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Pristimerin is a natural triterpenoid that has received much attention from medicinal chemists for its multiple biological activities. However, structural modifications of pristimerin, especially those aimed at discovering antitumor agents, are relatively limited. In this study, two series of pristimerin derivatives containing phenyloxazole and quinoxaline moieties, respectively, were designed via the scaffold hopping strategy. The target compounds were synthesized and analyzed for their cytotoxic activities in vitro using the MTT assay. The most potent cytotoxic compound ( 21o ) significantly inhibited the proliferation of MCF-7 cells with an IC 50 value of 2.0 μM, 1.5-fold more potent than pristimerin (IC 50 = 3.0 μM). Compared with pristimerin, compound 21o displayed the greatest improvement in selectivity (25.7-fold) against the MCF-7 and MCF-10A cell lines. Transmission electron microscopy, monodansylcadaverine and DCFH-DA staining, Western blotting, and different inhibitor assays were performed to elucidate the mechanism of action of compound 21o . Compound 21o induced autophagy-mediated cell death in MCF-7 cells by activating the ROS/JNK signaling pathway. Therefore, incorporating a quinoxaline substructure into pristimerin could be advantageous for enhancing its cytotoxic activity. Compound 21o may serve as a lead compound for developing new therapies to treat breast cancer.

Background/aim: As an antagonist of bone morphogenetic protein (BMP), Noggin facilitates osteolytic bone metastases from breast cancer. The present study aimed to further dissect its role in oestrogen receptor (ER) positive breast cancer., Materials and Methods: Noggin expression in ER positive breast cancer cell lines (MCF-7 and T-47D) was determined under conditions of oestrogen deprivation and treatment with 17-β-oestradiol (E 2 ). Activation of Smad1/5/8 in the oestrogen-regulated Noggin was examined using recombinant human BMP7 (rhBMP7) and a BMP receptor inhibitor (LDN-193189). The influence of Noggin on cellular functions was evaluated in MCF-7 and T-47D cell lines. Responses to tamoxifen and chemotherapy drugs were determined in MCF-7 and T-47D cells with Noggin over-expression using MTT assay., Results: Noggin expression was negatively correlated with ERα in breast cancers. Noggin was up-regulated upon oestrogen deprivation, an effect that was eliminated by E 2 Furthermore, increased levels of phosphorylated Smad1/5/8 were observed in the oestrogen-deprived MCF-7 and T-47D cells, which was prevented by E 2 and LDN-193189, respectively. BMP7-induced Noggin expression and activation of Smad1/5/8 was also prevented by E 2 and LDN-193189. Noggin over-expression resulted in an increase in the proliferation of both MCF-7 and T-47D cells. MCF-7 and T-47D cells over-expressing Noggin exhibited a good tolerance to tamoxifen (TAM), DTX, and 5-FU, but the percentage of viable cells was higher compared with the controls., Conclusion: Noggin expression can be repressed by oestrogen through inference with the BMP/Smad signalling. Over-expression of Noggin promotes the proliferation of MCF-7 and T-47D cells, contributing to drug resistance., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Claudin-10 (CLDN10) is a membrane integral protein. It is one of the widely expressed tight junctional claudins with functions not well defined. In the present study, the expression profile and its role in cerebral endothelial cells and in the interaction between breast cancer and endothelial cells were investigated., Materials and Methods: CLDN10 expression was examined in a wide range of cell types. Brain endothelial cell models with or without CLDN10 expression were generated using the hCMEC/D3 cell line and used to test the barrier and permeability functions. Transendothelial drug delivery and invasion were also evaluated., Results: hCMEC/D3 cells express high levels of CLDN10, compared with peripheral endothelial cells, mesothelial cells, fibroblasts, and breast cancer cells, which were either negative or expressed low levels of CLDN10. Knockdown of CLDN10 in hCMEC/D3 cells resulted in impaired tight junctions as seen by reduced transendothelial electric resistance and paracellular permeability. It also accelerated invasion of breast cancer cells through the endothelial cell layer. CLDN10 knockdown in hCMEC/D3 cells led to an increase in transendothelial chemodrug delivery. Furthermore, the SRC kinase inhibitor (AZM475271) was able to decrease the impedance and increase the paracellular permeability in cerebral endothelial cells., Conclusion: Cerebral endothelial cells express high levels of CLDN10, a protein regulating barrier function and thereby drug permeability and cancer invasiveness in brain endothelial cells, suggesting that it is a novel therapeutic target for the treatment of brain metastasis-related diseases., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Capillary morphogenesis gene 2 (CMG2) is involved in prostate and breast cancer progression. This study aimed to investigate sex hormone receptor-mediated regulation of CMG2 in breast and prostate cancer, and its implication in disease progression., Materials and Methods: Expression of CMG2, oestrogen receptor (ER) and androgen receptor (AR) was determined in breast and prostate cancer cell lines, respectively, using real-time quantitative PCR (QPCR) and western blot. Association between CMG2 and sex hormone receptors was analysed in a number of transcriptome datasets. Immunochemical staining was performed in tissue microarrays of breast cancer (BR1505D) and prostate cancer (PR8011A). CMG2 expression was determined in 17β-oestradiol treated breast cancer cells and AR over-expressing prostate cancer cells., Results: CMG2 was found to be inversely correlated with sex hormone receptors in breast and prostate cancer. Lower expression of CMG2 was associated with a poor prognosis in ER (+) breast cancer but not ER (-) tumours. Both ER (+) breast cancer cell lines and AR (+) prostate cancer cell lines presented lower expression of CMG2, which was increased following sex hormone deprivation. Exposure to 17-β-oestradiol and AR over-expression repressed CMG2 expression in breast cancer and prostate cancer cell lines, respectively., Conclusion: CMG2 is inversely correlated with ER and AR status in breast and prostate cancer, respectively. ER and AR mediate repression of CMG2 expression in corresponding cancerous cells., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The development of cancer was determined by not only the intrinsic properties of cancer cells, but also the communication between cancer cells and tumor microenvironment (TME). We applied ESTIMATE and CIBERSORT algorithms to calculate the immune/stromal component and tumor-infiltrating immune cells (TICs) in TME of BC. The results showed that immune component in TME predicted patients' survival and associated with progression of BC. Differentially expressed genes (DEGs) were primarily enriched in immune-related activities. Finally, CCL19 was acquired which shared the leading nodes in PPI network and was associated with patients' survival. High expression of CCL19 predicted better prognosis and participated in progression of BC. Genes in CCL19 up-regulated group were enriched in immune-related activities and these functions might depend on the communications between CCL19 and multiple TICs in TIME. In conclusion, CCL19 functioned as a potential prognostic biomarker and a modulator of TIME in BC through communicating with various TICs.

Phosphatidylinositol transfer protein membrane-associated 1 (PITPNM1) contains a highly conserved phosphatidylinositol transfer domain which is involved in phosphoinositide trafficking and signaling transduction under physiological conditions. However, the functional role of PITPNM1 in cancer progression remains unknown. Here, by integrating datasets of The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer (METABRIC), we found that the expression of PITPNM1 is much higher in breast cancer tissues than in normal breast tissues, and a high expression of PITPNM1 predicts a poor prognosis for breast cancer patients. Through gene set variation analysis (GSEA) and gene ontology (GO) analysis, we found PITPNM1 is mainly associated with carcinogenesis and cell-to-cell signaling ontology. Silencing of PITPNM1, in vitro, significantly abrogates proliferation and colony formation of breast cancer cells. Collectively, PITPNM1 is an important prognostic indicator and a potential therapeutic target for breast cancer.

Notch receptors (Notch1-4) play critical roles in tumorigenesis and metastasis of malignant tumors, including breast cancer. Although abnormal Notch activation is related to various tumors, the importance of single receptors and their mechanism of activation in distinct breast cancer subtypes are still unclear. Previous studies by our group demonstrated that Notch3 may inhibit the emergence and progression of breast cancer. PTEN is a potent tumor suppressor, and its loss of function is sufficient to promote the occurrence and progression of tumors. Intriguingly, numerous studies have revealed that Notch1 is involved in the regulation of PTEN through its binding to CBF-1, a Notch transcription factor, and the PTEN promoter. In this study, we found that Notch3 and PTEN levels correlated with the luminal phenotype in breast cancer cell lines. Furthermore, we demonstrated that Notch3 transactivated PTEN by binding CSL-binding elements in the PTEN promoter and, at least in part, inhibiting the PTEN downstream AKT-mTOR pathway. Notably, Notch3 knockdown downregulated PTEN and promoted cell proliferation and tumorigenesis. In contrast, overexpression of the Notch3 intracellular domain upregulated PTEN and inhibited cell proliferation and tumorigenesis in vitro and in vivo. Moreover, inhibition or overexpression of PTEN partially reversed the promotion or inhibition of cell proliferation induced by Notch3 alterations. In general, Notch3 expression positively correlated with elevated expression of PTEN, ER, lower Ki-67 index, and incidence of involved node status and predicted better recurrence-free survival in breast cancer patients. Therefore, our findings demonstrate that Notch3 inhibits breast cancer proliferation and suppresses tumorigenesis by transactivating PTEN expression.

Aurora B is a serine/threonine kinase that has been implicated in regulating cell proliferation in distinct cancers, including breast cancer. Here we show that Aurora B expression is elevated in basal-like breast cancer (BLBC) compared with other breast cancer subtypes. This high level of expression seems to correlate with poor metastasis-free survival and relapse-free survival in affected patients. Mechanistically, we show that elevated Aurora B expression in breast cancer cells activates AKT/GSK3β to stabilize Snail1 protein, a master regulator of epithelial-mesenchymal transition (EMT), leading to EMT induction in a kinase-dependent manner. Conversely, Aurora B knock down by short-hairpin RNAs (shRNAs) suppresses AKT/GSK3β/Snail1 signaling, reverses EMT and reduces breast cancer metastatic potential in vitro and in vivo. Finally, we identified a specific OCT4 phosphorylation site (T343) responsible for mediating Aurora B-induced AKT/GSK3β/Snail1 signaling and EMT that could be attenuated by Aurora B kinase inhibitor treatment. These findings support that Aurora B induces EMT to promote breast cancer metastasis via OCT4/AKT/GSK3β/Snail1 signaling. Pharmacologic Aurora B inhibition might be a potential effective treatment for breast cancer patients with metastatic disease.

Background/aim: In this study, we aimed to investigate the prognostic role of a previously identified panel of 10 stem cell markers stratified against the catalytic subunit of telomerase (hTERT) in human breast cancer., Materials and Methods: The mRNA copy numbers of these genes were determined using real time quantitative PCR in 124 breast cancer tissues and adjacent non-cancerous tissues. Relations between mRNA levels and survival were analysed using Kaplan-Meier plots and Cox regression analysis., Results: Five genes (BMI1, NES, POU5F1, ALDH1A2 and CDKN1A) correlated with survival when stratified with hTERT and predicted overall (Wilcoxon: p=0.004; Cox: p=0.006) and disease-free (Wilcoxon: p<0.000; Cox: p=0.000) survival., Conclusion: This panel of genes stratified by hTERT could open new avenues for the development of new prognostic tools, as well as for the identification of new research directions regarding breast oncogenesis., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Breast cancer (BC) is a type of malignant tumor originating from the epithelial tissue of the mammary gland, and about 20% of breast cancers are human epidermal growth factor receptor 2 positive (HER2+), which is a subtype with more aggression. Recently, HER2-positive breast cancer is often accompanied by poor prognosis of patients, and targeted therapy showed a promising prospect. To combat this disease, novel therapeutic targets are still needed. Adenylate kinase 4 (AK4) is a member of the adenylate kinase family and is expressed in the mitochondrial matrix. AK4 is involved in multiple cellular functions such as energy metabolism homeostasis. Interestingly, AK4 was observed highly expressed in several tumor tissues, and the involvement of AK4 in cancer development was generally revealed. However, the possible role of AK4 on the growth and development of breast cancer is still unclear. Here, we investigated the possible functions of AK4 on the progression of HER2-positive breast cancer. We found the high expression of AK4 in HER2-positive breast cancer tissues from patients who received surgical treatment. Additionally, AK4 expression levels were obviously correlated with clinical-pathological features, including pTNM stage ( P = 0.017) and lymph node metastasis ( P = 0.046). We mechanically confirmed that AK4 depletion showed the obvious impairment of cell proliferation and invasion in MCF7 and MDA-MB-231 cells. AK4 also facilitates tumor growth and metastasis of HER2-positive breast cancer in vivo. In conclusion, we identified and mechanically confirmed that AK4 is a novel therapeutic target of HER2-positive breast cancer., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Jie Zhang et al.)

Purpose: We evaluated the effect of younger age on recurrence risk in Chinese women diagnosed with T1N0M0 breast cancer (BC), using propensity score matching (PSM) analysis., Methods: We included 365 women who were diagnosed with T1N0M0 BC between 2003 and 2016, and who received surgery at our center. They were classified as younger (≤40 years) and older (>40 years). We used PSM to balance clinicopathologic characteristics between the two age groups. Survival was analyzed by the Kaplan-Meier method, before and after PSM., Results: Over a median follow-up period of 79 months, 54 patients developed recurrences. Before PSM, younger patients had worse recurrence-free survival (RFS) than older patients. Significantly worse RFS was seen in younger patients with HER2 + BC compared with their older counterparts. Younger patients had higher rates of locoregional recurrence rather than metastasis, especially in the first 5 years after diagnosis. After PSM, the two age groups still significantly differed in 5-year RFS., Conclusion: Among PSM pairs with T1N0M0 BC, with equal baselines and treatment conditions, we found that patients who presented at younger ages had worse outcomes, independently of other pathological features. Younger patients with BC may require more individualized therapy to improve their prognosis., (© 2019 Wiley Periodicals, Inc.)

Background: Telomerase reverse transcriptase (TERT) has a well-known role in carcinogenesis due to its functions in inducing cell immortality and preventing senescence. In this study, the relationships between TERT and a panel of known stem cell markers was examined in order to direct future enquiries into the role of 'stem-ness' in human breast cancer., Materials and Methods: Breast cancer tissues (n=124) and adjacent normal tissues (n=30) underwent reverse transcription and quantitative polymerase chain reaction. Transcript levels were analyzed for the correlation with that of TERT., Results: A significant direct correlation was found in cancerous tissue between TERT and BMI1 proto-oncogene polycomb ring finger 4 (BMI1; n=88, p<0.001), nestin (NES; n=88, p<0.001), POU domain, class 5, transcription factor 1 (POU5F1; n=88, p<0.001), aldehyde dehydrogenase 1 family member A2 (ALDH1A2; n=87, p=0.0298), cyclin-dependent kinase inhibitor 1A (CDKN1A; n=88, p<0.001), integrin subunit beta 1 (ITGNB1; n=88, p<0.001), integrin subunit alpha 6 (ITGA6; n=88, p<0.001), cluster of differentiation antigen 24 (CD24; n=88, p=0.0114), MET proto-oncogene (MET; n=78, p<0.001) and noggin (NOG; n=88, p<0.001)., Conclusion: The evidence presented in this article of possible interactions between TERT and a discrete subset of known stem cell markers would significantly contribute to further enquiries regarding clonal dynamics in the context of human breast cancer., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Immunotherapy provides the best approach to reduce the high mortality of metastatic breast cancer (BC). We demonstrate a chemo-immunotherapy approach, which utilizes a liposomal carrier to simultaneously trigger immunogenic cell death (ICD) as well as interfere in the regionally overexpressed immunosuppressive effect of indoleamine 2,3-dioxygenase (IDO-1) at the BC tumor site. The liposome was constructed by self-assembly of a phospholipid-conjugated prodrug, indoximod (IND), which inhibits the IDO-1 pathway, followed by the remote loading of the ICD-inducing chemo drug, doxorubicin (DOX). Intravenous injection of the encapsulated two-drug combination dramatically improved the pharmacokinetics and tumor drug concentrations of DOX and IND in an orthotopic 4T1 tumor model in syngeneic mice. Delivery of a threshold ICD stimulus resulted in the uptake of dying BC cells by dendritic cells, tumor antigen presentation and the activation/recruitment of naı̈ve T-cells. The subsequent activation of perforin- and IFN-γ releasing cytotoxic T-cells induced robust tumor cell killing at the primary as well as metastatic tumor sites. Immune phenotyping of the tumor tissues confirmed the recruitment of CD8 + cytotoxic T lymphocytes (CTLs), disappearance of Tregs, and an increase in CD8 + /FOXP3 + T-cell ratios. Not only does the DOX/IND-Liposome provide a synergistic antitumor response that is superior to a DOX-only liposome, but it also demonstrated that the carrier could be effectively combined with PD-1 blocking antibodies to eradicate lung metastases. All considered, an innovative nano-enabled approach has been established to allow deliberate use of ICD to switch an immune deplete to an immune replete BC microenvironment, allowing further boosting of the response by coadministered IDO inhibitors or immune checkpoint blocking antibodies.

Background: Cyclin-dependent kinase 2-associated protein 1 (CDK2AP1) interacts with CDK2AP2, modulates the actions of transforming growth factor-B1, cyclin-dependent kinase 2 and retinoblastoma protein, and closely interacts with micro-RNA21 and micro-RNA25. Our objective was to determine if CDK2AP1 mRNA expression levels were consistent with tumour-suppressive functions in breast cancer., Materials and Methods: A total of 134 samples were analysed. CDK2AP1 mRNA levels were measured using quantitative polymerase chain reaction (RT-PCR) and normalised against glyceraldehyde 3-phosphate dehydrogenase mRNA. Levels in breast cancer and adjacent non-cancerous breast tissue were analysed against pathological and clinical parameters (TNM staging, survival over a 10-year follow-up period)., Results: Normalised CDK2AP1 expression was 38-fold higher in adjacent non-cancerous breast tissue than in breast cancer. CDK2AP1 expression in disease-free patients at 10 years was more than threefold that of patients who died of breast cancer. However, neither of these differences in expression levels reached statistical significance. CDK2AP1 mRNA levels were higher in TNM1 compared to TNM3 (p=0.016) and with TNM4 (p=0.016). There were no significant associations between CDK2AP1 expression and estrogen receptor status, tumour grade and tumour type. There was no significant difference in overall survival between patients with high and those with low CDK2AP1 mRNA levels after a median follow-up of 10 years (Kaplan-Meier analysis, p=0.872)., Conclusion: To our knowledge, this is the first study in the literature to examine the mRNA expression of CDK2AP1 in human breast cancer over a long-term follow-up period. A compelling relationship exists between high CDK2AP1 mRNA expression and lower TNM classification of breast cancer, which is consistent with CDK2AP1 having a tumour-suppressive function., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Primary breast diffuse large B-cell lymphoma (PB-DLBCL) is an uncommon extranodal non-Hodgkin's lymphoma (NHL), which was traditionally treated with anthracycline-containing regimens followed by consolidative radiation therapy (RT) to add therapeutic benefits. The introduction of anti-CD20 antibody rituximab for the treatment of B-cell NHLs has significantly improved the clinical outcome of these malignant diseases. It is unclear, however, whether consolidative RT could still add therapeutic benefits for PB-DLBCL patients treated with rituximab. To answer this important question, we used the Surveillance, Epidemiology, and End Results (SEER) database to evaluate the impact of RT on the clinical outcomes of PB-DLBCL patients in the rituximab era. Information on patient age, year of diagnosis, stage, race, laterality, and RT status for PB-DLBCL patients diagnosed between 2001 and 2014 were extracted. Kaplan-Meier survival curves were plotted, and log-rank test was used to compare the potential survival difference. Multivariate analysis using Cox proportional hazards model was employed to determine the impact of RT and other factors such as age, race, tumor laterality, stage, and year of diagnosis on survival. Among the 386 patients identified, the median follow-up time was 45 months (range, 0-167 months); the median age was 64 years (range, 19-93 years); 33.9% of the patients were younger than 60 years of age; 69.9% of the patients were stage I; 79.0% were white; 51.8% received RT. The 5-year OS and cause-specific survival (CSS) for the whole cohort were 72.3% and 82.5%, respectively. The 5-year OS was significantly superior for patients who received RT compared to those who did not receive RT (78.1% vs. 66.0%, P = 0.031). In multivariable analysis, RT remained significantly associated with improved OS (P = 0.026). In summary, our study suggests that RT still adds significant therapeutic benefits for patients with PB-DLCBL in the rituximab era., (© 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Bone morphogenetic proteins (BMPs) belong to the TGF-β super family, and are essential for the regulation of foetal development, tissue differentiation and homeostasis and a multitude of cellular functions. Naturally, this has led to the exploration of aberrance in this highly regulated system as a key factor in tumourigenesis. Originally identified for their role in osteogenesis and bone turnover, attention has been turned to the potential role of BMPs in tumour metastases to, and progression within, the bone niche. This is particularly pertinent to breast cancer, which commonly metastasises to bone, and in which studies have revealed aberrations of both BMP expression and signalling, which correlate clinically with breast cancer progression. Ultimately a BMP profile could provide new prognostic disease markers. As the evidence suggests a role for BMPs in regulating breast tumour cellular function, in particular interactions with tumour stroma and the bone metastatic microenvironment, there may be novel therapeutic potential in targeting BMP signalling in breast cancer. This review provides an update on the current knowledge of BMP abnormalities and their implication in the development and progression of breast cancer, particularly in the disease-specific bone metastasis., (© 2017 The authors.)

Background/aim: Programmed death 1 (PD1) and its ligand programmed death ligand 1 (PDL1) form a pathway which when activated is thought to result in suppression of antitumor adaptive responses, influencing antitumor immunity. With potential targeted therapies emerging against PDL1, we investigated the clinical significance of mRNA expression levels of PD1 and PDL1 in our breast cancer cohort to explore its association with disease progression and prognosis. Previous studies evaluating the expression of PD1 and PDL1 (mRNA or protein) and its association with prognosis in breast cancer showed both positive and negative correlations and hence remain controversial., Materials and Methods: Quantitative polymerase chain reaction was used to determine transcript expression levels of PD1 and PDL1 in a cohort consisting of primary breast cancer tissues (n=127) and matching non-neoplastic background tissues (n=33) with available clinical and pathological information. Two-sample two-tailed t-test, Kaplan-Meier survival analysis and Wilcoxon tests were performed., Results: Significant PDL1 transcript level reductions were seen in patients who developed metastases, as well as those who had local recurrence, compared to patients who remained disease-free. Higher PDL1 transcript levels were also associated with better overall and disease-free survival. Significantly higher transcript expression levels of PD1 were found in tumor tissue, whilst a general increase in PDL1 expression was found in tumor tissues, although this did not reach statistical significance., Conclusion: Our study demonstrates higher levels of expression of PDL1 are associated with favorable clinical outcome., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Accumulating evidence has shown that hypoxia plays a key role in regulating proliferation of breast cancer cells. However, the mechanism of how hypoxia regulates breast cancer remains unclear. We sought to investigate if hypoxia regulated proliferation through circular RNA., Materials and Methods: Western blot was used to detect hypoxia-inducible factor 1 alpha (HIF1α) levels in breast cancer cells under hypoxic conditions. Candidate circular RNAs (circRNAs) were selected and quantified by quantitative real-time polymerase chain reaction (qRT-PCR) after hypoxia induction. CCK8 assay was used to investigate the changes of proliferation after interfering circDENND4C and HIF1a., Results: In breast cancer cells, circDENND4C was increased under hypoxic conditions and decreased after knocking-down HIF1α. In addition, knocking-down circDENND4C inhibited proliferation of breast cancer cells in a hypoxic environment. Finally, tumors with a large size had higher circDENND4C expression levels than those of small size., Conclusion: CircDENND4C is a HIF1α-associated circRNA promoting the proliferation of breast cancer cells under hypoxia., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Constitutive activation of Sonic hedgehog (SHH) has been observed in different types of cancers. In the present study, expressional profiling of SHH in a breast cancer cohort (n=150) of a Pakistani population and its association with different molecular subtypes have been explored., Materials and Methods: qRT-PCR and IHC were performed for expression analysis of SHH and its association with ER, PR, HER2 and Ki-67 were also statistically analyzed., Results: A significant over-expression of SHH was observed in tumor tissues in comparison to their respective controls (p<0.0001). A strong positive correlation was seen between SHH and proliferation marker (r=0.635, p=0). SHH expression was significantly high among patients with advanced tumor grade, stage, nodal involvement and metastasis. Furthermore, both luminal-B and triple-negative subtypes of cohort showed increased expression of SHH., Conclusion: Based on these findings, SHH may be used as a potential biomarker for breast carcinogenesis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: In the current study, the role of plexin B1 in breast cancer metastasis was explored., Materials and Methods: Freshly-excised tumours along with background tissues of affected patients (n=121) were collected from Pakistani hospitals and processed for RNA isolation and cDNA synthesis. Using quantitative polymerase chain reaction, expression of plexin B1 was evaluated and correlated with clinicopathological parameters. Furthermore, involvement of plexin B1 in metastasis was explored by generating gene knockdown in MDA-MB-231 and MCF-7 breast cancer cells., Results: Poorly-differentiated tumours showed low plexin B1 expression in comparison to well-differentiated ones. Similarly, reduced plexin B1 expression correlated positively with advanced tumour stage and metastasis. Loss of plexin B1 significantly reduced cell adhesion in comparison with respective control cell lines (p<0.05). Knockdown of plexin B1 in MDA-MB-231 cells led to a remarkable increase in cell motility in contrast to the respective control., Conclusion: Loss of plexin B1 expression might play a pivotal role in enhancing the metastatic potential of breast cancer cells., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Activin and its antagonist follistatin (FST) have been implicated in several solid tumours. This study investigated the role of FST in breast cancer., Materials and Methods: FST expression was examined using reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (qPCR) and immunohistochemistry in a cohort of breast cancer samples. Expression was correlated to pathological and prognostic parameters in our patient cohort. FST was overexpressed in MCF-7 cells and assays for growth and invasion were performed., Results: FST is expressed in breast tissue, in the cytoplasm of mammary epithelial cells. Expression was decreased in breast cancer tissue in comparison to normal mammary tissue. Over-expression of FST in vitro led to significantly increased growth rate and reduced invasion. Higher FST associates with lower-grade tumours and better survival., Conclusion: Our results suggest a role for FST as a suppressor of invasion and metastasis in breast cancer., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: MicroRNA-7 (miR-7) has been observed as a potent tumour suppressor in multiple cancer types including breast cancer. The aim of this study was to investigate the response sensitivities of metastatic breast cancer cells to miR-7 and the roles of miR-7 in the interaction of endothelial cells and metastatic cancer cells., Methods: Expression profile of miRNAs in a breast cancer specimen cohort and breast cancer cells were determined using real-time quantitative miRNA assays. Effect of the altering expression of miR-7 on migration, invasion, proliferation, interaction and underlying molecular mechanism of breast cancer cells and endothelial cells was investigated after treatment with the synthesised mimic of miR-7. Luciferase activity analysis was performed to validate Wave-3 as a novel target of miR-7., Results: miR-7 expression was negatively correlated with the stage, grade and survival of the breast cancer patients. There was also differential expression of miRNAs including miR-7 in the breast cancer cells. The synthesised mimic of miR-7 inhibits the motility and wound healing potential of breast cancer cells. The highly metastatic MDA-MB-231 cells are more sensitive to the miR-7 treatment than the poorly invasive MCF-7 cells. Treatment with miR-7 downregulated the expression of EGFR, IGF1R and Wave3 in MDA-MB-231 cells but not in MCF-7 cells. In addition, we further demonstrated that miR-7 inhibited the proliferation, migration and invasion of endothelial cells. And more importantly, miR-7 suppressed the homing and migration of endothelial cells to more aggressive tumour cell conditions., Conclusions: Given the dual inhibitory effect of miR-7 on metastatic breast cancer cells alone and the interaction of endothelial cells with the tumour-conditioned microenvironment, we suggest miR-7 may be a new therapeutic candidate for its capacity not only to prevent breast cancer cell spreading but also to inhibit tumour-associated angiogenesis in the metastatic breast cancer.

Phospholipase C (PLC) regulates a number of cellular behaviours including cell motility, cell transformation, differentiation and cell growth. PLC plays a regulatory role in cancer cells partly by acting as signalling intermediates for cytokines such as EGF and interleukins. The current study examined the expression of the PLC isozymes in human breast cancer and corresponding clinical relevance. Transcript levels of human PLC-α, -β1, -δ, -ε, and -γ1 in human breast cancer tissues were quantitatively determined by real-time PCR. Immunochemical staining was performed for PLC-δ. The clinical relevance was analysed with clinic pathological information. Mammary tissues widely expressed PLC-α, -β1, -δ, -ε, and -γ1. Significantly high levels of PLC -β1 and -ε were seen in breast cancer tissues in comparison with normal mammary gland tissues. PLC-γ1 however, showed marginally low levels in tumour tissues. No significant difference was seen in the expression of the PLC isozymes in tumours with lymph node metastases. Moderately and poorly differentiated breast tumours (grade 2 and grade 3) had significantly higher levels of PLC-γ1, compared with well differentiated tumours. High levels of PLC-δ were significantly correlated with a shorter disease-free survival. The altered expression of other isozymes had no correlation with the survival. It is concluded that mammary tissues differentially expressed PLC isozymes. These isozymes have certain implications in the disease development and progression, with PLC-δ showing a significant correlation with shorter disease-free survival.

Background: Susceptible barcode recognition plays an important role in the diagnosis and treatment of complex diseases. Numerous approaches have been proposed to identify risky barcodes involved in the progress of complex diseases. However, some methods only consider differences in barcode frequencies between the control and disease groups; as such, these methods may be partial or even wrong. For example, some barcodes with a high risk ratio yield a low frequency on cases or exhibit a high frequency on controls, which may unreasonable from a statistical point., Results: In our study, a stricter criteria, maximum discrepancy and maximum constituency, is designed to evaluate each barcode and ant colony algorithm is used to search combination space of epistasis. For complex diseases with multi-subtypes, our method can list several potential barcodes contributing to different subtypes of complex diseases. Another contribution of this work is to introduce a method for determining the length of barcodes and excluding noisy barcodes whose frequencies are abnormal. In addition, common pathogenic genes shared by different risky barcodes are also recognized, which may provide key clue for further study, such as gene function analysis., Conclusions: Experimental results reveal that our method can find multiple risky barcodes whose risk ratio and odds ratio are >1. These barcodes could be related to different subtypes of complex diseases.

Bone metastases associated with breast cancer remain a clinical challenge due to their associated morbidity, limited therapeutic intervention and lack of prognostic markers. With a continually evolving understanding of bone biology and its dynamic microenvironment, many potential new targets have been proposed. In this chapter, we discuss the roles of well-established bone markers and how their targeting, in addition to tumour-targeted therapies, might help in the prevention and treatment of bone metastases. There are a vast number of bone markers, of which one of the best-known families is the bone morphogenetic proteins (BMPs). This chapter focuses on their role in breast cancer-associated bone metastases, associated signalling pathways and the possibilities for potential therapeutic intervention. In addition, this chapter provides an update on the role receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) play on breast cancer development and their subsequent influence during the homing and establishment of breast cancer-associated bone metastases. Beyond the well-established bone molecules, this chapter also explores the role of other potential factors such as activated leukocyte cell adhesion molecule (ALCAM) and its potential impact on breast cancer cells' affinity for the bone environment, which implies that ALCAM could be a promising therapeutic target.

Na + /H + exchanger regulatory factor 1 (NHERF1) has been reported to interact with post-synaptic density protein/Drosophila disc large tumour suppressor/zonula occludens 1 protein (PDZ) binding proteins by its two PDZ domains. These associations have effects on cellular signal transductions. NHERF1 has also been indicated as a cancer-related gene in several solid tumour types. We identified a novel mutation (A190D), of the PDZ2 domain of NHERF1 in breast cancer tissues. NHERF1 A190D mutation abolished NHERF1 modulation of proliferation and migration. In this study, we found that NHERF1 A190D mutation increased nuclear localisation of the protein compared to wild-type NHERF1. It has been reported that YES-associated protein (YAP) interacts with NHERF1. Here we found that NHERF1 A190D mutation increased the binding affinity between NHERF1 and YAP, which inhibited the phosphorylation of YAP. These data suggest that wild-type NHERF1 acts as a tumour suppressor, while NHERF1 A190D mutation abolishes the tumour-suppressive effect in cancer cells, due to A190D mutation-mediated nuclear NHERF1 translocation and induction of YAP phosphorylation., (Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Human CUB and Sushi multiple domains 1 (CSMD1) is a membrane-bound complement inhibitor suggested to act as a putative tumor suppressor gene, since allelic loss of this region encompassing 8p23 including CSMD1 characterizes various malignancies. Here, we assessed the role of CSMD1 as a tumor suppressor gene in the development of breast cancer in vitro and in vivo. We found that human breast tumor tissues expressed CSMD1 at lower levels compared to that in normal mammary tissues. The decreased expression of CSMD1 was linked to a shorter overall survival of breast cancer patients. We also revealed that expression of CSMD1 in human breast cancer cells BT-20 and MDA-MB-231 significantly inhibited their malignant phenotypes, including migration, adhesion and invasion. Conversely, stable silencing of CSMD1 expression in T47D cells enhanced cancer cell migratory, adherent and clonogenic abilities. Moreover, expression of CSMD1 in the highly invasive MDA-MB-231 cells diminished their signaling potential as well as their stem cell-like properties as assessed by measurement of aldehyde dehydrogenase activity. In a xenograft model, expression of CSMD1 blocked the ability of cancer cells to metastasize to secondary sites in vivo, likely via inhibiting local invasion but not the extravasation into distant tissues. Taken together, these findings demonstrate the role of CSMD1 as a tumor suppressor gene in breast cancer.

Epirubicin is widely used for the therapy of various breast cancers. However, it has serious adverse side effects, particularly cardiotoxicity, which can cause irreversible damage in patients. Paeonol, an active component from Moutan Cortex, enhances antitumor activity of antineoplastics and reduces toxicities induced by chemotherapeutics. In this study, we investigated the anticancer activity of Paeonol in combination with Epirubicin against breast cancer and the alleviated effect of Paeonol on cardiotoxicity induced by Epirubicin. The apoptosis results and the coefficient of drug interaction values suggested significantly synergistic in combination of Paeonol and Epirubicin to 4T1 and MCF-7 cells. We further examined antitumor activities of Paeonol or/and Epirubicin in vivo in BALB/c mice and found that co-treatment of Paeonol and Epirubicin had a synergistic inhibitory effect on tumor growth and enhanced apoptosis in tumors in vivo compared with Epirubicin alone. Increased apoptosis was associated with the activation of apoptosis-related proteins including PARP, Bax, caspase 3, and inhibition of p38/JNK/ERK MAPKs. Moreover, Paeonol exhibited a mitigative effect on Epirubicin-induced cardiotoxicity through suppressing NF-kB pathway. In conclusion, Paeonol (a) enhanced the antitumor activity of Epirubicin in a synergistic manner against breast cancer cells via inhibiting p38/JNK/ERK MAPKs and (b) alleviated Epirubicin-induced cardiotoxicity by suppressing NF-kB pathway. These findings suggest that combination of Paeonol and Epirubicin is potentially applicable for breast cancer treatment.

G protein-coupled estrogen receptor (GPER) plays an important role in mediating the effects of estradiol. High levels of GPER have been implicated to associate with the malignant progress of invasive breast cancer (IBC). However, the mechanisms by which GPER protein levels were regulated remain unclear. In this study, PDZ protein Na+/H+ exchanger regulatory factor (NHERF1) was found to interact with GPER in breast cancer cells. This interaction was mediated by the PDZ2 domain of NHERF1 and the carboxyl terminal PDZ binding motif of GPER. NHERF1 was demonstrated to facilitate GPER expression at post-transcriptional level and improve GPER protein stability by inhibiting the receptor degradation via ubiquitin-proteasome pathway in a GPER/NHERF1 interaction-dependent manner. In addition, GPER protein levels are positively associated with NHERF1 protein levels in a panel of estrogen receptor (ER)-positive breast cancer cells. Furthermore, analysis of clinical IBC data from The Cancer Genome Atlas (TCGA) showed no significant difference in GPER mRNA levels between ER-positive IBC and normal breast tissues. However, gene set enrichment analysis (GSEA) showed that GPER signaling is ultra-activated in ER-positive IBC when compared with normal and its activation is positively associated with NHERF1 mRNA levels. Taken together, our findings identify NHERF1 as a new binding partner for GPER and its overexpression promotes protein stability and activation of GPER in ER-positive IBC. Our data indicate that regulation of GPER stability by NHERF1 may contribute to GPER-mediated carcinogenesis in ER-positive IBC.

The oncogenic role of ectopic expression of Na+/H+ exchanger regulatory factor 1 (NHERF1) was recently suggested. Here, we show that NHERF1 was upregulated in high grades compared with low grades. Increased NHERF1 expression was correlated with poor prognosis and poor survival. NHERF1 expression was higher in the nucleus of cancer cells than in contiguous non- mammary epithelial cells. A novel mutation, namely NHERF1 Y24S, was identified in human breast cancer tissues and shown to correspond to a conserved residue in the PDZ-I domain of NHERF1. Truncation and mutation of the PDZ-I domain of NHERF1 increased the nuclear distribution of the NHERF1 protein, and this redistribution was associated with the malignant phenotype of breast cancer cells, including growth, migration, and adhesion. The present results suggest a role for NHERF1 in the progression of breast cancer mediated by the nuclear distribution of the NHERF1 protein, as determined by the truncation or key site mutation of the PDZ-I domain., Competing Interests: The Authors do not have any conflicts of interest.

Background: Fibroblast activation protein-alpha (FAPα) is a type-II integral membrane serine protease and is expressed in most stromal fibroblasts. Recent studies showed that FAPα is also expressed in certain cancer cells but its role is still uncertain., Materials and Methods: We analyzed the non-enzymatic activity of FAPα in breast cancer cells by introducing an enzymatic mutant FAPα (FAPS624A), in which the serine catalytic triad was destroyed. FAPα overexpression and knockdown cells were generated as controls., Results: Cellular growth and motility were markedly increased in MCF-7 cells overexpressing FAPα and enzymatic-mutant FAPS624A. This is consistent with observations in FAPα-silenced BT549 cells. Western blotting showed activation of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and matrix metalloproteinase (MMP) 2/9 in both wild-type FAPα-overexpressing and enzymatic-mutant FAPS624A-overexpressing cells., Conclusion: Promotion of cellular growth and motility is independent of the enzymatic activity of FAPα in breast cancer cells. The PI3K/AKT and MMP2/9 signaling pathways might be involved in such regulation., (Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.)

Purpose: The role of postmastectomy radiotherapy (PMRT) in clinically node-positive, stage II-III breast cancer patients with pathological negative nodes (ypN0) after neoadjuvant chemotherapy (NAC) remains controversial., Methods: A total of 1560 clinically node-positive, stage II-III breast cancer patients treated with NAC and mastectomy who achieved ypN0 between 1998 and 2009 in the National Cancer Database were analyzed. The effects of PMRT on overall survival (OS) for the entire cohort and multiple subgroups were evaluated. Imputation and propensity score matching were used as sensitivity analyses to minimize biases., Results: Of the entire 1560 eligible patients, 903 (57.9%) received PMRT and 657 (42.1%) didn't. At a median follow-up of 56.0 months, no statistical difference was observed for OS between two groups by univariate and multivariate analyses (P = 0.120; HR 1.571, 95% CI 0.839-2.943). On subgroup analyses, PMRT significantly improved OS in patients with clinical stage IIIB/IIIC disease, T3/T4 tumor, or residual invasive breast cancer after NAC (P < 0.05). This improvement in OS remained significant after sensitivity analyses for the propensity score-matched patients., Conclusions: This study demonstrated that PMRT showed a heterogeneous effect in clinically node-positive, stage II-III breast cancer patients with ypN0 following NAC. PMRT improved OS for patients with clinical stage IIIB/IIIC disease, T3/T4 tumor, or residual invasive breast tumor after NAC. In the absence of definitive conclusions from prospective studies, including the ongoing NSABP B-51 trial, our findings may help identify specific groups of women with clinically node-positive, stage II-III breast cancers who could benefit from PMRT after NAC., Competing Interests: The authors have declared no conflicts of interest.

Background/aim: Mouse double minute 2 (MDM2) and prostate-specific membrane antigen (PSMA) are currently under investigation as individual therapeutic targets due to their overexpression in many cancer types, as well as their pro-tumorigenic effect on cells. Recently, knockdown of PSMA was linked to a decrease in MDM2 and matrix metalloproteinase 2 (MMP2) and an increase in MMP3 and MMP13 expression. We aimed to assess the link between PSMA, MDM2 and the MMPs in metastatic breast cancer cell lines., Materials and Methods: Real-time quantitative polymerase chain reaction (PCR) and western blotting were used to assess siRNA-mediated knockdown of MDM2 and PSMA in MDA-MB-231 and ZR-75.1 breast cancer cells. Assays to assess the growth, adhesion, migration and invasion of the cells following siRNA treatment were undertaken. MMP and tissue inhibitor of matrix metalloproteinases (TIMP) levels were assessed via quantitative PCR., Results: Knockdown of MDM2 resulted in a decrease in PSMA expression levels and vice versa; although this trend was not replicated at the protein level. Knockdown of each of the molecules resulted in a decrease in growth, adhesion, migration and invasive ability of breast cancer cells. Both knockdowns led to a decrease in MMP2 and an increase in MMP3, -10 and -13 gene expression., Conclusion: MDM2 and PSMA may co-regulate the expression of certain MMPs and, thus, the functionality of cells in metastatic breast cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Aggressive surgical removal of the primary tumour is the preferred treatment, but with tumour progression, some tumours cannot be completely removed surgically. Anaesthetics are administered to facilitate surgery. However, anaesthetics act as a potential factor in tumour recurrence or metastasis., Materials and Methods: Normal breast cells and cancer breast cells were treated with different doses of muscle-relaxant anaesthetics. The effects on breast cancer cell invasion, adhesion and migration of these anaesthetics were then investigated using in vitro models., Results: With increasing dose of rocuronium bromide and suxamethonium chloride CRS, the number of MCF-10A and MCF-7 cells, but not that of MDA-MB-231 cells, decreased. There was almost no difference in the number of cells when the three cell lines were treated with different doses of vecuronium bromide. The study also demonstrated that rocuronium bromide promoted the invasion, adhesion and growth of MDA-231 cells, while suxamethonium chloride CRS had no effect. Interestingly, vecuronium bromide did not affect the motility and invasion of breast cancer cells significantly., Conclusion: An understanding of the effect of anaesthetics and their impact on tumour metastasis is important, thus using an appropriate aesthetic strategy could improve long-term survival in some patients., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: A Disintegrin and Metalloprotease Domain 29 (ADAM29) is involved in many important physiological processes. Recent studies have demonstrated that ADAM29 is a susceptibility locus showing traits as a risk factor for breast cancer under genome-wide significance, however, the clinical relevance and cellular function of ADAM29 in breast cancer have not been reported., Materials and Methods: In this study, we assessed the expression levels of ADAM29 in a cohort of human breast cancer specimens. We also used MDA-MB-231 cells with differing ADAM29 expression and assessed the influence of ADAM29 and its mutations on the MDA-MB-231 cell line., Results: Increased transcript expression of ADAM29 was observed in breast cancer tissues compared to normal ones. The expression of ADAM29 and its mutations in different domains significantly influenced proliferation, migration and invasion of breast cancer cells in vitro., Conclusion: ADAM29 may represent a prognostic factor in human breast cancer, as well as a novel molecular candidate to be used as a therapeutic target., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background/aim: The ribosomal S6 protein kinase (RSK) family is an important effector of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) that could influence tumour metastasis by phosphorylating proteins in both the nuclear and cytoplasmic compartments. Aberrant expression of RSK is evident in certain malignancies but the role played by RSK in breast cancer is still not clear. This study aimed to examine the expression of RSK in human breast cancer specimens and its role to breast cancer metastasis., Materials and Methods: The expression of RSK1 to -3 were separately examined in human breast cancer tissues (normal, n=33; cancer, n=112) using quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemistry. Migration and adhesion of breast cancer cells treated with the RSK inhibitor SL0101 were investigated by electric cell impedance sensing (ECIS). The effect on growth and invasion of RSK1-3 was then investigated using in vitro models., Results: The clinical data and immunohistochemistry revealed that expression of RSK1 and RSK3 were less in tumour tissues than normal. mRNA expression of RSK2 was negatively correlated with grade, TNM staging, and survival rate. SL0101 inhibited adhesion of the MCF-7 and MDA-231 breast cancer cell lines. SL0101 suppressed MDA-231 invasion and the alternate RSK inhibitor BRD7389 inhibited the invasion of MCF-7 and MDA-231 cells., Conclusion: RSK1 and 3 but not RSK2 are down-regulated in breast tumour and are associated with disease progression. RSK may be a key component in the progression and metastasis of breast cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: The mitochondrial protein translocase of inner mitochondrial membrane 17 homolog A (TIMM17A) has been identified as a biomarker of breast cancer. The present study aimed to investigate the biological role of TIMM17A in human breast cancer cells., Materials and Methods: Anti-TIMM17A transgenes were stably transfected into MDA MB-231 and MCF-7 breast cancer cell lines. The impact of TIMM17A knock-down on cell migration and invasion were evaluated using the respective cell models., Results: Reducing the expression of TIMM17A in breast cancer cells resulted in reduction of cell migration using electric cell-substrate impedance sensing. It was also found that reduction of TIMM17A expression resulted in reduction of cell invasion compared to vector control., Conclusion: TIMM17A has a profound impact on the cellular function of breast cancer cells. A decrease of TIMM17A expression is associated with the reduction of the aggressiveness of breast cancer cells. TIMM17A, therefore, has potential in prognosis and treatment of breast cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Osteoprotegrin (OPG), receptor activator of nuclear factor κB (RANK) and RANK ligand (RANKL) are signal transducers which have pleiotropic actions. Each tumour necrosis factor receptor superfamily member has unique structural attributes which directly couples them to signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically linked OPG, RANK and RANKL to increasing tumour burden, metastatic bone involvement and estrogen status. This study aimed to establish the potential implications of targeting endogenously produced OPG and RANK in the osteotropic breast cancer cell line MDA-MB‑231 in vitro. Subsequently this study also aimed to explore the potential links between these molecules with regards to hepatocyte growth factor (HGF) signalling and extracted bone proteins (BME). OPG and RANK expression was successfully suppressed using hammerhead ribozyme technology. Subsequently effects were explored in MDA-MB‑231 cell proliferation, matrix adhesion, migration and invasion in vitro function assays. Reduced OPG expression resulted in increased breast cancer cell migration and invasion. These increases, particularly invasion, appeared to however be reduced under the influence of the exogenous stimuli (HGF and BME). In contrast, suppression of RANK in MDA-MB‑231 breast cancer cells resulted in decreased cancer cell proliferation, matrix-adhesion, motility and invasion with little cumulative effect being noted after the addition of exogenous stimuli. The complexity of the bone environment underpins the vast number of soluble factors and signalling pathways which can influence osteotropic cancer behaviour and progression. Further work into elucidating all the pathways affected could potentially lead to better identification of those patients most at risk.

Background: The aim of the current study was to examine the role of semaphorin 3C (SEMA3C) in breast cancer., Materials and Methods: SEMA3C transcripts expressed by breast tissues were determined using real-time polymerase chain reaction (PCR). Knock-down of SEMA3C was performed in MDA-MB-231 and MCF-7 breast cancer cell lines using anti-SEMA3C hammerhead ribozyme transgenes. The effect of SEMA3C knockdown on cancer cells was determined using in vitro cellular function assays., Results: Higher SEMA3C transcript levels were significantly associated with poor differentiation of cancer cells, and transcript levels were significantly reduced in oestrogen receptor-positive tumours. Knock-down of SEMA3C expression resulted in a decrease in cell proliferation, adhesion and invasion of breast cancer cells., Conclusion: Higher SEMA3C expression is correlated with tumour differentiation. Inhibition of SEMA3C reduces adhesion and invasion of breast cancer cells. This suggests that SEMA3C may play a significant role in morphological changes of cancer cells, leading to enhanced growth and dissemination., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Most cases of prostate and breast cancer metastasis occur to the bone, and are responsible for the majority of cancer-related deaths. Osteocytes constitute over 90% of adult bone cells. They orchestrate bone remodelling through determining osteoclast activity and affecting osteoblasts. The osteocyte lacuno-canalicular network is also intimately associated with the blood vessel network in the bone matrix. However, the roles of osteocytes in cancer cell invasion and metastasis remain unknown., Materials and Methods: In this study, we investigated the effects of early osteocytes on the behaviour of breast and prostate cancer cells. The proliferation of cultured cells was assessed using the AlamarBlue assay. The electric cell-substrate impedance sensing (ECIS) system was used to measure spreading, attachment and migratory behaviour of cancer cells in response to conditioned medium (CM) from mouse osteocytes. Other cell assays, including in vitro wound healing and transwell migration/invasion assays, were also applied to evaluate the effect of osteocytes on cancer cells., Results: We found that CM from osteocytes from both monolayer and three-dimensional (3D) cultures, stimulated proliferation of DU145 and PC3 prostate cancer cells but not LNCaP cells compared to control medium. Osteocyte CM also stimulated proliferation of MDA-MB-231 and MCF-7 breast cancer cells. However, osteocyte CM promoted the migration and adhesion of PC3 and DU145 in prostate cancer cells but had the reverse effect on PZHPV7, a normal prostate epithelial cell line. In the breast cancer cells studied, osteocyte CM inhibited post-wound migration of MCF-7 and ZR-75.1 cells but not MDA-MB-231 cells. Moreover, osteocyte CM stimulated transwell chemotactic migration of MDA-MB-231 cells but not of MCF-7 and ZR-75.1 cells., Conclusion: Osteocytes play diverse roles in the proliferative and migratory potential of breast and prostate cancer cells that may be associated with cancer-specific bone metastasis and requires further investigation., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) has been reported to interact with many cancer-related proteins. We recently identified a novel NHERF1 mutation (E43G) in breast tumours., Materials and Methods: The candidates of NHERF1 mutation were identified in breast cancer tissues by polymerase chain reaction and DNA sequencing. Wild-type NHERF1 and E43G mutation were expressed in NHERF1-knockdown cells (MCF7ΔNHERF1) and low-NHERF1-expressing cells (SKMES-1). The effects of mutated NHERF1 on cell functions were examined using in vitro methods. Glutathione S-transferase pull-down assays and western blotting were performed to study the effects of NHERF1 mutation on its interaction with cancer-related proteins., Results: Compared to wild-type NHERF1, expression of the mutated NHERF1 failed to suppress malignant traits in cancer cells, attenuated interaction of NHERF1 protein with epidermal growth factor receptor (EGFR), and inactivated its inhibition of EGF-induced Akt and extracellular regulated protein kinases (ERK) activation., Conclusion: The results show the causal role of NHERF1 in the regulation of the EGFR pathway and the progression of breast cancer., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Prostate apoptosis response-4 (PAR4) plays an important role in apoptosis and survival of cancer cells. The current study aimed to further elucidate its role in breast cancer., Materials and Methods: PAR4 expression in human breast cancer tissue was examined using quantitative polymerase chain reaction (qPCR) and immunohistochemical staining (IHC). Plasmids containing full-length human PAR4 coding sequence were used to overexpress PAR4 in breast cancer cells and the effect on cellular functions was examined using both in vitro functional assays and an in vivo murine model., Results: Patients with low PAR4 transcript levels had poorer overall survival. PAR4 expression may be associated with differential expression of oestrogen receptors α and β in the tumours. Overexpression of PAR4 in MDA-MB-231 cells resulted in reduced cell growth and invasion, and also reduced in vivo tumour growth., Conclusion: Decreased PAR4 expression in breast cancer is associated with shorter survival. PAR4 suppresses growth and invasiveness of breast cancer cells., (Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Objectives. Osteopontin (OPN) is overexpressed in breast cancers, while its clinical and prognostic significance remained unclear. This study aimed to assess the prognostic value of OPN, especially its splice variants, in breast cancers. Methods. Data were extracted from eligible studies concerning the OPN and OPN-c expression in breast cancer patients and were used to calculate the association between OPN/OPN-c and survival. Two reviewer teams independently screened the literatures according to the inclusion and exclusion criteria based on quality evaluation. Following the processes of data extraction, assessment, and transformation, meta-analysis was carried out via RevMan 5.3 software. Results. A total of ten studies involving 1,567 patients were included. The results demonstrated that high level OPN indicated a poor outcome in the OS (HR = 2.22, 95% CI: 1.23-4.00, and P = 0.008; random-effects model) with heterogeneity (I (2) = 62%) of breast cancer patients. High level OPN-c appeared to be more significantly associated with poor survival (HR = 2.14, 95% CI: 1.51-3.04, and P < 0.0001; fixed-effects model) with undetected heterogeneity (I (2) = 0%). Conclusions. Our analyses indicated that both OPN and OPN-c could be considered as prognostic markers for breast cancers. The high level of OPN-c was suggested to be more reliably associated with poor survival in breast cancer patients.

Recently, research has shed new light on the role of Prohibitin (PHB) in cancer pathogenesis across an array of cancer types. Important mechanisms for PHB have been unveiled in several cancers, especially with regard to the androgen independent state of prostate cancer (PC) and oestrogen dependent breast cancer. However, PHB is often overlooked due to its complex but subtle roles within the cell. Having gathered both historical and current research exploring PHB's role in different cancer types including prostate and breast, here we aim to pair this information with its molecular properties in the hope of translating this information into a clinical perspective, thus discussing its possible use in future cancer therapy., (Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Treatment of malignant disease is of paramount importance in modern medicine. In 2012, it was estimated that 162,000 people died from cancer in the UK which illustrates a fundamental problem. Traditional treatments for cancer have various drawbacks, and this creates a considerable need for specific, molecular targets to overcome cancer spread. Epithelial protein lost in neoplasm (EPLIN) is an actin-associated molecule which has been implicated in the development and progression of various cancers including breast, prostate, oesophageal and lung where EPLIN expression is frequently lost as the cancer progresses. EPLIN is important in the regulation of actin dynamics and has multiple associations at epithelial cells junctions. Thus, EPLIN loss in cancer may have significant effects on cancer cell migration and invasion, increasing metastatic potential. Overexpression of EPLIN has proved to be an effective tool for manipulating cancerous traits such as reducing cell growth and cell motility and rendering cells less invasive illustrating the therapeutic potential of EPLIN. Here, we review the current state of knowledge of EPLIN, highlighting EPLIN involvement in regulating cytoskeletal dynamics, signalling pathways and implications in cancer and metastasis.

Background: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown., Methods: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4., Results: Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients., Conclusion: Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

Interleukin 21 (IL21) is a cytokine produced predominantly by cluster of differentiation 4 (CD4+) T-cells and natural killer T-cells. There exists evidence that IL21 is implicated in various immunological processes through its specific receptor (IL21R). However, the participation of IL21 in the pathogenesis of solid tumors is not fully conclusive. In the present study, we demonstrated that there was differential expression of IL21R in breast cancer cells using reverse transcription-polymerase chain reaction (RT-PCR), western blotting and sequence analysis. The expression of IL21R was stronger in MDA-231 cells, weaker in MCF7 but negative in ZR-75.1 cells. The invasion and migratory capacity of IL21R+ MDA-231 cells was enhanced by IL21 in a dose-dependent manner. After IL21R was knocked-down by siRNA gene silencing, the response of MDA-231 to treatment with IL21 was attenuated. We found that siRNA silencing of IL21R also spontaneously suppressed cell proliferation. However, IL21 had no additional effect on the proliferation of MDA-231 cells. We also found that IL21R was involved in signaling pathways of matrix metalloproteinases (MMPs), that are crucial for spreading and migration of metastatic MDA231 cells. In conclusion, we unveiled the roles of IL21R in breast cancer cells, which enhances our knowledge on immunological regulation of cancer cells through the axis of IL21 and its receptor., (Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.)

Involvement of semaphorin 4D (Sema4D) and the receptor proteins of the plexins B family (plexin-B1, -B2 and -B3) in solid tumours suggests they play a role in breast cancer. In the present study, the expression of Sema4D and plexin-Bs was examined in a breast cancer cohort. The expression of Sema4D and plexin-Bs was examined in 147 tumours together with 22 normal mammary tissues using quantitative PCR along with clinicopathological patient data, as well as in MCF-7 and MDA-MB-231 cell lines treated with selective oestrogen receptor modulators (SERMs). The expression of Sema4D, plexin-B1 and -B2 was markedly reduced in tumours with local recurrence, compared to the patients that remained disease-free. The reduced Sema4D expression was associated with poorer disease-free survival (median, 111.6 months, 95% CI, 96.5-126.7), compared to the patients with a higher expression (median, 144.0 months; 95% CI, 130.8-157.3; p=0.033). A reduced expression of plexin-B1 was observed in tumours with poorer differentiation and was associated with poorer overall and disease-free survival. No similar association was identified in relation to plexin-B2 and -B3. A higher expression of Sema4D and plexin-B1 was observed in the ERα-positive tumours compared to the ERα-negative tumours. The expression of these molecules was largely regulated in breast cancer cells exposed to SERMs. A decreased expression of Sema4D, plexin-B1 and -B2 was associated with local recurrence and poor prognosis. Response to SERMs indicated potential perspectives of these molecules in clinical assessment and management of diseases.

The death-associated protein 3 (DAP3) is a highly conserved phosphoprotein involved in the regulation of autophagy. A previous clinical study by our group suggested an association between low DAP3 expression and clinicopathological parameters of human breast cancer. In the present study, we intended to determine the role of DAP3 in cancer cell behaviour in the context of human breast cancer. We developed knockdown sub-lines of MCF7 and MDA-MB-231, and performed growth, adhesion, invasion assays and electric cell-substrate impedance sensing (ECIS) studies of post-wound migration of the cells. In addition, we studied the mRNA expression of caspase 8 and 9, death ligand signal enhancer (DELE), IFN-β promoter stimulator 1 (IPS1), cyclin D1 and p21 in the control and knockdown sub-lines. The knockdown sub-lines of MCF7 and MDA-MB-231 had significantly increased adhesion and decreased growth when compared to the controls. Furthermore, invasion and migration were significantly increased in the MDA-MB-231DAP3kd cells vs. the controls. The expression of caspase 9 and IPS1, known components of the apoptosis pathway, were significantly reduced in the MCF7DAP3kd cells (p=0.05 and p=0.003, respectively). We conclude that DAP3 silencing contributes to breast carcinogenesis by increasing cell adhesion, migration and invasion. It is possible that this may be due to the activity of focal adhesion kinase further downstream of the anoikis pathway. Further research in this direction would be beneficial in increasing our understanding of the mechanisms underlying human breast cancer.

Estrogen receptor (ER)-positive breast cancer patients treated with tamoxifen are known to have an elevated risk of subsequent endometrial cancer. However, it is unclear if ER-negative patients also have a higher risk of endometrial cancer. This population-based study aims to evaluate whether breast cancer patients with distinctive ER and PR status possess differential risks in developing delayed endometrial malignancy. Data were obtained from the Surveillance, Epidemiology, and End Results program (1992-2009). Standardized incidence ratio (SIR) was calculated as the observed cases of endometrial cancers among breast cancer survivors compared with the expected cases in the general population. Data were stratified by latency periods, race, age, and calendar year of breast cancer diagnosis. We identified 2044 patients who developed a second primary endometrial cancer among 289,933 breast cancer survivors. The overall SIRs for subsequent endometrial cancers were increased in all of the four subtypes (ER+PR+, ER+PR-, ER-PR+, and ER-PR-) of breast cancer. SIR was increased for all latency periods except for the initial 6-11 months after breast cancer diagnosis. Stratifying by age of diagnosis, elevated SIRs in all ER/PR groups were statistically significant among patients diagnosed with breast cancer after the age of 40. Demographically, non-Hispanic whites had increased SIRs in all subtypes of breast cancer, while Hispanic whites had no statistically elevated SIRs. Here we showed that patients with invasive breast cancer have a higher risk of developing subsequent endometrial cancer regardless of ER or PR status. The increased risk among hormone receptor-negative breast cancer survivors raises concerns whether common etiological factors among these breast cancer subtypes increase the susceptibility to develop endometrial cancer. Lower threshold for routine endometrial cancer surveillance may be warranted.