Proteins In Breast Cancer Cells Research Articles

A circular RNA circ-DNMT1 enhances breast cancer progression by activating autophagy.

Circular RNAs are a large group of noncoding RNAs that are widely expressed in mammalian cells. Genome-wide analyses have revealed abundant and evolutionarily conserved circular RNAs across species, which suggest specific physiological roles of these species. Using a microarray approach, we detected increased expression of a circular RNA circ-Dnmt1 in eight breast cancer cell lines and in patients with breast carcinoma. Silencing circ-Dnmt1 inhibited cell proliferation and survival. Ectopic circ-Dnmt1 increased the proliferative and survival capacities of breast cancer cells by stimulating cellular autophagy. We found that circ-Dnmt1-mediated autophagy was essential in inhibiting cellular senescence and increasing tumor xenograft growth. We further found that ectopically expressed circ-Dnmt1 could interact with both p53 and AUF1, promoting the nuclear translocation of both proteins. Nuclear translocation of p53 induced cellular autophagy while AUF1 nuclear translocation reduced Dnmt1 mRNA instability, resulting in increased Dnmt1 translation. From here, functional Dnmt1 could then translocate into the nucleus, inhibiting p53 transcription. Computational algorithms revealed that both p53 and AUF1 could bind to different regions of circ-Dnmt1 RNA. Our results showed that the highly expressed circular RNA circ-Dnmt1 could bind to and regulate oncogenic proteins in breast cancer cells. Thus circ-Dnmt1 appears to be an oncogenic circular RNA with potential for further preclinical research.

PO-234 Transwell co-culture with human breast adipocytes alters the proteome expression profiles of MCF-7 and MDA-MB-231 human breast cancer cells

IntroductionBreast tumours grow within adipose tissue, and stromal adipocytes are likely the first cell type encountered by breast cancer cells as they metastasize. Adipocytes at the invasive edge of human breast tumours exhibit a modified phenotype, and consequently these adipocytes have been named Cancer Associated Adipocytes (CAA). Previous in vitro research has shown that co-culture with CAA promotes breast cancer cell resistance to radiation and some therapeutic agents, as well as breast cancer cell invasiveness both in vitro and in vivo. The aim of the current research was to identify cellular proteins that are differentially regulated in breast cancer cells co-cultured with CAA.Material and methodsThis study collected human breast adipose tissue samples, from which pre-adipocytes were isolated, differentiated into mature adipocytes, and co-cultured with hormone receptor positive (MCF-7) and negative (MDA-MB-231) human breast cancer cells for 3 days in a transwell co-culture system. The proteomes of co-cultured and control breast cancer cells were compared quantitatively using iTRAQ labelling and LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed by Western blotting.Results and discussionsThis study identified and quantified a total of 1126 and 1218 proteins expressed in MCF-7 and MDA-MB-231 cells, respectively. Of these, 85 in MCF-7 and 63 in MDA-MB-231 had a fold change >1.5 between co-culture and control breast cancer cell samples. Overall, MCF-7 cells had more proteins downregulated (n=53) than upregulated (n=32) after co-culture, whereas, more proteins were upregulated (n=51) than downregulated (n=12) in MDA-MB-231 cells. Enrichment analysis revealed an upregulation of proteins involved in metabolic pathways, namely TCA cycle proteins in MCF-7 cells and glycolysis proteins in MDA-MB-231 cells. The metabolic enzyme PGK1 was upregulated in response to co-culture with CAA in both cell lines, and is associated with poorer overall survival and resistance to the chemotherapy agent paclitaxel in breast cancer. Western blotting results validated mass spectrometry ratios for three candidate proteins.ConclusionOverall, this study describes for the first time the local effect CAA have on the expression of proteins in breast cancer cells in vitro, and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA.

Abstract 2360: Deregulated APA and cancer specific APA isoforms

Abstract Certain aspects of diagnosis, prognosis, and treatment of cancer patients are still important challenges to be addressed. We developed a pipeline to uncover patterns of alternative polyadenylation (APA), a hidden complexity in cancer transcriptomes, to further accelerate efforts to discover novel cancer genes and pathways. Here, we found a significant shift in usage of poly(A) signals in six common tumor types compared to normal tissues. We further defined specific subsets of APA events to efficiently classify cancer types/subtypes. Triple negative breast cancers, for example, have specific 3'UTR length alterations where the significant majority are shortening events (70%, 113 of 165) of mostly proliferation-related transcripts compared with normal breast tissue. Such shortening events correlate with increased protein levels and relapse free survival of patients, suggesting functional significance of isoform variability. In line with this isoform diversity, we also detected deregulated expression of mRNA polyadenylation complex proteins in breast cancer cells. Of note, APA proteins are responsive to proliferative signals including estrogen and epidermal growth factor, suggesting a potential explanation to 3'-end isoform diversity in cancer cells. Overall, our study offers a computational and experimental approach for use of APA in novel gene discovery and classification in common tumor types, with important implications in basic research, biomarker discovery, and precision medicine approaches. Citation Format: Oguzhan Begik, Melda Ercan, Harun Cingoz, Tolga Can, Merve Oyken, Ayse Elif Erson-Bensan. Deregulated APA and cancer specific APA isoforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2360.

Elucidation of Therapeutic Peptide Binding Partners from Isolated Mitochondria.

CT20p is a protein derived from the C-terminus of Bax. It has selective cytotoxicity for cancer cells, such as the sensitive triple-negative MDA-MB-231 breast adenocarcinoma cells, but not normal cells like the resistant MCF-10A epithelial breast cells. To understand the reason for the peptide’s selective toxicity, a "pull-down" experiment with biotinylated CT20p (biotin-CT20p) and whole-cell protein lysates from breast cancer and normal cells were performed. These studies revealed that CT20p binds to a cytosolic protein called chaperonin-containing TCP-1 (CCT), a molecular chaperone that folds actin and tubulin. However, this method could not detect possible rare interactions made by CT20p with mitochondrial proteins. To determine whether CT20p is associated with mitochondrial proteins as part of the mechanism by which it induces cell death, mitochondrial protein lysates from MDA-MB-231 and MCF-10A cells were isolated and a streptavidin-agarose pulldown procedure using biotin-CT20p was performed. Protein interactions were visualized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining. The results of the experimental procedure showed that biotin-CT20p did not "pull down" any observable mitochondrial proteins from the sensitive MDA-MB-231 cells, indicating that the peptide may not interact with mitochondrial proteins in breast cancer cells. Rather, the interactions observed with biotin-CT20p were with mitochondrial proteins derived from resistant MCF-10A cells, indicating that these interactions were not driving the cancer-selective cell death process. The absence of CT20p-associated proteins from the mitochondrial lysates of MDA-MB-231 breast cancer cells supports the hypothesis that CT20p, unlike the parent protein, Bax, exerts its cytotoxic effects via a cytosolic protein.

Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

BackgroundAs crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer.MethodsWe performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR.ResultsWe determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential.ConclusionsThese results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.

MiR-29c plays a suppressive role in breast cancer by targeting the TIMP3/STAT1/FOXO1 pathway

BackgroundmiR-29c has been associated with the progression of many cancers. However, the function and mechanism of miR-29c have not been well investigated in breast cancers.MethodsReal-time quantitative PCR was used to assess expression of miR-29c and DNMT3B mRNA. Western blot and immunochemistry were used to examine the expression of DNA methyltransferase 3B (DNMT3B) protein in breast cancer cells and tissues. The functional roles of miR-29c in breast cancer cells such as proliferation, migration, invasion, colony formation, and 3D growth were evaluated using MTT, transwell chambers, soft agar, and 3D Matrigel culture, respectively. In addition, the luciferase reporter assay was used to check if miR-29c binds the 3′UTR of DNMT3B. The effects of miR-29c on the DNMT3B/TIMP3/STAT1/FOXO1 pathway were also examined using Western blot and methyl-specific qPCR. The specific inhibitor of STAT1, fludarabine, was used to further check the mechanism of miR-29c function in breast cancer cells. Studies on cell functions were carried out in DNMT3B siRNA cell lines.ResultsThe expression of miR-29c was decreased with the progression of breast cancers and was closely associated with an overall survival rate of patients. Overexpression of miR-29c inhibited the proliferation, migration, invasion, colony formation, and growth in 3D Matrigel while knockdown of miR-29c promoted these processes in breast cancer cells. In addition, miR-29c was found to bind 3′UTR of DNMT3B and inhibits the expression of DNMT3B, which was elevated in breast cancers. Moreover, the protein level of TIMP3 was reduced whereas methylation of TIMP3 was increased in miR-29c knockdown cells compared to control. On the contrary, the protein level of TIMP3 was increased whereas methylation of TIMP3 was reduced in miR-29c-overexpressing cells compared to control. Knockdown of DNMT3B reduced the proliferation, migration, and invasion of breast cancer cell lines. Finally, our results showed that miR-29c exerted its function in breast cancers by regulating the TIMP3/STAT1/FOXO1 pathway.ConclusionThe results suggest that miR-29c plays a significant role in suppressing the progression of breast cancers and that miR-29c may be used as a biomarker of breast cancers.

EFEMP2 Mediates GALNT14-Dependent Breast Cancer Cell Invasion

N-Acetylgalactosaminyltransferase-14 (GALNT14) is a member of acetylgalactosaminyltransferases family. We have shown that GALNT14 could promote breast cancer cell invasion. However, the underlying molecular mechanism is unclear. Here, using yeast two hybrid, we find that EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) interacts with GALNT14. Both in vitro and in vivo binding assays show that EFEMP2 is associated with GALNT14. Moreover, we find that GALNT14 mediates glycosylation of EFEMP2. EFEMP2 significantly increased the invasion ability of breast cancer cells including MCF-7 and MBA-MD-231 cells, and this phenomenon is suppressed by knockdown expression of GALNT14. In addition, the GALNT14-dependent O-glycosylation of EFEMP-2 regulates the stability of EFEMP-2 protein in breast cancer cells. Taken together, our results demonstrate a novel molecular mechanism underlying breast cancer invasion.

A Mucin1 C-terminal Subunit-directed Monoclonal Antibody Targets Overexpressed Mucin1 in Breast Cancer.

Background: Mucin1 (MUC1) is a highly glycosylated transmembrane protein that has gained attention because of its overexpression in various cancers. However, MUC1-targeted therapeutic antibodies have not yet been approved for cancer therapy. MUC1 is cleaved to two subunits, MUC1-N and MCU1-C. MUC1-N is released from the cell surface, making MUC1-C a more reasonable target for cancer therapy. Therefore, we produced a monoclonal antibody (anti-hMUC1) specific to the extracellular region of MUC1-C and evaluated its effects in vitro and in vivo.Methods: We produced a monoclonal antibody (anti-hMUC1) using a purified recombinant human MUC1 polypeptide and our novel immunization protocol. The reactivity of anti-hMUC1 was characterized by ELISA, western blotting and immunoprecipitation analyses. The localization of the antibody in the breast cancer cells after binding was determined by confocal image analysis. The effects of the antibody on the growth of cells were also investigated. We injected anti-hMUC1 and performed in vivo tracing analysis in xenograft mouse models. In addition, expression of MUC1 in tissue sections from patients with breast cancer was assessed by immunohistochemistry with anti-hMUC1.Results: The anti-hMUC1 antibody recognized recombinant MUC1 as well as native MUC1-C protein in breast cancer cells. Anti-hMUC1 binds to the membrane surface of cells that express MUC1 and is internalized in some cancer cell lines. Treatment with anti-hMUC1 significantly reduced proliferation of cells in which anti-hMUC1 antibody is internalized. Furthermore, the anti-hMUC1 antibody was specifically localized in the MUC1-expressing breast cancer cell-derived tumors in xenograft mouse models. Based on immunohistochemistry analysis, we detected significantly higher expression of MUC1 in cancer tissues than in normal control tissues. Conclusion: Our results reveal that the anti-hMUC1 antibody targets the extracellular region of MUC1-C subunit and may have utility in future applications as an anti-breast cancer agent.

A hederagenin saponin isolated from Clematis ganpiniana induces apoptosis in breast cancer cells via the mitochondrial pathway.

Natural plants offer a treasure trove of resources for anticancer drug development. Clematis are widely used in Traditional Chinese Medicine. However, studies on the active substances in Clematis are scarce. In the present study, four monomer compounds were successfully extracted from this species and their inhibitory effects on the growth of breast cancer cells were investigated using bioactivity tests. Among them, Clematis hederagenin saponin (CHS) belongs to the class of triterpenoid saponins. CHS showed cytotoxic effects on breast cancer cells in a dose- and time-dependent manner. The compound also induced apoptosis in breast cancer cells in a time-dependent manner. Further investigation into the underlying mechanisms of apoptosis induction in breast cancer cells showed that the compound significantly reduced mitochondrial Apaf-1 and cytochrome c proteins in breast cancer cells. In addition, it upregulated the activities of caspase-3 and −9. In conclusion, CHS induced apoptosis in breast cancer cells through regulation of the mitochondrial apoptosis pathway. The results suggest that the hederagenin saponin extracted from Clematis ganpiniana offers great potential as a novel anti-breast cancer drug.

TRAIL DR5-CTSB crosstalk participates in breast cancer autophagy initiated by SAHA

To investigate the ability of SAHA-induced TRAIL DR5-CTSB crosstalk to initiate the breast cancer autophagy, RTCA assay was performed to assess the effect of SAHA on breast cancer cells, and western blot and ELISA were used to verify the inductive effects on expression of CTSB. Breast cancer cells were transfected with TRAIL DR5 siRNA to block the function of TRAIL DR5. Cell viability and apoptosis of breast cancer cells were analyzed using a muse cell analyzer. The distribution of LC3-II in TRAIL DR5-silenced breast cancer cells treated with SAHA was observed by immunofluorescence microscopy, the mRNA levels of autophagy-related genes were detected by RNA microarray, and the activity of autophagy-related signaling pathways was screened by MAPK antibody array. Results indicated that SAHA did indeed repress the growth of breast cancer cell lines with inducing CTSB expression. Western blot and ELISA results indicated that TRAIL DR5 was involved in the expression of CTSB in SAHA-induced breast cancer cells. Cell viability and apoptosis assays showed that the inactivation of TRAIL DR5 can significantly inhibit the effects of SAHA. An immunofluorescence assay indicated that, with SAHA treatment, MDA-MB-231 and MCF-7 cells underwent apparent morphological changes. While SAHA was added in the TRAIL-DR5 blocked cells, the distribution of LC3-II signal was dispersed, the intensity of fluorescence signal was weaker than that of SAHA alone. RNA array indicated that SAHA significantly increased mRNA expression of autophagy marker LC3A/B whereas the change was significantly reversed in TRAIL DR5-silenced cells. The results of MAPK antibody array showed that SAHA and TRAIL DR5 could affect the activity of AKT1, AKT2, and TOR protein in breast cancer cells. These results provide more evidence that SAHA may stimulate TRAIL DR5-CTSB crosstalk, influence the activity of downstream TOR signalling pathway mainly through the AKTs pathway, and initiate the autophagy of breast cancer cells.

DHEA increases epithelial markers and decreases mesenchymal proteins in breast cancer cells and reduces xenograft growth

Breast cancer is one of the most common neoplasias and the leading cause of cancer death in women worldwide. Its high mortality rate is linked to a great metastatic capacity associated with the epithelial-mesenchymal transition (EMT). During this process, a decrease in epithelial proteins expression and an increase of mesenchymal proteins are observed. On the other hand, it has been shown that dehydroepiandrosterone (DHEA), the most abundant steroid in human plasma, inhibits migration of breast cancer cells; however, the underlying mechanisms have not been elucidated.In this study, the in vitro effect of DHEA on the expression pattern of some EMT-related proteins, such as E-cadherin (epithelial), N-cadherin, vimentin and Snail (mesenchymal) was measured by Western blot and immunofluorescence in MDA-MB-231 breast cancer cells with invasive, metastatic and mesenchymal phenotype. Also, the in vivo effect of DHEA on xenograft tumor growth in nude mice (nu−/nu−) and on expression of the same epithelial and mesenchymal proteins in generated tumors was evaluated.We found that DHEA increased expression of E-cadherin and decreased N-cadherin, vimentin and Snail expression both in MD-MB-231 cells and in the formed tumors, possibly by DHEA-induced reversion of mesenchymal phenotype. These results were correlated with a tumor size reduction in mouse xenografts following DHEA administration either a week earlier or concurrent with breast cancer cells inoculation.In conclusion, DHEA could be useful in the treatment of breast cancer with mesenchymal phenotype.

The mutant p53-ID4 complex controls VEGFA isoforms by recruiting lncRNA MALAT1.

The abundant, nuclear-retained, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been associated with a poorly differentiated and aggressive phenotype of mammary carcinomas. This long non-coding RNA (lncRNA) localizes to nuclear speckles, where it interacts with a subset of splicing factors and modulates their activity. In this study, we demonstrate that oncogenic splicing factor SRSF1 bridges MALAT1 to mutant p53 and ID4 proteins in breast cancer cells. Mutant p53 and ID4 delocalize MALAT1 from nuclear speckles and favor its association with chromatin. This enables aberrant recruitment of MALAT1 on VEGFA pre-mRNA and modulation of VEGFA isoforms expression. Interestingly, VEGFA-dependent expression signatures associate with ID4 expression specifically in basal-like breast cancers carrying TP53 mutations. Our results highlight a key role for MALAT1 in control of VEGFA isoforms expression in breast cancer cells expressing gain-of-function mutant p53 and ID4 proteins.

Membrane protein stoichiometry studied in intact mammalian cells using liquid-phase electron microscopy.

Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets.

Jerantinine A induces tumor-specific cell death through modulation of splicing factor 3b subunit 1 (SF3B1)

Precursor mRNA (pre-mRNA) splicing is catalyzed by a large ribonucleoprotein complex known as the spliceosome. Numerous studies have indicated that aberrant splicing patterns or mutations in spliceosome components, including the splicing factor 3b subunit 1 (SF3B1), are associated with hallmark cancer phenotypes. This has led to the identification and development of small molecules with spliceosome-modulating activity as potential anticancer agents. Jerantinine A (JA) is a novel indole alkaloid which displays potent anti-proliferative activities against human cancer cell lines by inhibiting tubulin polymerization and inducing G2/M cell cycle arrest. Using a combined pooled-genome wide shRNA library screen and global proteomic profiling, we showed that JA targets the spliceosome by up-regulating SF3B1 and SF3B3 protein in breast cancer cells. Notably, JA induced significant tumor-specific cell death and a significant increase in unspliced pre-mRNAs. In contrast, depletion of endogenous SF3B1 abrogated the apoptotic effects, but not the G2/M cell cycle arrest induced by JA. Further analyses showed that JA stabilizes endogenous SF3B1 protein in breast cancer cells and induced dissociation of the protein from the nucleosome complex. Together, these results demonstrate that JA exerts its antitumor activity by targeting SF3B1 and SF3B3 in addition to its reported targeting of tubulin polymerization.

Metformin induces degradation of mTOR protein in breast cancer cells.

Activation of mTOR is implicated in the development and progression of breast cancer. mTOR inhibition exhibited promising antitumor effects in breast cancer; however, its effect is compromised by several feedback mechanisms. One of such mechanisms is the upregulation of mTOR pathway in breast cancer cells. Despite the established role of mTOR activation in breast cancer, the status of total mTOR protein and its impact on the tumor behavior and response to treatment are poorly understood. Besides, the mechanisms underlying mTOR protein degradation in normal and cancer breast cells are still largely unknown. We and others found that total mTOR protein level is elevated in breast cancer cells compared to their nonmalignant counterparts. We have detected defective proteolysis of mTOR protein in breast cancer cells, which could, at least in part, explain the high level of mTOR protein in these cells. We show that metformin treatment in MCF‐7 breast cancer cells induced degradation of mTOR and sequestration of this protein in a perinuclear region. The decrease in mTOR protein level in these cells correlated positively with a concomitant inhibition of proliferation and migration potentials of these cells. These findings provided a novel mechanism for the metformin action in breast cancer treatment. Understanding the proteolytic mechanism responsible for mTOR level in breast cancer may pave the way for improving the efficacy of breast cancer treatment regimens and mitigating drug resistance as well as providing a basis for potential novel therapeutic modalities for breast cancer.

Chaperonin Containing TCP-1 Protein Level in Breast Cancer Cells Predicts Therapeutic Application of a Cytotoxic Peptide.

Metastatic disease is a leading cause of death for patients with breast cancer, driving the need for new therapies. CT20p is a peptide previously discovered by our group that displays cancer-specific cytotoxicity. To design the optimal therapeutic use of the peptide, we identified the intracellular target of CT20p in breast cancer cells, correlating expression patterns of the target with susceptibility to CT20p. Using polymeric nanoparticles to deliver CT20p, we assessed cytoskeletal changes, cell migration, adhesion, and viability in cells treated with the peptide. Protein pull-down experiments, coupled to mass spectrometry, enabled identification of the peptide's intracellular target. Biochemical and histologic techniques validated target identity in human cell lines and breast cancer tissue microarrays and revealed susceptibility patterns to CT20p. Chaperonin containing TCP-1 (CCT) was identified as the intracellular target of CT20p. Cancer cells susceptible to CT20p had increased CCT, and overexpression of CCTβ, a subunit of the CCT complex, enhanced susceptibility to CT20p. Susceptible cells displayed reduced tubulin, a substrate of CCT, and inhibition of migration upon CT20p treatment. CCTβ levels were higher in invasive ductal carcinomas than in cancer adjacent tissues and increased with breast cancer stage. Decreased breast cancer patient survival correlated with genomic alternations in CCTβ and higher levels of the chaperone. Increased CCT protein in breast cancer cells underlies the cytotoxicity of CT20p. CCT is thus a potential target for therapeutic intervention and serves as a companion diagnostic to personalize the therapeutic use of CT20p for breast cancer treatment. Clin Cancer Res; 22(17); 4366-79. ©2016 AACR.

Metformin treatment decreases mTOR mRNA level in MCF-7 breast cancer cells

Besides the involvement of mTOR activity in several cancer conditions, evidence exists that increased total mTOR protein level might be linked to some cancer conditions such as colorectal carcinoma. The increase in total mTOR protein level in colon cancer was found to be associated with enhanced tumor progression and poor prognosis. Total mTOR protein level is elevated in breast cancer cells compared to their nonmalignant counterparts. High mTOR protein level in breast cancer cells could be attributed to decreased mTOR protein degradation, increased mTOR protein expression, or both. Increased protein expression may involve an increase in the gene expression.Here, we investigated the possibility of increased MTOR gene expression as a potential underlying cause of the elevated total mTOR protein in breast cancer cells. Our results suggest that transcription of MTOR gene is increased in the estrogen receptor positive (ER+) MCF-7 breast cancer cells compared to other breast cell lines. DNA sequencing of the MTOR promoter identified sequence variations in MCF-7 cells, which could be potentially involved in upregulation of mTOR expression. Among these variations is a truncation of guanine thymine dinucleotide (GT) n repeat region in MCF-7 cells, which might be possibly implicated in the elevated transcription of MTOR gene in these cells. Moreover, our results revealed that metformin treatment, profoundly decreased mTOR mRNA levels in MCF-7 breast cancer cells.In conclusion, unraveling the potential mechanisms involved in the regulation of mTOR expression in breast cancer cells could provide an avenue for optimizing the efficacy of breast cancer treatment regimens.

Anacardic Acid, Salicylic Acid, and Oleic Acid Differentially Alter Cellular Bioenergetic Function in Breast Cancer Cells

Anacardic acid is a dietary and medicinal phytochemical that inhibits breast cancer cell proliferation and uncouples oxidative phosphorylation (OXPHOS) in isolated rat liver mitochondria. Since mitochondrial-targeted anticancer therapy (mitocans) may be useful in breast cancer, we examined the effect of anacardic acid on cellular bioenergetics and OXPHOS pathway proteins in breast cancer cells modeling progression to endocrine-independence: MCF-7 estrogen receptor α (ERα)+ endocrine-sensitive; LCC9 and LY2 ERα+, endocrine-resistant, and MDA-MB-231 triple negative breast cancer (TNBC) cells. At concentrations similar to cell proliferation IC50 s, anacardic acid reduced ATP-linked oxygen consumption rate (OCR), mitochondrial reserve capacity, and coupling efficiency while increasing proton leak, reflecting mitochondrial toxicity which was greater in MCF-7 compared to endocrine-resistant and TNBC cells. These results suggest tolerance in endocrine-resistant and TNBC cells to mitochondrial stress induced by anacardic acid. Since anacardic acid is an alkylated 2-hydroxybenzoic acid, the effects of salicylic acid (SA, 2-hydroxybenzoic acid moiety) and oleic acid (OA, monounsaturated alkyl moiety) were tested. SA inhibited whereas OA stimulated cell viability. In contrast to stimulation of basal OCR by anacardic acid (uncoupling effect), neither SA nor OA altered basal OCR- except OA inhibited basal and ATP-linked OCR, and increased ECAR, in MDA-MB-231 cells. Changes in OXPHOS proteins correlated with changes in OCR. Overall, neither the 2-hydroxybenzoic acid moiety nor the monounsaturated alky moiety of anacardic acid is solely responsible for the observed mitochondria-targeted anticancer activity in breast cancer cells and hence both moieties are required in the same molecule for the observed effects. J. Cell. Biochem. 117: 2521-2532, 2016. © 2016 Wiley Periodicals, Inc.

Proteomic Analysis of Epithelial to Mesenchymal Transition (EMT) Reveals Cross-talk between SNAIL and HDAC1 Proteins in Breast Cancer Cells

Epithelial to mesenchymal transition (EMT)(1) occurs naturally during embryogenesis, tissue repair, cancer progression, and metastasis. EMT induces cellular and microenvironmental changes resulting in loss of epithelial and acquisition of mesenchymal phenotypes, which promotes cellular invasive and migratory capabilities. EMT can be triggered by extracellular factors, including TGF-β, HGF, and EGF. Overexpression of transcription factors, such as SNAIL, SLUG, ZEB1/2, and TWIST1, also induces EMT and is correlated to cancer aggressiveness. Here, the breast adenocarcinoma cell line MCF7 was transduced with SNAIL to identify specific mechanisms controlled by this transcription factor during EMT. Overexpression of SNAIL led to EMT, which was thoroughly validated by molecular, morphological, and functional experiments. Subcellular proteome enrichment followed by GEL-LC-MS/MS was performed to provide extensive protein fractionation and in-depth proteomic analysis. Quantitative analysis relied on a SILAC strategy, using the invasive breast cancer cell line MDA-MB-231 as a reference for quantitation. Subsets of proteins enriched in each subcellular compartment led to a complementary list of 4289 proteins identified with high confidence. A subset of differentially expressed proteins was validated by Western blot, including regulation in specific cellular compartments, potentially caused by protein translocation. Protein network analysis highlighted complexes involved in cell cycle control and epigenetic regulation. Flow cytometry analysis indicated that SNAIL overexpression led to cell cycle arrest in G0/G1 phases. Furthermore, down-regulation of HDAC1 was observed, supporting the involvement of epigenetic processes in SNAIL-induced EMT. When HDAC1 activity was inhibited, MCF7 not only apparently initiated EMT but also up-regulated SNAIL, indicating the cross-talk between these two proteins. Both HDAC1 inhibition and SNAIL overexpression activated the AKT pathway. These molecular mechanisms appear to be essential to EMT and therefore for cancer metastasis. Specific control of such epigenetic processes might then represent effective approaches for clinical management of metastatic cancer.

Nuclear distribution of eIF3g and its interacting nuclear proteins in breast cancer cells.

Eukaryotic translation initiation factor 3 subunit g (eIF3g) is a core subunit of the eukaryotic translation initiation factor 3 complex, and is important in the initiation of translation. It is also involved in caspase-mediated apoptosis, and is upregulated in multidrug-resistant cancer cells. In the present study, the nuclear distribution of eIF3g was determined by performing co-immunoprecipitation of proteins that potentially interact with eIF3g in the nucleus. Mass spectrometry characterization showed that three proteins, heterogeneous nuclear ribonucleoprotein U/scaffold attachment factor A, HSZFP36/zinc finger protein 823 and β-actin, were among the candidate eIF3g-interacting proteins in the nucleus. The protein-protein interaction was further confirmed by cross-linking and a glutathione S-transferase pull-down assay, followed by western blotting. The co-localization of these proteins was determined by confocal microscopy. These findings provide novel insight into the possible functions of eIF3g in the nucleus and serves as an important first step for further investigation of the roles of eIF3g in cancer development.