Clinical significance of XRCC1 gene expression in human breast cancer and its cell and molecular mechanisms北大核心CSCD

Objective: To investigate the expression of X-ray repair cross complementing 1 (XRCC1) in human breast cancer and its relationship with the clinical characteristics, and to analyze the effects of XRCC1 over-expression on the proliferation and migration of breast cancer MB-231 cells and the molecular mechanism. Methods: The expression level of XRCC1 mRNA in breast cancer cell lines and human breast cancer tissues was detected by real-time fluorescent quantitative PCR. The expression of XRCC1 protein in human breast cancer tissues was detected by immunohistochemistry. The relationship between the expression of XRCC1 protein and the clinicopathological characteristics of breast cancer patients was analyzed. The pcDNA3.1(+)-Flag-XRCC1 plasmids were transfected into breast cancer MB-231 cells for the overexpression of XRCC 1 gene. Then the proliferation activity was detected by CCK-8 and soft agar plate clone formation assay. The cell cycle and apoptosis were detected by FCM method. The cell migration and invasion were detected by Transwell chamber assay. The expressions of cell cycle-, apoptosis- and migration-related proteins were detected by Western blotting. Results: The expression level of XRCC1 mRNA was significantly decreased in most breast cancer cell lines (all P < 0.001). As compared with the normal mammary epithelium and the paired adjacent breast tissues, the expression levels of XRCC1 mRNA and protein were downregulated in human breast cancer tissues (all P < 0.001). The expression level of XRCC1 mRNA was positively correlated with the prognosis of breast cancer patients (γ 2 =0.052, P =0.046), and XRCC1 protein expression was correlated with tumor diameter, lymph node metastasis, histological grade and TNM stage (all P < 0.05). After the overexpression of XRCC 1 gene, the proliferation, colony formation, invasion and migration of breast cancer MB-231 cells were significantly inhibited (all P < 0.01), the cell cycle was significantly blocked in G1 phase (P < 0.001), and the apoptosis rate was significantly increased (P < 0.001). Furthermore, the expressions of p21, p27, Bax, cleaved caspase-3 and E-cadherin were significantly upregulated (all P < 0.001), while the expressions of cyclin-dependent kinase 4/6 (CDK4/6), cyclin D1, Bcl-2, N-cadherin and vimentin were down-regulated (all P < 0.001) in MB-231 cells with XRCC1 overexpression. Conclusion: XRCC1 expression is down-regulated in breast cancer cell lines and tissues, and its expression level is positively correlated with the prognosis of breast cancer patients. Restoring XRCC 1 gene expression can inhibit cell growth, migration and invasion, and can induce apoptosis. So XRCC1 may be a potential tumor suppressor regulating the occurrence and development of human breast cancer. © 2019 by TUMOR.     

Overexpression of β1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases

Introduction  

Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the α4 chain from predominantly laminin-9 (α4β2γ1) to predominantly laminin-8 (α4β1γ1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast.     

Down‐regulation of gelsolin expression in human breast ductal carcinoma in situ with and without invasion

Expression of gelsolin, an actin filament regulatory protein, in human breast ductal carcinoma in situ (DCIS) was analyzed by immunohistochemistry using a monoclonal antibody. Formalinfixed paraffinembedded tissues from 59 pure DCIS specimens and 33 DCIS specimens with associated invasive components were evaluated for gelsolin reactivity and compared to eight normal breast cases and 76 invasive breast cancers. The proportion of cases exhibiting negative/low expression of gelsolin in the epithelium was as follows – normal, 0%; pure DCIS, 56%; DCIS associated with invasion, 58% in the DCIS component and 66% in the invasive component; invasive carcinoma, 70%. These data demonstrate that downregulation of gelsolin expression in breast epithelium frequently parallels progression to malignancy. Testing gelsolin expression (normal vs. negative/low levels) in the DCIS lesions for associations with patient age or any of the following histopathologic parameters revealed no significant (95% probability level) correlations – tumor size; pathologic (Van Nuys system) grade; nuclear grade; necrosis; presence of histologic calcifications; presence or type of adjacent benign lesions; architectural histologic pattern; and mammographic extent. Gelsolin loss was more commonly associated with mammographic soft tissue lesions as compared to calcified lesions (P = 0.009). A positive trend of borderline significance (P = 0.06) found in the DCIS with invasion group was a correlation between downregulated gelsolin expression in the DCIS component and size (< versus 15mm) of the invasive tumor. In conclusion, reduced gelsolin protein is detectable in at least half of breast lesions which have progressed to DCIS. The trend between increasing gelsolin loss and malignant progression from normal epithelium to DCIS to invasive breast cancer (P < 0.0001) suggests additional investigation is needed to determine the potential of altered gelsolin expression as a marker for prognosis and for therapeutic interventions in breast cancer.     

Enzyme histochemical pattern in human tumours—II. Oxidoreductases in carcinoma of the colon and the breast

The activity of a number of oxidoreductases in carcinomas of the breast, colon and rectum was studied enzyme histochemically and compared with the activity in adjacent normal epithelium. In both mammary and colonic carcinoma, the activity of the NADPH-tetrazolium reductase system was found to be elevated. In colonic and rectal carcinoma there seems to be a strong tendency of the catabolic enzymes (NADH-tetrazolium reductase, succinic acid dehydrogenase, and α-glycerophosphate oxidase) towards decreased activity. Breast cancers, however, have a tendency toward an elevated catabolism.     

Detection of human epidermal growth factor receptor 2 protein and gene in fine needle aspiration cytology specimens and tissue sections from invasive breast cancer: can cytology specimens take the place of tissue sections?

Overexpression of HER2 protein and HER2 gene amplification in breast cancer are prognostic factors for the response to specific medical treatments such as trastuzumab, endocrine therapy, and chemotherapy. Whereas HER2 expression and gene amplification are generally examined in tissue sections, we investigated whether specimens from fine needle aspiration cytology (FNAC) are adequate for these analyses. HER2 protein overexpression and HER2 gene amplification were assessed in both FNAC specimens and tissue sections from 58 cases of invasive breast cancer. Immunohistochemistry assay for HER2 protein expression was performed according to the HercepTest protocol, and HER2 gene amplification was examined with the Spot-light CISH (chromogenic in situ hybridization) Detection kit. There was a significant positive correlation between assessments of HER2 protein status in the cytology specimens and tissue sections. The sensitivity, specificity, and accuracy of HER2 gene amplification detection in cytology specimens in relation to those in tissue sections were 84.0% (21/25 cases), 87.9% (29/33 cases), and 86.2% (50/58 cases), respectively. FNAC specimens are suitable for detection of HER2 overexpression and HER2 gene amplification in invasive breast cancer.     

Wild-type genotypes of BRCA1 gene SNPs combined with micro-RNA over-expression in mammary tissue leading to familial breast cancer with an increased risk of distant metastases’ occurrence

The objectives of this study were to identify specific long noncoding RNAs (lncRNAs) in laryngeal squamous cell carcinoma (LSCC) and to clarify the function of cisplatin and paclitaxel on the confirmed laryngeal cancer lncRNAs. Fifty-four pairs of laryngeal tumor and adjacent normal tissue were collected. Candidate lncRNAs were searched in authorized databases. The significant lncRNAs were identified and confirmed through high-output real-time PCR. Chemotherapy assay evaluated the influences of cisplatin and paclitaxel on the significant lncRNAs. Thirty-seven cancer-related candidate lncRNAs were selected. Three up-expressed and two down-expressed significant lncRNAs were identified and confirmed. The expressions of lncRNA CDKN2B-AS1, HOTAIR and MALAT1 were dramatically reduced with the increasing concentration of cisplatin and paclitaxel and also lengthening of the treatment duration. Cisplatin and paclitaxel have target function on significant lncRNAs in LSCC, which presents novel molecular targets to cure LSCC patients and also leads an orientation for developing new drugs.     

Recovery of anoikis in Src-transformed cells and human breast carcinoma cells by restoration of the SIRP α1/SHP-2 signaling system

Src kinase dysregulation contributes to cancer progression but mechanistic understanding for this contribution remains incomplete. Signal regulatory protein α1 (SIRPα1) is a tumor suppressor that is constitutively suppressed in v-Src-transformed cells, where restoration of SIRPα1 expression inhibits anchorage-independent growth. In this study, we investigated the role of the protein tyrosine phosphatase-2 (SHP-2) in SIRPα1 activity. SHP-2 suppression resulted in a blockade of SIRPα1-mediated inhibition of anchorage-independent growth. Notably, we found that SIRPα1 did not act in v-Src-transformed cells by triggering cell growth arrest but by eliciting a suspension-selective apoptosis (anoikis), and that SHP-2 was required for this effect. Furthermore, we found that SHP-2 was crucial for recovery of stress fiber and focal contact formation by SIRPα1 in v-Src-transformed cells. Finally, we found that SIRPα1/SHP-2 signaling regulates anoikis in human breast carcinoma cells with activated c-Src. Taken together, our findings define SHP-2 as an essential component of tumor suppression and anoikis mediated by SIRPα1 in human breast carcinoma cells as well as in v-Src-transformed cells.     

Detection of HER-2/neu gene amplification in breast carcinomas using quantitative real-time PCR — A comparison with immunohistochemical and FISH results

The aim of our study was to evaluate the value of quantitative real-time-PCR (qPCR) in the determination of HER-2/neu amplification status of human breast carcinomas by comparing qPCR, FISH and immunohistochemistry results from the same samples. A total of 210 breast carcinomas were examined. Ready-to-use CB11 antibody was applied to detect HER-2/neu oncoprotein expression. In 76 out of 210 cases FISH was performed, and 162 cases were investigated with qPCR. Seventy-five tumors were 2+ or 3+ positive with immunohistochemistry, while 135 samples were either completely negative or 1+. In 45 cases results from all three methods were available. Out of these, in twenty negative and sixteen positive cases both FISH and qPCR led to similar results. The mean qPCR amplification ratio in the concordant positive cases was 5.424 while in the qPCR+/FISH- group the mean ratio was 2.765. Out of 121 samples with scores of 0 or 1+ immunohistochemical result, analyzed also with qPCR, 26 showed HER-2/neu gene amplification. In these cases the mean amplification ratio was 2.53. Comparison of FISH and qPCR together with immunohistochemistry shows that qPCR is more sensitive to detect HER-2/neu gene amplification in tumors scored as 2+ with immunohistochemistry, but the diagnostic cut-off ratio should be defined above 2.7 to avoid high number of false positive cases. Amongst the immunohistochemistry score 2+ cases, 10 of 18 showed gene amplification by qPCR while 10 of 26 by FISH. In conclusion, a well calibrated HER-2/neu qPCR assay may serve as useful alternative to FISH in breast cancer patients.     

DNA polymerase β deficiency is linked to aggressive breast cancer: A comprehensive analysis of gene copy number,mRNA and protein expression in multiple cohorts

Short arm of chromosome 8 is a hot spot for chromosomal breaks, losses and amplifications in breast cancer. Although such genetic changes may have phenotypic consequences, the identity of candidate gene(s) remains to be clearly defined. Pol β gene is localized to chromosome 8p12‐p11 and encodes a key DNA base excision repair protein. Pol β may be a tumour suppressor and involved in breast cancer pathogenesis. We conducted the first and the largest study to comprehensively evaluate pol β in breast cancer. We investigated pol β gene copy number changes in two cohorts (n = 128 & n = 1952), pol β mRNA expression in two cohorts (n = 249 & n = 1952) and pol β protein expression in two cohorts (n = 1406 & n = 252). Artificial neural network analysis for pol β interacting genes was performed in 249 tumours. For mechanistic insights, pol β gene copy number changes, mRNA and protein levels were investigated together in 128 tumours and validated in 1952 tumours. Low pol β mRNA expression as well as low pol β protein expression was associated high grade, lymph node positivity, pleomorphism, triple negative, basal‐like phenotypes and poor survival (ps < 0.001). In oestrogen receptor (ER) positive sub‐group that received tamoxifen, low pol β protein remains associated with aggressive phenotype and poor survival (ps < 0.001). Artificial neural network analysis revealed ER as a top pol β interacting gene. Mechanistically, there was strong positive correlation between pol β gene copy number changes and pol β mRNA expression (p < 0.0000001) and between pol β mRNA and pol β protein expression (p < 0.0000001). This is the first study to provide evidence that pol β deficiency is linked to aggressive breast cancer and may have prognostic and predictive significance in patients.     

Lack of CD151/integrin α3β1 complex is predictive of poor outcome in node-negative lobular breast carcinoma: opposing roles of CD151 in invasive lobular and ductal breast cancers

Background:

The proposed involvement of CD151 in breast cancer (BCa) progression is based on findings from studies in invasive ductal carcinoma (IDC). The IDC and invasive lobular carcinoma (ILC) represent distinct disease entities. Here we evaluated clinical significance of CD151 alone and in association with integrin α3β1 in patients with ILC in context of the data of our recent IDC study.

Methods:

Expression of CD151 and/or integrin α3β1 was evaluated in ILC samples (N=117) using immunohistochemistry. The findings were analysed in relation to our results from an IDC cohort (N=182) demonstrating a prognostic value of an expression of CD151/integrin α3β1 complex in patients with HER2-negative tumours.

Results:

Unlike in the IDCs, neither CD151 nor CD151/α3β1 complex showed any correlation with any of the ILC characteristics. Lack of both CD151 and α3β1 was significantly correlated with poor survival (P=0.034) in lymph node-negative ILC N(−) cases. The CD151⁻/α3β1⁻ patients had 3.12-fold higher risk of death from BCa in comparison with the rest of the ILC N(−) patients.

Conclusions:

Biological role of CD151/α3β1 varies between ILC and IDC. Assessment of CD151/α3β1 might help to identify ILC N(−) patients with increased risk of distant metastases.     

Mutation and protein expression analysis of CYP1A1 gene—a study on female breast cancer cases from India

Increased risk may be associated with exposure to genotoxic agents during breast development because the undifferentiated ductal elements of breast are more susceptible to the action of genotoxic early in life and thus an impairment in Cytochrome P 4501A1 (CYP1A1) may contribute to the development of breast cancer. Therefore, we carried out the population-based study in a total of 105 Indian female breast cancer cases with equal normal adjacent controls. A total of 20 samples (20/105, 19.04 %) showed final mutations in the exon 7 of the CYP1A1 gene where 5 cases harbored frame shift mutation (deletion of G nucleotide), and the remaining were missense mutation observed in 15 cases of breast cancer with significant association to histological grade (chi square ?7.20, p?=?0.02), tumor stage (chi square ?6.36, p?=?0.01), menopausal stage (chi square ?9.76, p?=?0.001), and ER status (chi square ?4.22, p?=?0.03). We further did protein expression analysis of CYP1A1 through immunohistochemistry where 66 cases showed down or no expression (+) (66/105, 62.85 %), 28 cases with moderate expression (++) (28/105, 26.66 %), and 11 cases with high expression (+++) (11/105, 10.47 %). Highly significant associations were observed between protein expression and clinico-pathological variables like Her 2 category (chi square =?31.73, p?<?0.0001) and tumor stage (chi square =?10.27, p?=?0.005). Importantly, mutation(s) of the type like deletion of A nucleotide and missense mutation (Gly > Val) exclusively showed low (+) or no expression for the CYP1A1 protein when studied in relation to each other. In summary, CYP1A1 may be associated with breast cancer and its down regulation may serve as an important tool in the field of biomarker study.     

Cryopreservation and autotransplantation of human ovarian tissue prior to cytotoxic therapy – A technique in its infancy but already successful in fertility preservation

Increasing survival rates in young cancer patients, new reproductive techniques and the growing interest in quality of life after gonadotoxic cancer therapies have placed fertility preservation as an important issue to oncologists, fertility specialists and patients.Several techniques are now available for fertility preservation in these patients. A new promising method is cryopreservation and transplantation of ovarian cortex. Ovarian tissue can be extracted by laparoscopy without any significant delay of gonadotoxic therapy. The tissue can be cryopreserved by specialised centres of reproductive medicine and transplanted in case the women experience premature ovarian failure (POF).This review summarises the European expertise on cryopreservation and transplantation of ovarian tissue, following around 30 reported transplantations globally, resulting in six live births and several ongoing pregnancies. It emphasises that fertility preservation by the cryopreservation of ovarian tissue is a new but already a successful clinical option, which can be considered for selected cancer patients.     

Tumor cell adhesion to frozen lymph node sections — a correlate of lymphatic metastasis in breast carcinoma models of human and rat origin

Summary The role of tumor cell adhesion in lymphatic metastasis of breast cancer was investigatedin vitro using a rat mammary carcinoma model of four cell lines with different metastatic phenotypes, two human breast cancer cell lines, and cryostast sections of normal rat or human lymph nodes, respectively. A positive correlation was found between the adhesion levels obtained with three metastatic rat mammary cell lines (TMT-081 > MT-100M & TMT-50) and a non-metastatic line MT-W9B, the latter being 3–4 fold less adhesive to the lymph node sections than the metastatic tumors. This selective adhesion was specific, as it was not found with cryostat sections of rat liver and brain. Enzyme assays indicated that cell surface glycoproteins bearing terminal -galactoside residues were involved in the adhesion of the rat tumors.Adhesion of the human breast carcinoma cells Hs578T to sections of human lymph nodes was significantly higher than that of the normal breast epithelial cell line Hs578Bst, and comparable to adhesion of a second breast carcinoma line, MCF-7. Moreover, Hs578T cells isolated from regional lymph nodes of tumor-bearing nude mice were significantly more adhesive to human lymph node sections than the parental line.Adhesion of both human and rat tumors could be partially blocked by the addition of the synthetic peptide GRGDSPK and by antibodies directed to the 1 chain of integrin, suggesting that an integrin receptor may played a role in the adhesion. The results suggest that tumor cell adhesion to cryostat sections of lymph nodes is a correlate of the malignant phenotype in mammary tumors of diverse origins, and could be used to delineate the adhesion factors mediating lymphatic metastasis.     

A novel small‐molecule activator of procaspase‐3 induces apoptosis in cancer cells and reduces tumor growth in human breast,liver and gallbladder cancer xenografts

Purpose

Procaspase‐3, a proenzyme of apoptotic executioner caspase‐3, is overexpressed in numerous tumors. We aimed to characterize a novel procaspase‐3 activator, WF‐210, which may have potential as an anticancer drug.

Experimental design

The procaspase‐3 activating ability, antitumor efficacy, mechanisms of action, and toxicity profiles of WF‐210 were investigated in vitro and in vivo, using normal cells, cancer cells, and mouse xenograft models. The role of procaspase‐3 in WF‐210‐induced apoptosis was explored by manipulating procaspase‐3 expression in cultured cells.

Results

WF‐210 activated procaspase‐3 with an EC50 of 0.95 μM, less than half that of its mother compound PAC‐1 (2.08 μM). The mechanism involved the chelation of inhibitory zinc ions, subsequently resulting in an auto‐activation of procaspase‐3. WF‐210 was more cytotoxic than PAC‐1 to human cancer cells, but less cytotoxic to normal cells. Cancer cells with high procaspase‐3 expression, like HL‐60 and U‐937, were particularly sensitive. WF‐210‐induced the apoptosis of HL‐60 and U‐937 cells by activating procaspases and promoting proteasome‐dependent degradation of XIAP and Survivin. The level of WF‐210‐induced apoptosis in cultured cells was related to the level of procaspase‐3 expression. Finally, WF‐210 was superior to PAC‐1 in retarding the in vivo growth of breast, liver and gallbladder xenograft tumors which overexpress procaspase‐3, and induced no substantial weight loss or neurotoxicity. WF‐210 and PAC‐1 had no effect on the growth of MCF‐7 xenograft tumors, which do not express procaspase‐3.

Conclusion

We identified WF‐210 as a potent small‐molecule activator of procaspase‐3. The favorable antitumor activity and acceptable toxicity profile of WF‐210 provide a strong rationale for its clinical evaluation in the treatment of tumors with high procaspase‐3 expression.