Subset Of Breast Cancer Cells Research Articles

The loss of B7-H4 expression in breast cancer cells escaping from T cell cytotoxicity contributes to epithelial-to-mesenchymal transition

BackgroundB7 homology 4 (B7-H4), a potential target for cancer therapy, has been demonstrated to inhibit T cell cytotoxicity in the early stages of breast cancer. However, B7-H4 manipulating breast tumor immune microenvironment (TIME) in the tumor progression remains unknown.MethodsWe engineered T cells with B7-H4-specific chimeric antigen receptors (CARs) and performed a T cell co-culture assay to characterize B7-H4 expression level in breast cancer cells escaping from T cell cytotoxicity. We generated B7-H4 knockout (KO) and overexpression (OE) breast cancer cells to determine the epithelial-to-mesenchymal transition (EMT) and stemness characteristics in vitro and in vivo, including tumor proliferation, migration, metastasis and chemoresistance. The Cancer Genome Atlas breast cancer database was accessed to investigate the correlation between B7-H4 expression levels and EMT characteristics in patients with breast cancer.ResultsOur result found that B7-H4 expression level was significantly reduced in a subset of breast cancer cells that escaped from the cytotoxicity of B7-H4 CAR-T cells. Compared with wild type cells, B7-H4 KO cells prompt EMT and stemness characteristics, including migration, invasion and metastasis, and OE cells vice versa. The increase in H3K27me3 in KO cells confirmed the epigenetic reprogramming of cancer stem cells. The IC50 of doxorubicin or oxaliplatin significantly increased in KO cells, which was in agreement with a decrease in OE cells. Moreover, a trend of downregulated B7-H4 from stage I to stage II breast cancer patients indicates that the low-expressing B7-H4 breast cancer cells escaping from TIME have spread to nearby breast lymph nodes in the cancer progression.ConclusionsOur study illuminates the novel role of renouncing B7-H4 in breast cancer cells through immune escape, which contributes to EMT processes and provides new insights for breast cancer treatments.

Abstract P3-05-42: Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis

Abstract Introduction. The CUB domain containing protein 1, also known as TRASK (Transmembrane and associated with Src kinases) or CD318, has been suggested as a biomarker for stem cells or stem like cells. The transmembrane CDCP1 protein is known to activate the Src kinase and has recently shown to play a key part in the aggressiveness of breast cancer cells including migration, invasiveness and possibly growth induced by the motogenic Hepatocyte Growth Factor (HGF) and its receptor MET (Kawase et al 2022), a process requires Rho-GEF, PI3K and STAT5. This appears to allow identification of a subset of cancer cells that are aggressive in their biological behaviours. Here, we examined the expression profile of CDCP1, SRC, the HGF/HGF receptor complex and HGF activation regulators in a cohort of breast cancer and attempt to establish if such protein axis is able to identify patients with high risk of poor clinical outcome. Methods. Using an establish breast cancer cohort which is comprised of both normal mammary tissues and breast cancer tissues freshly obtained after surgery, we quantified the gene transcripts of CDCP1/CD318 and examined its clinical and pathological relevance. Integrated analysis was conducted for CDCP1 and the expression profile of SRC, PI3K, HGF, the HGF receptor cMET, HGF activator (HGFA), HGF activation regulators including HAI-1, HAI2, matriptase-1 and matriptase-2, and STAT family members. This was done against the clinical outcome of the patients as well as the clinical and pathological factors. Results. Breast cancer tissues expressed high levels of CDCP1 compared with normal mammary tissues. CDCP1 itself had a weak yet significant value in predicting the overall survival of the patients (p=0.047). The expression levels of these CDCP1 related molecules aren’t significantly correlated with CDCP1. However, integrated analyses revealed that CDCP1, together with its pathway regulators SRC, HGF, the HGF receptor (MET) and the HGF activation regulators matriptases form a power prognostic indicator for the clinical outcome of the patients (p< 0.0001, HR=1.8 for overall survival (OS) and p=0.002, HR=1.5 for disease-free survival (DFS)). This integrated profile has also identified subgroups of patients with highly favourable and very poor clinical outcomes over a ten-year follow-up period, with the respective survival at 100% and 36% respectively. The predictive power for OS and DFS is highly independent of other clinical and pathological factors in a multivariate analysis (p=0.003 and p=0.017 respectively). The predictive power is applicable to ER negative and HER2 positive tumours. Discussion. CDCP1/CD318, a factor known to stimulate cancer cell aggressiveness, together with its pathway kinase SRC and newly identified extracellular activator HGF and the HGF regulators forms a significant independent prognostic factor. It identifies subgroups of patients with favourable and poor prognosis, allowing consideration of targeted therapies for the patients. Reference Kawase N, Sugihara A, Kajiwara K, Hiroshima M, Akamatsu K, Nada S, Matsumoto K, Ueda M, Okada M. SRC kinase activator CDCP1 promotes hepatocyte growth factor-induced cell migration/invasion of a subset of breast cancer cells. J Biol Chem, 2022, 298:101630. doi: 10.1016/j.jbc.2022.101630 Citation Format: Yiming Yang, Xuefei Dong, Tracey A. Martin, Lin Ye, Jane Lane, Andrew J. Sanders, QingPing Dou, Eleri Davies, Wen G. Jiang. Identification of subpopulation of breast cancer patients with poor clinical outcome using CUB domain containing protein-1 (CDCP1)/CD318 and its interactive proteins SRC and the HGF axis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-42.

SRC kinase activator CDCP1 promotes hepatocyte growth factor–induced cell migration/invasion of a subset of breast cancer cells

Cancer invasion and metastasis are the major causes of cancer patient mortality. Various growth factors, including hepatocyte growth factor (HGF), are known to promote cancer invasion and metastasis, but the regulatory mechanisms involved are not fully understood. Here, we show that HGF-promoted migration and invasion of breast cancer cells are regulated by CUB domain–containing protein 1 (CDCP1), a transmembrane activator of SRC kinase. In metastatic human breast cancer cell line MDA-MB-231, which highly expresses the HGF receptor MET and CDCP1, we show that CDCP1 knockdown attenuated HGF-induced MET activation, followed by suppression of lamellipodia formation and cell migration/invasion. In contrast, in the low invasive/nonmetastatic breast cancer cell line T47D, which had no detectable MET and CDCP1 expression, ectopic MET expression stimulated the HGF-dependent activation of invasive activity, and concomitant CDCP1 expression activated SRC and further promoted invasive activity. In these cells, CDCP1 expression dramatically activated HGF-induced membrane remodeling, which was accompanied by activation of the small GTPase Rac1. Analysis of guanine nucleotide exchange factors revealed that ARHGEF7 was specifically required for CDCP1-dependent induction of HGF-induced invasive ability. Furthermore, immunofluorescence staining demonstrated that CDCP1 coaccumulated with ARHGEF7. Finally, we confirmed that the CDCP1-SRC axis was also crucial for HGF and ARHGEF7-RAC1 signaling in MDA-MB-231 cells. Altogether, these results demonstrate that the CDCP1-SRC-ARHGEF7-RAC1 pathway plays an important role in the HGF-induced invasion of a subset of breast cancer cells.

Metastasis-initiating cells induce and exploit a fibroblast niche to fuel malignant colonization of the lungs

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke phenotypic changes in lung fibroblasts, forming a supportive metastatic niche. Colonization of the lungs confers an inflammatory phenotype in metastasis-associated fibroblasts. Specifically, IL-1α and IL-1β secreted by breast cancer cells induce CXCL9 and CXCL10 production in lung fibroblasts via NF-κB signaling, fueling the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells, which exhibits tumor-initiating ability when co-transplanted with fibroblasts and has high JNK signaling that drives IL-1α/β expression. Importantly, disruption of the intercellular JNK-IL-1-CXCL9/10-CXCR3 axis reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for the molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.

349P - Polo-like kinase 1 inhibitor onvansertib synergizes with paclitaxel in breast cancer carrying p53 mutation

BackgroundSomatic mutation in TP53 gene (mutp53) is a strong prognostic marker in breast cancer. Triple negative breast cancer (TNBC) is characterized by up to 80% mutp53 and the greatest overall genomic instability among subtypes. Polo-like kinase 1 (PLK1) regulates progression of cells through the G2 phase of the cell cycle. We hypothesize that mutp53 in the context of breast cancer can predict synergy to paclitaxel [p] plus onvansertib [o], an orally available highly selective Plk1 inhibitor. MethodsDrugs were tested in a set of mutp53 breast cancer cells including TNBC SUM149 and SUM159, luminal SUM52 and T47D, and wtp53 TNBC SUM1315, luminal MCF7, and normal breast MCF10A. We performed cell proliferation assays in order to determine the IC50 (Inhibition Concentration of 50%) for each drug. Synergism was determined using the Chou-Talalay method, by determining the combination index (CI). Apoptosis was determined by PARP cleavage proteins detection in cells synchronized with double thymidine block. For the in vivo experiments, mice were injected with 5x106 SUM159 cells and randomly assigned to experimental condition (control, o, p, o+p). The primary endpoint was tumor volume (TV) measured longitudinally. We performed one-sided two-samples Wilcoxon test comparing the means of the adjusted TV measured at 10 days for p and o+p. ResultsTNBC cells were more sensitive to single agent o compared to luminal cells. Breast cell lines with mutp53 (SUM149, SUM159, SUM52 and T47D) were found to have Combination Index (CI) < 1 when treated with o+p. Conversely, in cell line with wtp53 (SUM1315, MCF7, and MCF10A), o+p did not show synergy, CI≥1. Western blot showed prolonged apoptosis for PLK1 inhibition plus taxane compared to PLK1 inhibitor alone, in SUM149 and SUM159. In the xenograft model SUM159, the adjusted 10-day TV for o+p was significantly less than TV for p, p-value = 0.0303. ConclusionsOnvansertib in combination with paclitaxel showed promising results in a subset of breast cancer cells and animal model characterized by the presence of mutp53. Our preclinical results confirm mutp53 as predictive biomarker of synergy and set the feasibility of performing a clinical trial with the combination o+p for mutp53 breast cancer patients. Legal entity responsible for the studyThe authors. FundingK12 Paul Calabresi funding, K12 CA157688. DisclosureM. Erlander: Leadership role, Chief Scientific Officer (CSO): Trovagene Oncology. All other authors have declared no conflicts of interest.

Protease Nexin I is a feedback regulator of EGF/PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness

Breast cancer is the most prevalent cancer in women worldwide, which remains incurable once metastatic. Breast cancer stem cells (BCSCs) are a small subset of breast cancer cells, which are the radical cause of drug resistance, tumor relapse, and metastasis in breast cancer. The extracellular serine protease inhibitor serpinE2, also named protease nexin-1 (PN-1), contributes to enhanced metastasis of cancer cells mainly by remodeling the tumor matrix. In this study, we found that PN-1 was up-regulated in breast cancer, which promoted cell invasion, migration and stemness. Furthermore, by using specific inhibitors, we discovered that epidermal growth factor (EGF) up-regulated PN-1 in breast cancer cells through cascade activation of epidermal growth factor receptor (EGFR) to the activation of protein kinase Cδ (PKCδ), mitogen-activated protein kinase (MEK) and extracellular signal-related kinase (ERK), which finally led to the up-regulation of early growth response protein 1 (EGR1). Moreover, EGF signaling was further activated as a feedback of PN-1 up-regulation through PN-1 blocking HtrA1. Taken together, our findings revealed a novel signaling axis that up-regulated PN-1 expression in breast cancer cells, and the new mechanism of PN-1-promoted breast cancer metastasis, which may provide new insights into identifying novel therapeutic targets for breast cancer.

A multiple breast cancer stem cell model to predict recurrence of T1\u20133, N0 breast cancer

BackgroundLocal or distant relapse is the key event for the overall survival of early-stage breast cancer after initial surgery. A small subset of breast cancer cells, which share similar properties with normal stem cells, has been proven to resist to clinical therapy contributing to recurrence.MethodsIn this study, we aimed to develop a prognostic model to predict recurrence based on the prevalence of breast cancer stem cells (BCSCs) in breast cancer. Immunohistochemistry and dual-immunohistochemistry were performed to quantify the stem cells of the breast cancer patients. The performance of Cox proportional hazard regression model was assessed using the holdout methods, where the dataset was randomly split into two exclusive sets (70% training and 30% testing sets). Additionally, we performed bootstrapping to overcome a possible biased error estimate and obtain confidence intervals (CI).ResultsFour groups of BCSCs (ALDH1A3, CD44+/CD24−, integrin alpha 6 (ITGA6), and protein C receptor (PROCR)) were identified as associated with relapse-free survival (RFS). The correlated biomarkers were integrated as a prognostic panel to calculate a relapse risk score (RRS) and to classify the patients into different risk groups (high-risk or low-risk). According to RRS, 67.81 and 32.19% of patients were categorized into low-risk and high-risk groups respectively. The relapse rate at 5 years in the low-risk group (2.67, 95% CI: 0.72–4.63%) by Kaplan-Meier method was significantly lower than that of the high-risk group (19.30, 95% CI: 12.34–26.27%) (p < 0.001). In the multiple Cox model, the RRS was proven to be a powerful classifier independent of age at diagnosis or tumour size (p < 0.001). In addition, we found that high RRS score ER-positive patients do not benefit from hormonal therapy treatment (RFS, p = 0.860).ConclusionThe RRS model can be applied to predict the relapse risk in early stage breast cancer. As such, high RRS score ER-positive patients do not benefit from hormonal therapy treatment.

The Role of ZNF143 in Breast Cancer Cell Survival Through the NAD(P)H Quinone Dehydrogenase 1⁻p53⁻Beclin1 Axis Under Metabolic Stress.

Autophagy is a cellular process that disrupts and uses unnecessary or malfunctioning components for cellular homeostasis. Evidence has shown a role for autophagy in tumor cell survival, but the molecular determinants that define sensitivity against autophagic regulation in cancers are not clear. Importantly, we found that breast cancer cells with low expression levels of a zinc-finger protein, ZNF143 (MCF7 sh-ZNF143), showed better survival than control cells (MCF7 sh-Control) under starvation, which was compromised with chloroquine, an autophagy inhibitor. In addition, there were more autophagic vesicles in MCF7 sh-ZNF143 cells than in MCF7 sh-Control cells, and proteins related with the autophagic process, such as Beclin1, p62, and ATGs, were altered in cells with less ZNF143. ZNF143 knockdown affected the stability of p53, which showed a dependence on MG132, a proteasome inhibitor. Data from proteome profiling in breast cancer cells with less ZNF143 suggest a role of NAD(P)H quinone dehydrogenase 1(NQO1) for p53 stability. Taken together, we showed that a subset of breast cancer cells with low expression of ZNF143 might exhibit better survival via an autophagic process by regulating the p53–Beclin1 axis, corroborating the necessity of blocking autophagy for the best therapy.

MiR-29a contributes to breast cancer cells epithelial\u2013mesenchymal transition, migration, and invasion via down-regulating histone H4K20 trimethylation through directly targeting SUV420H2

Breast cancer is the most prevalent cancer in women worldwide, which remains incurable once metastatic. Breast cancer stem cells (BCSCs) are a small subset of breast cancer cells which are essential in tumor formation, metastasis, and drug resistance. microRNAs (miRNAs) play important roles in the breast cancer cells and BCSCs by regulating specific genes. In this study, we found that miR-29a was up-regulated in BCSCs, in aggressive breast cancer cell line and in breast cancer tissues. We also confirmed suppressor of variegation 4–20 homolog 2 (SUV420H2), which is a histone methyltransferase that specifically trimethylates Lys-20 of histone H4 (H4K20), as the target of miR-29a. Both miR-29a overexpression and SUV420H2 knockdown in breast cancer cells promoted their migration and invasion in vitro and in vivo. Furthermore, we discovered that SUV420H2-targeting miR-29a attenuated the repression of connective tissue growth factor (CTGF) and growth response protein-1 (EGR1) by H4K20 trimethylation and promoted the EMT progress of breast cancer cells. Taken together, our findings reveal that miR-29a plays critical roles in the EMT and metastasis of breast cancer cells through targeting SUV420H2. These findings may provide new insights into novel molecular therapeutic targets for breast cancer.

Pharmacological Inhibition of PARP6 Triggers Multipolar Spindle Formation and Elicits Therapeutic Effects in Breast Cancer.

: PARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity. SIGNIFICANCE: These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.

Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Breast cancer stem cells (BCSCs), a small subset of breast cancer cells with stem cell-like properties, are essential in tumor formation, metastasis, resistance to anticancer therapies, and cancer recurrence. MicroRNAs (miRNAs) are involved in tumorigenicity by regulating specific oncogenes and tumor-suppressor genes, and their roles in BCSCs are becoming apparent. A novel, 3-dimensional (3D), semisolid culture system was established to culture MCF-7 spheroid cells with high percentage of BCSCs. The differences in miRNA expression among the MCF-7 parental cells, BCSC-enriched MCF-7 spheroid cells, and CD44+/CD24- MCF-7 cells were evaluated by miRNA microarray, and the high expression of miR-210 in MCF-7 spheroid cells and CD44+/CD24- MCF-7 cells was verified by quantitative RT-PCR. MCF-7 cells were cultured in a hypoxic chamber to detect the effect of hypoxia on miR-210 expression and the stemness of the cells. The 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl tetrazolium bromide (MTT), transwell, and sphere-formation assays were performed to detect the proliferation, migration, and self-renewal ability of miR-210-overexpressed MCF-7 cells and MCF-7 spheroid cells with miR-210 knocked down. The target of miR-210 was validated with a dual-luciferase reporter assay and Western blotting. In vivo xenograft assay and metastasis assay were performed to study the effects of miR-210 targeting E-cadherin on BCSCs growth and lung metastasis, and the tumors were assessed by immunohistochemistry and immunofluorescence. We developed a novel 3D, semisolid culture system to culture MCF-7 spheroid cells, which are enriched in BCSCs, and found, by performing miRNAs expression profiling, miR-210 was up-regulated in those cells compared with MCF-7 parental cells. High miR-210 expression was also detected in CD44+/CD24- MCF-7 cells and human CD44+/CD24- breast cancer cells, which was demonstrated to be partially due to the hypoxic microenvironment around BCSCs in MCF-7 spheroids or solid tumors. Ectopic expression of miR-210 in MCF-7 cells promoted their migration, invasion, proliferation, and self-renewal in both in vitro and in vivo studies. We further reported that miR-210 suppressed E-cadherin expression by targeting the open reading frame region of E-cadherin mRNA and by up-regulation of E-cadherin transcription repressor, Snail. Accordingly, E-cadherin overexpression compromises the migration, invasion, proliferation, and self-renewal ability of miR-210-overexpressed MCF-7 both in vitro and in vivo. These findings reveal a novel regulatory pathway centered on hypoxia-mediated miR-210 targeting of E-cadherin, which contributes to the properties and breast tumorigenesis of BCSCs.-Tang, T., Yang, Z., Zhu, Q., Wu, Y., Sun, K., Alahdal, M., Zhang, Y., Xing, Y., Shen, Y., Xia, T., Xi, T., Pan, Y., Jin, L. Up-regulation of miR-210 induced by a hypoxic microenvironment promotes breast cancer stem cells metastasis, proliferation, and self-renewal by targeting E-cadherin.

Internalization of CD239 highly expressed in breast cancer cells: a potential antigen for antibody-drug conjugates

Antibody–drug conjugates (ADCs) are attractive in cancer therapy because they can directly bind to cancer cells and provide anticancer activity. To kill cancer cells with ADCs, the target antigens are required not only to be highly and/or selectively expressed on cancer cells but also internalized by the cells. CD239, also known as the Lutheran blood group glycoprotein (Lu) or basal cell adhesion molecule (B-CAM), is a specific receptor for laminin α5, a major component of basement membranes. Here, we show that CD239 is strongly expressed in a subset of breast cancer cells and internalized into the cells. We also produced a human single-chain variable fragment (scFv) specific to CD239 fused with human IgG1 Fc, called C7-Fc. The binding affinity of the C7-Fc antibody is similar to that of mouse monoclonal antibodies. Although the C7-Fc antibody alone does not influence cellular functions, when conjugated with a fragment of diphtheria toxin lacking the receptor-binding domain (fDT), it can selectively kill breast cancer cells. Interestingly, fDT-bound C7-Fc shows anticancer activity in CD239-highly positive SKBR3 cells, but not in weakly positive cells. Our results show that CD239 is a promising antigen for ADC-based breast cancer therapy.

Phosphorylation of Sox2 at Threonine 116 is a Potential Marker to Identify a Subset of Breast Cancer Cells with High Tumorigenecity and Stem-Like Features

We have previously identified a novel phenotypic dichotomy in breast cancer (BC) based on the response to a SRR2 (Sox2 regulatory region 2) reporter, with reporter responsive (RR) cells being more tumorigenic/stem-like than reporter unresponsive (RU) cells. Since the expression level of Sox2 is comparable between the two cell subsets, we hypothesized that post-translational modifications of Sox2 contribute to their differential reporter response and phenotypic differences. By liquid chromatography-mass spectrometry, we found Sox2 to be phosphorylated in RR but not RU cells. Threonine 116 is an important phosphorylation site, since transfection of the T116A mutant into RR cells significantly decreased the SRR2 reporter luciferase activity and the RR-associated phenotype. Oxidative stress-induced conversion of RU into RR cells was accompanied by Sox2 phosphorylation at T116 and increased Sox2-DNA binding. In a cohort of BC, we found significant correlations between the proportion of tumor cells immuno-reactive with anti-phosphorylated Sox2T116 and a high tumor grade (p = 0.006), vascular invasion (p = 0.001) and estrogen receptor expression (p = 0.032). In conclusion, our data suggests that phosphorylation of Sox2T116 contributes to the tumorigenic/stem-like features in RR cells. Detection of phospho-Sox2T116 may be useful in identifying a small subset of tumor cells carrying stem-like/tumorigenic features in BC.

Molecular docking based screening of Noggin inhibitors.

Noggin (NOG) a BMP (bone morphogenetic protein) antagonist plays a key role in preferentially driving a subset of breast cancer cells towards the bone and causing osteolytic lesions leading to severe pain and discomfort in the patients. Owing to its role in bone metastasis, NOG could be promising molecular target in bone metastasis and that identifying small molecule inhibitors could aid in the treatment. Towards identifying cognate inhibitors of NOG, structure based virtual screen was employed. A total of 8.5 million ligands from e-molecule database were screened at a novel binding site on NOG identified by the Sitemap tool, employing GLIDE algorithm. Potential eight molecules were selected based on the Glide score, binding mode and H-bond interactions. Free energy of binding was calculated using Molecular mechanics based MMGBSA and the obtained energy was used in the prioritizing the compounds with the similar structures and glide score. Further, the compounds were evaluated for their druggability employing physico-chemical property analysis. Our study helped in identifying novel potential NOG inhibitors that can further be validated using in-vivo and in-vitro studies and these molecules can also be employed as tool compounds to study the functions of BMP.

Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer.

PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

Ultrasensitive Quantitation of Plasma Membrane Proteins via isRTA.

Quantitation of plasma membrane proteins (PMPs) is fundamental and frequently performed daily in the lab. However, challenged by the inherent/interacting heterostructures and complex surroundings of the PMPs in lipid membrane, quantitative techniques for PMP often require complex treatments (e.g., labeling, isolation, purification, and determination), and the sensitivity is usually not satisfactory. To address this problem, we have proposed a novel method that enables quantitation of PMPs with extremely high sensitivity, in an easier-to-manipulate and more streamlined way. This method is based on the design of an in situ rolling cycling replication-templated amplification strategy (isRTA). In fact, two rounds of DNA cascade isothermal amplifications have been conducted. The first round of amplification can provide templates for the second round of amplification; thus, significant enhancement of quantitative signals can be achieved. In this way, PMPs are quantified with ultrahigh sensitivity; as few as 25 copies of PMPs can be detected per cell. Moreover, the advantages of isRTA have been demonstrated by simultaneous identification of several PMP biomarkers (MUC1, EpCAM, and HER2) that are expressed over a wide distribution range on breast cancer cells. The precise typing of breast cancer cell subsets is thus possible because of the "quantitative-to-qualitative" strategy. Therefore, the unprecedented sensitivity and high usability of the isRTA method may present significant prospects for delving into membrane proteins and their related biofunctions in many research fields.

Immune modulation by a cellular network of mesenchymal stem cells and breast cancer cell subsets: Implication for cancer therapy

The immune modulatory properties of mesenchymal stem cells (MSCs) are mostly controlled by the particular microenvironment. Cancer stem cells (CSCs), which can initiate a clinical tumor, have been the subject of intense research. This review article discusses investigative studies of the roles of MSCs on cancer biology including on CSCs, and the potential as drug delivery to tumors. An understanding of how MSCs behave in the tumor microenvironment to facilitate the survival of tumor cells would be crucial to identify drug targets. More importantly, since CSCs survive for decades in dormancy for later resurgence, studies are presented to show how MSCs could be involved in maintaining dormancy. Although the mechanism by which CSCs survive is complex, this article focus on the cellular involvement of MSCs with regard to immune responses. We discuss the immunomodulatory mechanisms of MSC-CSC interaction in the context of therapeutic outcomes in oncology. We also discuss immunotherapy as a potential to circumventing this immune modulation.

Loss of Tpm4.1 leads to disruption of cell-cell adhesions and invasive behavior in breast epithelial cells via increased Rac1 signaling.

Here we report the identification and characterization of a novel high molecular weight isoform of tropomyosin, Tpm4.1, expressed from the human TPM4 gene. Tpm4.1 expression is down-regulated in a subset of breast cancer cells compared with untransformed MCF10A breast epithelial cells and in highly metastatic breast cancer cell lines derived from poorly metastatic MDA-MD-231 cells. In addition, patients with invasive ductal breast carcinoma show decreased TPM4 expression compared with patients with ductal breast carcinoma in situ, and low TPM4 expression is associated with poor prognosis. Loss of Tpm4.1 using siRNA in MCF10A cells increases cell migration in wound-healing and Boyden chamber assays and invasion out of spheroids as well as disruption of cell-cell adhesions. Down-regulation of Tpm4.1 in MDA-MB-231 cells leads to disruption of actin organization and increased cell invasion and dissemination from spheroids into collagen gels. The down-regulation of Tpm4.1 induces Rac1-mediated alteration of myosin IIB localization, and pharmacologic inhibition of Rac1 or down-regulation of myosin IIB using siRNA inhibits the invasive phenotypes in MCF10A cells. Thus Tpm4.1 plays an important role in blocking invasive behaviors through Rac1-myosin IIB signaling and our findings suggest that decreased expression of Tpm4.1 might play a crucial role during tumor progression.

A preliminary study of the role of extracellular -5'- nucleotidase in breast cancer stem cells and epithelial-mesenchymal transition.

Tumor stem cell theory may well explain a variety of malignant behaviors of tumors. Cells undergoing epithelial-mesenchymal transition (EMT) share many characteristics with tumor stem cells. Our previous studies showed that extracellular -5'- nucleotidase (CD73), one of the important surface markers of mesenchymal stem cells, may promote growth and metastasis of breast cancer cells both in vivo and in vitro. In this study, we assessed breast cancer stem cell (BCSC) markers [acetaldehyde dehydrogenase (ALDH)+ and CD44+CD24-] in various breast cancer cell lines with flow cytometry after overexpression (by lentivirus infection) or suppression (by siRNA interference) of CD73. We measured CD73 expression in breast cancer mammospheres with real-time PCR and western blots. Finally, we examined the expression of CD73 and EMT markers in different breast cancer cell lines, as well as in mammary cells (MCF10A) that underwent EMT induced by transforming growth factor beta (TGF-β). We found that CD73 positively correlated with ALDH+ or CD44+CD24- subsets of breast cancer cells. CD73 was expressed more in breast cancer mammospheres than in adherent cells. CD73 and mesenchymal marker expression was higher in breast cancer cells with more malignant features, while CD73 was lower in low malignant breast cancer cells with higher epithelial markers. Furthermore, CD73 expression increased during the process of TGF-β-induced EMT. Our results indicate that CD73 may play an important role in BCSCs.

Abstract A42: The role of M1 and M2 macrophage in the cycling phase of breast cancer

Abstract Breast cancer malignancy is the second leading cause of death among women. The majority of deaths associated with cancer, from solid tumors are the result of metastasis and not from the primary tumor site Clinical evidence indicates that breast cancer (BC) cells (BCC) can exist in dormancy within the bone marrow (BM), for &amp;gt;10 years. Methods by which BCCs adapt dormancy as well as reversed dormancy into metastatic cells are highly significant for the development of new and safe therapies. This research focuses on the BM because it is the preferred organ for dormant BCCs. Furthermore, in several cases, the BM has been identified as a source of BCCs during clinical resurgence. A subset of BCCs with tumor initiating property (cancer stem cells, CSCs) has been identified as those responsible for dormancy and chemoresistance. We will take advantage of our working hierarchy of BCCs to determine how M2 macrophage (MΦ) facilitates dormancy or clinical resurgence into metastatic cells. The mechanisms by which dormancy occurs followed by later resurgence into metastatic role for the BM microenvironment to facilitate CSC dormancy. CSCs can form gap junctional intercellular communication (GJIC) with stroma, by release of miRNA, which induces cycling quiescence. MΦ are classified into two subsets, either M1 or M2 type. M1 are mostly pro-inflammatory (classically activated to kill microorganisms and tumor cells), while M2 are non-inflammatory (alternatively activated to promote tissue repair). M2 MΦ is one cell type that comprises the stromal compartment inside the BM hematopoietic microenvironment. We studied how activated MSCs can influence M1 or M2 polarization. Our supporting studies indicate that M2 MΦ can arrest BCCs in G1/G0, while M1 MΦ can induce cell cycle progression. Activated MSCs transitioned M2 into M1 phenotype whereas inactivated MSCs maintains M2 MΦ phenotype. Here we present how the transitional changes in M2 to M1 MΦ affect the behavior of the CSCs within the BM stromal compartment. Citation Format: Nykia D. Walker, Michael Elias, Steve Greco, Pranela Rameshwar. The role of M1 and M2 macrophage in the cycling phase of breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A42.