,CIDEB,GNMT,and GPAM.Consequently,drug and xenobiotics metabolism pathways are significantly suppressed suggesting a decrease in liver detoxification capacity.In correspondence with the RNA-seq data analysis,we observed a significant upregulation of DNAJB1 and HSP90AB1 as well as significant downregulation of CYP39A1 in the blood plasma of severe COVID-19 patients with liver dysfunction.CONCLUSION Severe COVID-19 patients appear to experience significant transcriptional shift that may ensue tissue remodeling,mitochondrial dysfunction and lower hepatic detoxification resulting in the clinically observed liver dysfunction.

Combination of Paracetamol and the Glutathione Depleting Agent Buthionine Sulfoximine Show Differential Effect on Liver Cancer Cells and Normal Hepatocytes

Background: Paracetamol exerts toxic effects on liver cells through its metabolism into N-acetyl-p-benzoquinone imine (NAPQI), which is detoxified by conjugation with cellular glutathione (GSH). Once GSH is depleted, NAPQI stimulates a range of oxidative reactions that result in cell necrosis. The aim of the present investigation is to find a new strategy that would selectively protect normal hepatic tissues and sensitize liver cancer cells to the toxic effects of paracetamol or its metabolite. This may lead to the development of a targeted therapy for liver cancer. Methods: The anti-proliferative effects of paracetamol and buthionine sulfoximine BSO (a glutathione depleting agent) alone and in combination on the liver cancer cells HepG2 and normal rat hepatocytes were investigated by sulphorhodamine-B assay. Effects on cell cycle regulation and induction of apoptosis were tested by flow cytometry. The level of prostaglandin expression was measured by ELISA. Results: The present study showed that both agents alone or in combination have anti-proliferative effects on both cell types. Surprisingly, BSO showed a cytoprotective effects on normal hepatocytes treated with high concentrations (1.75 and 2 mM) of paracetamol. This was confirmed by cell cycle analysis that recorded decreased fraction of sub-G1 cells indicating reduction of apoptosis in normal hepatocytes. Analysis of prostaglandin E2 revealed differential effects of paracetamol on normal and liver cancer cells. A significant increase in PGE2 level over the control was observed in normal hepatocytes whereas a significant decrease was seen in HepG2 cells after treatment with paracetamol. Conclusion: These results indicate that combination of paracetamol/BSO has differential effects on liver cancer cells and normal hepatocytes, which opens the avenue for a new effective and selective combination for management of liver cancer.