Quantitative Characterization of Cell Behaviors through Cell Cycle Progression via Automated Cell Tracking

Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor beta, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.

In normal and cancer cells, successful cell division requires accurate duplication of chromosomal DNA. All cells require a multiprotein DNA duplication system (replisomes) for their existence. However, death of normal cells in our body occurs through the apoptotic process. During apoptotic process several crucial genes are downregulated with the upregulation of caspase pathways, leading to ultimate degradation of genomic DNA. In metastatic cancer cells (SKBR-3, MCF -7, and MDA-462), this process is inhibited to achieve immortality as well as overexpression of the enzymes for the synthesis of marker molecules. It is believed that the GSL of the lacto family such as LeX, SA-LeX, LeY, Lea, and Leb are markers on the human colon and breast cancer cells. Recently, we have characterized that a few apoptotic chemicals (cis-platin, L-PPMP, D-PDMP, GD3 ganglioside, GD1b ganglioside, betulinic acid, tamoxifen, and melphalan) in low doses kill metastatic breast cancer cells. The apoptosis-inducing agent (e.g., cis-platin) showed inhibition of DNA polymerase/helicase (part of the replisomes) and also modulated (positively) a few glycolipid-glycosyltransferase (GSL-GLTs) transcriptions in the early stages (within 2h after treatment) of apoptosis. These Lc-family GSLs are also present on the surfaces of human breast and colon carcinoma cells. It is advantageous to deliver these apoptotic chemicals through the metastatic cell surfaces containing high concentration of marker glycolipids (Lc-GSLs). Targeted application of apoptotic chemicals (in micro scale) to kill the cancer cells would be an ideal way to inhibit the metastatic growth of both breast and colon cancer cells. It was observed in three different breast cancer lines (SKBR-3, MDA-468, and MCF-7) that in 2h very little apoptotic process had started, but predominant biochemical changes (including inactivation of replisomes) started between 6 and 24h of the drug treatments. The contents of replisomes (replisomal complexes) during induction of apoptosis are not known. It is known that DNA helicase activities (major proteins catalyze the melting of dsDNA strands) change during apoptotic induction process. Previously DNA Helicase-III was characterized as a component of the replication complexes isolated from carcinoma cells and normal rapid growing embryonic chicken brain cells. Helicase activities were assayed by a novel method (combined immunoprecipitation-ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of nicked "ACT-DNA," by determining values obtained from +/- aphidicolin added to the incubation mixtures. Very little is known about the stability of the "replication complexes" (or replisomes) during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during replication, repair, and recombination processes. In all three breast carcinoma cell lines (SKBR-3, MCF-7, and MDA-468), a common trend, decrease of activities of DNA polymerase-alpha and Helicase-III (estimated and detected with a polyclonal antibody), was observed, after cis-platin- and L-PPMP-induced apoptosis. Previously our laboratory has documented downregulation (within 24-48h) of several GSL-GLTs with these apoptotic reagents in breast and colon cancer cells also. Perhaps induced apoptosis would improve the prognosis in metastatic breast and colon cancer patients.

Background: Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates poor prognosis. Patients with BCLM have a 5-year survival rate of 8%. Specific treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must clear space among hepatocytes, the primary cell population in the liver, during metastatic formation. Cell competition is a developmentally conserved process that explains how organs or primary tumors form when cells within those tissues have varying fitness and compete with one another. This competition can result in the survival and expansion of one cell population, and the induction of cell death through apoptosis in the other. We hypothesized that MCC use cell competition with hepatocytes to create space and expand within the liver. Objectives: In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods: We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes, initially using balb/c derived breast cancer (4T1) and hepatocyte (H2.35) cells and syngeneic mouse models of liver metastasis. These models directly capture MCC and hepatocyte interactions, and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we next performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generated a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells. This dataset includes 3264 primary breast cancer cells across 3 models of aggressive breast cancers, encompassing HER2+ and TNBC subtypes, as well as 526 embryonic mammary gland cells. Using this platform, we identify genes enriched in both breast cancer and the developing mammary gland. Next, we validate genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determine the impact of target genes on cell competition in our models. Results/Discussion: Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice livers with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified 5 potential targets that could drive breast cancer metastasis and cell competition. Using our competition models, perturbation of SerpinE2, a protease inhibitor which was one of the top gene targets, successfully modulated competition between MCC and hepatocytes. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild type conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with wild-type (WT) MCC. These findings suggest that hepatocytes cannot outcompete or kill WT MCC, but they can outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Our findings suggest that restoring the competitiveness of the liver parenchyma could be a new treatment modality for treating liver metastases. Citation Format: Katherine Lake, Sakshi Mohta, Clayton Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Christine Zhang, Jacob Pena, Christine Hodgdon, Julia Maues, Elizabeth Chen, Isaac Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-01.

MicroRNAs (miRNAs) regulate the progression of human malignancy by targeting oncogenes or tumor suppressors, which are 12 promising targets for cancer treatment. Increasing evidence has suggested the aberrant expression and tumor-suppressive function of miR-1298 in cancers, however, the regulatory mechanism of miR-1298 in breast cancer (BC) remains unclear. Here, our findings showed that miR-1298 was down-regulated in BC tissues and cell lines. Lower level of miR-1298 was significantly correlated with the advanced progression of BC patients. Experimental study showed that overexpression of miR-1298 inhibited the proliferation, induced apoptosis and cell cycle arrest in BC cells. The in vivo xenograft mice model showed that highly expressed miR-1298 significantly reduced the tumor growth and metastasis. Further mechanism analysis revealed that miR-1298 bound the 3′-untranslated region (UTR) of a disintegrin and metalloproteinase 9 domain (ADAM9) and suppressed the expression of ADAM9 in BC cells. ADAM9 was overexpressed in BC tissues and inversely correlated with miR-1298. Down-regulation of ADAM9 induced apoptosis and cell cycle arrest of BC cells. Moreover, ectopic expression of ADAM9 by transiently transfecting with vector encoding the full coding sequence of ADAM9 attenuated the inhibitory effects of miR-1298 on the proliferation and cell cycle progression of BC cells. Collectively, our results illustrated that miR-1298 played a suppressive role in regulating the phenotype of BC cells through directly repressing ADAM9, suggesting the potential application of miR-1298 in the therapy of BC.

Insulin-like growth factors (IGFs) and insulin signaling via the type I IGF receptor (IGF1R) and insulin receptor (IR) respectively, are potent activators of PI3K/Akt/mTOR. Drugs targeting IGF1R and the related IR were tested clinically including in combination with mTOR inhibitors. Inhibition of mTOR was not effective as inhibition of mTOR relieved the negative feedback loop regulating levels of the adaptor protein, insulin receptor substrate 1 (IRS-1), that mediates proliferative effects of IGFs and insulin and rapamycin enhanced phosphorylation of Akt. The ribosomal protein S6 kinase (S6K) phosphorylates IRS-1 on serine residues and targets it for proteasomal degradation, and this negative feedback regulation is important in attenuating IGF and insulin signaling. Cyclin dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. CDK4/6 inhibitors have recently been approved for treatment of estrogen receptor positive (ER+), Her2- advanced breast cancers and these CDK4/6 inhibitors such as palbociclib block phosphorylation of retinoblastoma (Rb). IGFs and insulin stimulate cell cycle progression and increase cyclin D1 levels in breast cancers. Therefore, we hypothesized that CDK4/6 inhibition can be combined with IGF1R/IR targeting to block mitogenic functions of IGF/insulin signaling in breast cancer as this would not relieve the negative feedback regulation of IGF and insulin signaling. Palbociclib blocked growth of ER+ parental MCF-7 and T47D breast cancer cells that respond to hormonal therapy including tamoxifen, a selective estrogen receptor modulator. Parental MCF-7 and T47D were more sensitive to palbociclib compared to matched cells with acquired resistance to tamoxifen (MCF-7/TamR and T47D/TamR). Palbociclib also blocked IGF-I and insulin stimulated entry into cell cycle leading to G0/G1 arrest in ER+ breast cancer cells. Combination of palbociclib with an IGF1R inhibitory antibody, but not IR antibody, was more effective in inhibiting growth of tamoxifen sensitive parental ER+ breast cancer cells. In contrast combination of palbociclib with an IR antibody was effective in inhibition of MCF-7/TamR cells. Unlike mTOR inhibitors that upregulated IRS-1 levels leading to increased phosphorylation of Akt through IGF1R/IR, palbociclib did not affect IRS-1 levels and did not enhance phosphorylation of Akt in ER+ and TNBC cells. Further, palbociclib also blocked growth and cell cycle progression of triple negative breast cancer (TNBC) cells. Our data show that combining IGF1R/IR inhibitors with palbociclib may be superior to combining them with mTOR inhibitors for ER+ breast cancer and palbociclib can be a potential therapeutic strategy for TNBC. These data implicate that cotargeting CDK4/6 and IR could be a therapeutic option for patients with endocrine resistant disease. Citation Format: Katelyn Hoff, Deepali Sachdev. CDK4/6 inhibition blocks effects of IGFs and insulin in estrogen receptor positive and triple negative breast cancers [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 2303.

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs determined a definite increment in tumor cell migration velocity, even with a marked enhancement of the migration directionality induced by CAFs. Our results demonstrate a reciprocal influence of mammary cancer and stromal cells on various adhesiveness/invasiveness features. In particular, an overall pro-tumoral/-invasive effect of CAFs on both well- and poorly differentiated mammary cancer cells was exteriorized by reduction of cell adhesion, induction of membrane fluidity, and migration velocity and directionality, along with a promotion of epithelial-mesenchymal transition.

Background Although breast cancer liver metastases (BCLM) are common events, with 40-50% of patients with metastatic breast cancer developing liver metastases, their development indicates particularly poor prognosis. Targeted treatments for liver metastases are virtually non-existent, partly because the key events of early liver metastasis formation remain unknown. The liver is a dense organ, and metastatic breast cancer cells (MCC) must first clear space among hepatocytes, the primary cell population in the liver, to form. Cell competition is a process that explains how organs or primary tumors form from two populations of competing cells with varied fitness levels. The expansion of one population of cells creates space through the induction of apoptosis in the other. Objectives In this study, we tested the hypothesis that MCC use cell competition with hepatocytes to create space and expand within the liver, resulting in hepatocyte apoptosis. Methods We develop new in-vitro and mouse lineage-traced models to assay cell competition between MCC and hepatocytes. These models directly capture MCC and hepatocyte interactions and we can assess modes of cell competition, including apoptosis, through immunostaining. Because prior literature suggests that the developing mammary gland informs both cell competition and metastasis, we performed a bioinformatics screen to identify potential signals that contribute to cell competition between MCC and hepatocytes. We generate a new single-cell RNA-seq dataset that incorporates information from the developing mammary gland and breast cancer cells and identify genes enriched in both. Next, we validated genes by testing for their high expression in MCC when compared to hepatocytes. Then, we determined the impact of target genes on cell competition in our models. Results/Discussion Our models demonstrate that MCC use cell competition to create space in the liver by inducing apoptosis in neighboring hepatocytes. MCC induce apoptosis in neighboring hepatocytes through cell competition in in-vitro co-cultures by 11.7-fold compared to MCC apoptosis. Hydrodynamic tail-vein engraftment in NOD-scid-gamma mice with MCC also resulted in increased neighboring hepatocyte apoptosis. Using our bioinformatics screen, we identified SerpinE2, a protease inhibitor, as a target that could drive breast cancer metastasis and cell competition. Knock-out of SerpinE2 in MCC did not affect their viability or growth but resulted in MCC apoptosis adjacent to hepatocyte neighbors by 16.9-fold when compared to wild-type (WT) conditions. Further, co-culture of MCC that lacked SerpinE2 with hepatocytes resulted in decreased hepatocyte apoptosis by 2.3-fold compared to hepatocytes co-cultured with WT MCC. These findings suggest that perturbation of SerpinE2 successfully modulates competition between MCC and hepatocytes: Hepatocytes can selectively outcompete and kill MCC that lack SerpinE2. Future work focused on the perturbation of SerpinE2 may lead to novel therapies for patients at risk for or with BCLM. Citation Format: Katherine E Lake, Sakshi Mohta, Clayton A Smith, Venkata Repaka, Lily Xu, Kaitlyn Saunders, Vrushali Pandit, Emily Goff, Jacob Pena, Luise Chinea, Luisa Froessl, Sangeetha M Reddy, Heather L McArthur, Christine Hodges, Julia Maues, Elizabeth Chen, Isaac S Chan. Treating liver metastases by reversing cell competition between metastatic cancer cells and hepatocytes [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A041.

BackgroundCDK11p58, a Ser/Thr kinase that belongs to the cell division cycle 2-like 1 (CDC2L1) subfamily, is associated with cell cycle progression, tumorigenesis and apoptotic signaling. CDK11p58 is also involved in the regulation of steroid receptors, such as androgen and estrogen receptors. We previously found that CDK11p58 was abnormally expressed in prostate cancer. However, its role in breast cancer remains unclear.MethodsCDK11p58 expression was evaluated by immunohistochemical staining in a tissue array. A Transwell assay was used to detect invasion and metastasis in breast cancer cells. The TaqMan® Metastasis Gene Expression Assay was used to search for potential downstream factors in the CDK11p58 signaling pathway. qRT-PCR was used to evaluate mRNA levels, and the dual luciferase array was used to analyze promoter activity. Western blotting was used to detect the protein level.ResultsCDK11p58 expression was negatively correlated with node status (P = 0.012), relapse status (P = 0.002) and metastasis status (P = 0.023). Kaplan-Meier survival curves indicated that the disease-free survival (DFS) was significantly poor in breast cancer patients with low CDK11 expression. Interestingly, using the breast cancer cell lines ZR-75-30 and MDA-MB-231, we found that CDK11p58 was capable of repressing the migration and invasion of ERα-positive breast cancer cells, but not ERα-negative breast cancer cells, in a kinase-dependent manner. Gene expression assays demonstrated that integrin β3 mRNA was dramatically repressed by CDK11p58, and luciferase results confirmed that the integrin β3 promoter was inhibited by CDK11p58 through ERα repression. The expression of integrin β3 was highly related to ERα signaling; ERα overexpression stimulated integrin β3 expression, whereas siRNA-mediated knockdown of ERα attenuated integrin β3 expression.ConclusionsThese data indicate that CDK11p58 is an anti-metastatic gene in ERα-positive breast cancer and that the regulation of integrin β3 by CDK11p58 via the repression of ERα signaling may constitute part of a signaling pathway underlying breast cancer invasion.

PurposeLong intergenic non-coding RNA 922 (LINC00922) plays a critical role in the progression of lung cancer. In this study, we aimed to quantify LINC00922 expression in breast cancer and determine its influence on the malignant behavior of breast cancer cells in vitro and in vivo. We also investigated the mechanism by which LINC00922 affects the progression of breast cancer.Materials and MethodsReverse transcription-quantitative polymerase chain reaction was performed to quantify LINC00922 expression in breast cancer tissues and cell lines. The cell counting kit-8 assay, flow cytometry, Transwell migration and invasion assays, and tumor model assays were performed to determine the effects of LINC00922 deficiency on breast cancer cell proliferation, apoptosis, migration and invasion in vitro, and tumor growth in vivo, respectively. Furthermore, bioinformatics analysis was performed to predict the potential target microRNA of LINC00922. The prediction was further evaluated using luciferase reporter and RNA immunoprecipitation assays.ResultsLINC00922 was clearly overexpressed in breast cancer tissues and cell lines. LINC00922 depletion restricted breast cancer cell proliferation, migration, and invasion but induced cell apoptosis in vitro. Additionally, its knockdown evidently repressed tumor growth of breast cancer cells in vivo. Mechanistically, LINC00922 was demonstrated to serve as a molecular sponge for miR-424-5p in breast cancer cells. Furthermore, brain-derived neurotrophic factor (BDNF) was verified as a direct target of miR-424-5p in breast cancer cells, and BDNF expression was found to be positively regulated by LINC00922 through sponging miR-425-5p. Rescue experiments further revealed that the influences on breast cancer cell proliferation, apoptosis, migration, and invasion induced by LINC00922 silencing were abrogated by increasing the output of the miR-424-5p/BDNF axis.ConclusionThe LINC00922/miR-424-5p/BDNF pathway is implicated in the acceleration of the malignant behavior of breast cancer cells. These findings suggest that this pathway is a promising novel molecular target in breast cancer therapy.

Cervical cancer is a common malignant gynecological tumor for females all over the world. Circular RNAs (circRNAs) are being found to have relevance to various human cancers, including cervical cancer. This study is designed to explore the role and mechanism of circRNA DDB1- and CUL4-associated factor 1 (circVPRBP, also known as hsa_circ_0065898) on the progression of cervical cancer. CircVPRBP, microRNA-106b-5p (miR-106b-5p), and tripartite motif-containing protein 3 (TRIM3) levels were determined by real-time quantitative PCR. Cell proliferative ability, apoptosis rate, cell cycle progression, migration, and invasion were detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, 5-ethynyl-2'-deoxyuridine, colony formation assay, flow cytometry, and transwell assays. Protein levels of matrix metallopeptidase 2 (MMP2) and matrix MMP9, and TRIM3 were measured by western blot assay. The binding relationship between miR-106b-5p and circVPRBP or TRIM3 was predicted by Starbase and then verified by a dual-luciferase reporter and RNA immunoprecipitation assays. The biological role of circVPRBP on cervical tumor growth was examined by the xenograft tumor model in vivo . CircVPRBP and TRIM3 were decreased, and miR-106b-5p was increased in cervical cancer tissues and cell lines. Furthermore, circVPRBP could suppress cell growth and metastasis of cervical cancer cells in vitro . Mechanically, circVPRBP could regulate TRIM3 expression by sponging miR-106b-5p. Also, circVPRBP upregulation repressed tumor growth of cervical cancer cells in vivo . CircVPRBP could inhibit the malignant biological behavior of cervical cancer cells by miR-106b-5p/TRIM3 axis, providing a promising therapeutic target for cervical cancer treatment.

Monitoring and tracking of cell motion is a key component for understanding disease mechanisms and evaluating the effects of treatments. Time-lapse optical microscopy has been commonly employed for studying cell cycle phases. However, usual manual cell tracking is very time consuming and has poor reproducibility. Automated cell tracking techniques are challenged by variability of cell region intensity distributions and resolution limitations. In this work, we introduce a comprehensive cell segmentation and tracking methodology. A key contribution of this work is that it employs multi-scale space-time interest point detection and characterization for automatic scale selection and cell segmentation. Another contribution is the use of a neural network with class prototype balancing for detection of cell regions. This work also offers a structured mathematical framework that uses graphs for track generation and cell event detection. We evaluated cell segmentation, detection, and tracking performance of our method on time-lapse sequences of the Cell Tracking Challenge (CTC). We also compared our technique to top performing techniques from CTC. Performance evaluation results indicate that the proposed methodology is competitive with these techniques, and that it generalizes very well to diverse cell types and sizes, and multiple imaging techniques.

BackgroundMicroRNAs (miRNAs) are involved in the progression of diverse human cancers. This work aimed to delve into how microRNA-135a-5p (miR-135a-5p) affects the biological behaviors of Breast Cancer (BC) cells. MethodsGene Expression Omnibus (GEO) datasets were used to analyze the expression differences of miR-135a-5p in cancer tissues of BC patients. Quantitative real-time PCR and western blot were conducted to detect miR-135a-5p and Bcl-2 Associated Athanogene (BAG3) expression levels in BC tissues and cells, respectively. The proliferation, migration, invasion, and cell cycle of BC cells were detected by cell counting kit-8 assay, BrdU assay, wound healing assay, transwell assay, and flow cytometry. The targeted relationship between miR-135a-5p and BAG3 mRNA 3′UTR predicted by bioinformatics was further testified by a dual-luciferase reporter gene assay. Pearson's correlation analysis was adopted to analyze the correlation between miR-135a-5p expression and BAG3 expression. The downstream pathways of BAG3 were analyzed by the LinkedOmics database. ResultsMiR-135a-5p was significantly down-regulated and BAG3 expression was significantly raised in BC tissues. MiR-135a-5p overexpression repressed the viability, migration and invasion of BC cells, and blocked cell cycle progression in G0/G1 phase while inhibiting miR-135a-5p worked oppositely. BAG3 was verified as a target of miR-135a-5p. Overexpression of BAG3 reversed the impacts of miR-135a-5p on the malignant biological behaviors of BC cells. The high expression of BAG3 was associated with the activation of the cell cycle, mTOR and TGF-β signaling pathways. ConclusionMiR-135a-5p regulates BAG3 to repress the growth, migration, invasion, and cell cycle progression of BC cells.

Background: Recent evidence presented the significant role of the microRNA-193 (miR-193) family in biological processes by the contribution of specific targeting, which mainly display as a tumor suppressor in various cancers. In the present study, we evaluated the effect of miR-193a-5p replacement on some metastasis gene expression in metastatic breast cancer (BC) cells. Methods: For this purpose, firstly, the quantitative real-time polymerase chain reaction (qRTPCR) was used to detect the miR-193a-5p expression in the MDA-MB-231 BC cell line. Subsequently, miR-193a-5p was transfected into the cells, and the expression levels of ROCK1 (Rho‑associated, coiled‑coil containing protein kinase 1), CXCR4 (Chemokine Receptor-4), CD44, and vimentin genes were evaluated by qRT-PCR. Results: The expression level of miR-193a-5p strongly reduced in MDA-MB-231 cells. Interestingly, the ROCK1 (P < 0. 001), CD44 (P < 0.0001), CXCR4 (P < 0. 001) and vimentin (P < 0. 001) expression levels significantly decreased following miR-193a-5p transfection in MDA-MB-231 BC cells. Conclusion: To conclude, it seems that miR-193a-5p restoration can attenuate the metastatic behavior of BC cells in vitro through decreased expression level of metastasis-related genes and may constitute an effective novel therapeutic strategy in miRNA-replacement therapy and treatment of metastatic breast adenocarcinoma in the future.

Estrogen (E2) stimulation promotes proliferation in estrogen receptor-positive (ER+) breast cancer, which accounts for 60-75% of all breast cancer. E2 signals through the ER via three pathways: the classical genomic pathway involving transcriptional activation of the ER, crosstalk between ER and other transcription factors, and “non-genomic” signaling cascades, which do not involve the transcriptional activity of the ER. This “non-genomic” pathway likely involves Src family kinases (SFKs) and has been implicated in cell cycle progression in fibroblasts engineered to express the ER (Castoria 1999). While not typically amplified or mutationally activated in ER+ breast cancer, Src is hyperactive in most breast cancers. To test whether SFK activity is required for G1/S cell cycle progression in breast cancer cells, we pretreated quiescent ER+ MCF7 cells with a selective SFK inhibitor (SU11333) prior to stimulation with E2. Inhibition of SFK activity blocked progression from G1 to S phase. Previous data from our laboratory has demonstrated that G1/S progression in response to peptide growth factor stimulation of quiescent fibroblasts is dependent upon SFK-mediated stabilization of myc mRNA (Bromann 2005). We therefore tested whether SFK activity also regulates myc expression in breast cancer cells. Our data suggest that SFK activity is required for accumulation of myc mRNA in quiescent MCF7s after E2 stimulation, and that E2 stimulated stabilization of myc mRNA is dependent on SFK activity. One potential myc mRNA stabilizer is IMP1 (IGF2BP1, ZBP1, CRDBP) an RNA binding protein that is primarily expressed during development and in stem cells, and re-expressed in some cancer types, including breast cancer. IMP1 has previously been shown to stabilize myc mRNA in stem cells as well as osteosarcoma cells. While the signaling pathways that mediate this stabilization have not been well studied, previous work has identified a Src phosphorylation site on IMP1 which regulates IMP1 function (Sasaki 2010). We hypothesized that IMP1 may play a role in E2-mediated stabilization of myc mRNA. Indeed, MCF7 cells require IMP1 to accumulate myc mRNA after E2 stimulation. In summary, E2 stimulation activates SFKs, which then promote G1/S cell cycle progression in part by stabilizing myc mRNA levels, through the activity of IMP1. Bromann et al JBC, 2005 Castoria et al. EMBO J, 1999 Sasaki et al. J Neurosci, 2010 Citation Format: Christopher Abdullah, Hasan Korkaya, Sara A. Courtneidge. Estrogen stimulates cell cycle progression dependent on Src kinase activity and IMP1 stabilization of myc mRNA in estrogen receptor-positive breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3300. doi:10.1158/1538-7445.AM2014-3300

95P - Targeting the ASCT2 glutamine uptake and metabolism pathway in triple-negative breast cancer