Analysis of Differential Gene Expression and Core Canonical Pathways Involved in the Epithelial to Mesenchymal Transition of Triple Negative Breast Cancer Cells by Ingenuity Pathway Analysis

Triple Negative Breast Cancer (TNBC) is a malignant form of cancer with very high mortality and morbidity. Epithelial to Mesenchymal Transition (EMT) is the most common pathophysiological change observed in cancer cells of epithelial origin that promotes metastasis, drug resistance and cancer stem cell formation. Since the information regarding differential gene expression in TNBC cells and cell signaling events leading to EMT is limited, this investigation was done by comparing transcriptomic data generated by RNA isolation and sequencing of a EMT model TNBC cell line in comparison to regular TNBC cells. RNA sequencing and Ingenuity Pathway Software Analysis (IPA) of the transcriptomic data revealed several upregulated and downregulated gene expressions along with novel core canonical pathways including Sirtuin signaling, Oxidative Phosphorylation and Mitochondrial dysfunction events involved in EMT changes of the TNBC cells.

Upfront Docetaxel with LH-RH Antagonist for Metastatic Hormone Sensitive Prostate Cancer Considering Epithelial to Mesenchymal Transition

Objective: Upfront docetaxel use for hormone naïve advanced prostate cancer is reported that it successfully delayed the progression to hormone refractory stage, though the adequate methodology to obtain the maximum effect is unclear. We investigated these issues from our experiences of upfront docetaxel use with LH-RH antagonist for metastatic hormone sensitive prostate cancer, aiming at the prevention of epithelial-mesenchymal transition (EMT) for apoptosis tolerance. Patients and Methods: Of 31 stage IV new prostate cancer patients treated with upfront docetaxel and LH-RH antagonist (Degarelix), 25 patients who could be followed more than 12 months (mean 36.2 months) were analyzed. Docetaxel was used two to three courses basically 75 mg/m2 dose initializing two weeks after the induction of first Degarelix. Results: The clinical course was divided clearly to two groups according to prostate specific antigen (PSA) values. Of 25 patients, 12 patient’s PSA did not decrease below 0.1 ng/ml within 6 months (group A) and gradually rose afterwards. PSA in another 13 patients (group B) decreased below 0.1 within 6 months and kept below 0.1 during the follow up period. Although statistically not significant, the initial group A’s PSA was higher than group B’s (average 1308 and 353 ng/ml), however, number of metastasis, Gleason sum, and bone metastatic extent of disease showed no difference between them. Among group B patients, 7 cases had only upfront docetaxel and hormonal therapy, and some of these patients showed only atrophic gland and fibrotic tissue at second prostate biopsy (specimens after more than two years of therapy), suggesting complete response. Conclusion: Our study suggested that PSA value at 6 months may predict the outcome of whole therapy. Patients showing PSA less than 0.1 ng/ml at 6 months and requiring no therapy other than docetaxel and hormone may be induced to complete response. Upfront docetaxel with LH-RH antagonist may prevent EMT for obtaining apoptosis tolerance, in case the patient does not have the castration-resistant clone at the beginning of the therapy (group B).

Epithelial to mesenchymal transition in the progression of tubulointerstitial fibrosis

Objective To review the mechanisms of epithelial to mesenchymal transition （EMT） and its role in the progression of tubulointerstitial fibrosis. Data sources The data used in this review were obtained mainly from the studies of EMT reported from 2000-2006. Study selection Relevant articles on studies of EMT in tubulointerstitial fibrosis were selected. Data were mainly extracted from the 45 articles listed in the reference section of this review. Results The process of EMT has gained wide recognition as candidate mechanism in progression of chronic fibrotic disorders. New markers were identified and facilitate the observation of EMT. EMT is regulated by many factors through activation of kinase-dependent signaling cascades. Recent findings suggest that EMT is a reversible process, which can be controlled by factors for their epithelial inducing activities. Conclusion Remarkable progresses of EMT research have been made recently. Preventing or reversing EMT is a promising strategy against renal fibrosis.

Follistatin Like 5 ( FSTL5) inhibits epithelial to mesenchymal transition in hepatocellular carcinoma

Background:Epithelial to mesenchymal transition(EMT)is a key process in determining distant metastasis and intra-hepatic dissemination of hepatocellular carcinoma(HCC).Follistatin(FST)family members are considered to be an attractive therapeutic targets and prognostic indicators in cancers.As a derivative of FST,Follistatin Like 5(FSTL5)may play a similar role in HCC cells.This study aimed to investigate the expression and function of FSTL5 in HCC and its role in EMT.Methods:FSTL5,E-cadherin and vimentin in HCC,and paracancerous tissues were detected by immunohistochemistry.Correlation of FSTL5 expression with overall survival was assessed.The proliferation and invasion of HCC cell lines SK-Hep1 and MHCC-LM3 were analyzed by cell counting kit-8 and Transwell assays.The expression of FSTL5,E-cadherin,and vimentin in HCC cells was examined by polymerase chain reaction and Western blot analysis.T-test was used to analyze the difference in proliferation and invasion ability between groups.The Spearman rank correlation test was used to detect the correlation between the expression of FSTL5 and E-cadherin or vimentin.Results:The expression of FSTL5 in HCC was lower than that in paracancerous tissues(9.97%vs.82.55%,χ2=340.15,P<0.001).Patients with high FSTL5 expression had a better prognosis(χ2=8.22,P=0.004)and smaller tumor diameter(χ2=45.52,P<0.001),less lymph node metastasis(χ2=5.58,P=0.02),earlier tumor node metastasis stage(χ2=11.29,P=0.001),a reduced number of tumors(χ2=5.05,P=0.02),lower alpha-fetoprotein value(χ2=24.36,P<0.001),more probability of hepatitis carrying(χ2=40.9,P<0.001),and better liver function grade(χ2=5.21,P=0.02).Immunohistochemistry showed that FSTL5 expression in HCC tissues was positively correlated with E-cadherin expression(r=0.38,P<0.001)and negatively correlated with vimentin expression(r=-0.385,P<0.001).Furthermore,over-expression of FSTL5 up-regulated the expression of E-cadherin and down-regulated the expression of vimentin in SK-Hep1(negative control[NC]vs.FSTL5-interfering group[Lv-FSTL5]:E-cadherin[t=45.03,P<0.001],vimentin[t=67,P<0.001])and MHCC-LM3(NC vs.Lv-FSTL5:E-cadherin[t=50,P<0.001],vimentin[t=72.75,P<0.001])cells at mRNA level.The same as protein level.In addition,the over-expression of FSTL5 inhibited the proliferation(NC vs.Lv-FSTL5:SK-Hep1,3 d[t=7.324,P=0.018],4 d[t=6.23,P=0.021],5 d[t=10.21,P=0.003];MHCC-LM3,3 d[t=4.32,P=0.037],4 d[t=7.49,P=0.012],5 d[t=9.3661,P=0.009])and invasion(NC vs.Lv-FSTL5:SK-Hep1,t=21.57,P<0.001;MHCC-LM3,t=18.04,P<0.001)of HCC cells.Conclusions:Down-regulation of FSTL5 may contribute to EMT of HCC,and FSTL5 is a potential target in the treatment of HCC.

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.

Pomegranate extract inhibits EMT in clear cell renal cell carcinoma in a NF-κB and JNK dependent manner

Objective:Clear cell renal cell carcinoma(ccRCC)is the most common subtype of renal cell carcinoma(RCC)and is characterized by biallelic inactivation of the von Hippel-Lindau(VHL)tumor suppressor gene.One effect of VHL inactivation is hypoxia inducible factor alpha(HIFa)-independent constitutive activation of nuclear factor kappa B(NF-κB)and c-jun N-terminal kinase(JNK).Both NF-κB and JNK drive ccRCC growth and epithelial to mesenchymal transition(EMT).The purpose of this study was to determine the biochemical effects of pomegranate juice extracts(PE)on RCC cell lines.Methods:The pre-clinical effects of PE on NF-κB,JNK,and the EMT phenotype were assayed,including its effect on proliferation,anchorage-independent growth,and invasion of pVHLdeficient RCCs.Results:PE inhibits the NF-κB and JNK pathways and consequently inhibits the EMT phenotype of pVHL-deficient ccRCCs.The effects of PE are concentration-dependent and affect not only biochemical markers of EMT(i.e.,cadherin expression)but also functional manifestations of EMT,such as invasion.These effects are manifested within days of exposure to PE when diluted 2000-fold.Highly dilute concentrations of PE(106 dilution),which do not impact these pathways in the short term,were found to have NF-κB and JNK inhibitory effects and ability to reverse the EMT phenotype following prolonged exposure.Conclusion:These findings suggest that PE may mediate inhibition growth of pVHL-deficient ccRCCs and raises the possibility of its use as a dietary adjunct to managing patients with active surveillance for small,localized,incidentally identified renal tumors so as to avoid more invasive procedures such as nephrectomy.

Shen Qi Wan attenuates renal interstitial fibrosis through upregulating AQP1

Renal interstitial fibrosis(RIF)is the crucial pathway in chronic kidney disease(CKD)leading to the end-stage renal failure.However,the underlying mechanism of Shen Qi Wan(SQW)on RIF is not fully understood.In the current study,we investigated the role of Aquaporin 1(AQP1)in SQW on tubular epithelial-to-mesenchymal transition(EMT).A RIF mouse model induced by adenine and a TGF-β1-stimulated HK-2 cell model were etablished to explore the involvement of AQP 1 in the protective effect of SQW on EMT in vitro and in vivo.Subsequently,the molecular mechanism of SQW on EMT was explored in HK-2 cells with AQP1 knockdown.The results indicated that SQW alleviated kidney injury and renal collagen deposition in the kidneys of mice induced by adenine,increased the protein expression of E-cadherin and AQP1 expression,and decreased the expression of vimentin andα-smooth muscle actin(α-SMA).Similarly,treatmement with SQW-containing serum significantly halted EMT process in TGF-β1 stimulated HK-2 cells.The expression of snail and slug was significantly upregulated in HK-2 cells after knockdown of AQP1.AQP1 knockdown also increased the mRNA expression of vimentin andα-SMA,and decreased the expression of E-cadherin.The protein expression of vimentin increased,while the expression of E-cadherin and CK-18 significantly decreased after AQP1 knockdown in HK-2 cells.These results revealed that AQP1 knockdown promoted EMT.Furthermore,AQP1 knockdown abolished the protective effect of SQW-containing serum on EMT in HK-2 cells.In sum,SQW attentuates EMT process in RIF through upregulation of the expression of AQP1.

Phenotypic plasticity in prostate cancer： role of ntrinsically disordered proteins

A striking characteristic of cancer ceLls is their remarkable phenotypic plasticity, which is the ability to switch states or phenotypes in response to environmental fluctuations. Phenotypic changes such as a partial or complete epithelial to mesenchymal transition （EMT） that play important roles in their survival and proliferation, and development of resistance to therapeutic treatments, are widely believed to arise due to somatic mutations in the genome. However, there is a growing concern that such a deterministic view is not entirely consistent with multiple lines of evidence, which indicate that stochasticity may also play an important role in driving phenotypic plasticity. Here, we discuss how stochasticity in protein interaction networks （PINs） may play a key role in determining phenotypic plasticity in prostate cancer （PCa）. Specifically, we point out that the key players driving transitions among different phenotypes （epithelial, mesenchymal, and hybrid epithelial/mesenchymal）, including ZEB1, SNAIl, OVOL1, and OVOL2, are intrinsically disordered proteins （IDPs） and discuss how plasticity at the molecular level may contribute to stochasticity in phenotypic switching by rewiring PINs. We conclude by suggesting that targeting iDPs implicated in EMT in PCa may be a new strategy to gain additional insights and develop novel treatments for this disease, which is the most common form of cancer in adult men.

Molecular characterization of circulating tumor cells in pancreatic ductal adenocarcinoma:potential diagnostic and prognostic significance in clinical practice

Background:The clinical value of heterogeneous sub-populations of circulating tumor cells(CTCs)in pancreatic ductal adenocarcinoma(PDAC)remains unclear.Methods:Peripheral blood samples were obtained from 67 PDAC patients.CTCs were isolated by employing CD45 negative enrichment technique and further characterized for epithelial to mesenchymal transition(EMT)or human equilibrative nucleoside transporter-1(hENT-1).The relationships between CTCs sub-phenotypes with clinicopathological factors or post-operative recurrence in PDAC patients were analyzed.Results:EMT related CTCs could be isolated and identified from the 81%of patients(54/67),and both the total count(median:5 vs.17/mL,P<0.0001)and M-CTC percentage(median:0.2 vs.0.345,P=0.0244)of CTCs could differentiate local/regional with metastatic disease.Multivariate analysis showed that both AJCC stage(P=0.025)and M-CTC percentage(P=0.001)were independent prognostic indicators of recurrence free survival(RFS)in resected patients.Moreover,Kaplan-Meier survival analysis showed that M-CTC after 2 courses of chemotherapy was significantly associated with inferior RFS(49.5 weeks vs.undefined,P=0.0288).No significant correlation in hENT-1 expression was found between CTCs and matched tumor tissues,and further multivariate analysis suggested hENT-1 expression in CTCs as independent prognostic factor for RFS(P=0.016).Patients with low hENT-1 expression in CTCs had decreased RFS(32 weeks vs.undefined,P=0.0337).Conclusions:CTCs could be the promising diagnostic biomarkers in PDAC patients,and phenotypic profiling of CTCs based on EMT or hENT-1 could help establish novel prognostic biomarkers in resected patients undergoing adjuvant gemcitabine-based chemotherapy.

Role of cellular reprogramming and epigenetic dysregulation in acquired chemoresistance in breast cancer

Acquired resistance to chemotherapy is a major limitation in clinical treatment for breast cancer.Accumulating evidence from in vitro,in vivo and clinical studies suggest that acquired chemoresistance is progressive,multifactorial and involve genetic and epigenetic aberrations.Among various mechanisms that contribute to chemoresistance,cellular reprogramming has extensively been implicated in breast cancer resistance lately.Cellular reprogramming events such as acquisition of epithelial to mesenchymal transition(EMT)and cancer stemness(CSCs)not only provide cancer cells with reversible phenotypic plasticity and survival advantage against cytotoxicity but also leads to aggressiveness,metastasis,clinical resistance,tumor recurrence and poor survival.The transient and reversible nature of cellular reprogramming processes and their controlled interaction with epigenetic regulatory complexes strongly support the involvement of dynamic epigenetic regulatory network in governing the cellular reprogramming and associated acquired chemoresistance.Further,epigenetic modulations are also gaining interest as promising interventions addressing the cancer cell reprogramming machinery to overcome acquired chemoresistance.This review discusses the previous reports and our recent findings that lead to current understanding of epigenetic dysregulation dictating the cellular reprogramming processes such as acquisition of EMT and CSCs phenotype and how they co-ordinate to establish acquired drug resistance in breast cancer.

Metastatic tumor cells- genotypes and phenotypes

BACKGROUND： Metastasis is the primary cause of mortality in cancer patients. Therefore, elucidating the genetics and epigenetics of metastatic tumor cells and the mechanisms by which tumor cells acquire metastatic properties constitute significant challenges in cancer research. OBJECTIVE： To summarize the current understandings of the specific genotype and phenotype of the metastatic tumor cells. METHOD and RESULT： In-depth genetic analysis of tumor cells, especially with advances in the next-generation sequencing, have revealed insights of the genotypes of metastatic tumor cells. Also, studies have shown that the cancer stem cell （CSC） and epithelial to mesenchymal transition （EMT） phenotypes are associated with the metastatic cascade. CONCLUSION： In this review, we will discuss recent advances in the field by focusing on the genomic instability and phenotypic dynamics of metastatic tumor cells.

Role of circulating tumor cells in future diagnosis and therapy of cancer

Circulating tumor cells(CTCs)have become a blistering topic of discussion for oncologists because of their tremendous potential in the diagnosis and treatment of cancer.Over the past few years,they have been doled with quite an amount of research in this area understanding that CTCs are shed from tumors and circulate in the bloodstream.This process can also occur at an early stage of cancer.The major limitation in isolation of CTCs is their availability in limited numbers.Hence,many techniques have been developed and are under continuous improvement to enhance their effi cacy of CTC isolation and enumeration.They have shown their potentiality to not just indicate the presence of a tumor but also to provide us with its core information.They have also proven to be useful in detecting minor subgroups of cells present in the primary tissue which might eventually be the cause of treatment resistance or relapse of the disease.Hence,detecting and characterizing CTCs can defi nitely become an inevitable step in treating solid tumor malignancies.In this review,we have tried to comprehend the basics of CTCs including isolation,detection,characterization,and molecular mechanism of their circulation in the blood stream.We have mostly focused on the signifi cance of CTCs in diagnosis and therapies of four most common types of cancers,namely,breast,prostate,lung,and colorectal.This review provides the coverage of most of the advancements with regards to different tumor malignancies and their probable use in predicting outcomes of the disease to realize the concept of personalized medicine.