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Small cell lung cancer (SCLC) is an aggressive neuroendocrine subtype of lung cancer with poor patient prognosis. However, the mechanisms that regulate SCLC progression and metastasis remain undefined. Here, we show that the expression of the slit guidance ligand 2 (SLIT2) tumor suppressor gene is reduced in SCLC tumors relative to adjacent normal tissue. In addition, the expression of the SLIT2 receptor, roundabout guidance receptor 1 (ROBO1), is upregulated. We find a positive association between SLIT2 expression and the Yes1 associated transcriptional regulator (YAP1)-expressing SCLC subtype (SCLC-Y), which shows a better prognosis. Using genetically engineered SCLC cells, adenovirus gene therapy, and preclinical xenograft models, we show that SLIT2 overexpression or the deletion of ROBO1 restricts tumor growth in vitro and in vivo. Mechanistic studies revealed significant inhibition of myeloid-derived suppressor cells (MDSCs) and M2-like tumor-associated macrophages (TAMs) in the SCLC tumors. In addition, SLIT2 enhances M1-like and phagocytic macrophages. Molecular analysis showed that ROBO1 knockout or SLIT2 overexpression suppresses the transforming growth factor beta 1 (TGF-β1)/β-catenin signaling pathway in both tumor cells and macrophages. Overall, we find that SLIT2 and ROBO1 have contrasting effects on SCLC tumors. SLIT2 suppresses, whereas ROBO1 promotes, SCLC growth by regulating the Tgf-β1/glycogen synthase kinase-3 beta (GSK3)/β-catenin signaling pathway in tumor cells and TAMs. These studies indicate that SLIT2 could be used as a novel therapeutic agent against aggressive SCLC.

Tumor-associated macrophages (TAM) are heterogeneous in nature and comprise antitumor M1-like (M1-TAM) or pro-tumor M2-like (M2-TAM) TAMs. M2-TAMs are a major component of stroma in breast tumors and enhance metastasis by reducing their phagocytic ability and increasing tumor fibrosis. However, the molecular mechanisms that regulate phenotypic plasticity of TAMs are not well known. Here we report a novel tumor suppressor Slit2 in breast cancer by regulating TAMs in the tumor microenvironment. Slit2 reduced the in vivo growth and metastasis of spontaneous and syngeneic mammary tumor and xenograft breast tumor models. Slit2 increased recruitment of M1-TAMs to the tumor and enhanced the ability of M1-TAMs to phagocytose tumor cells in vitro and in vivo. This Slit2-mediated increase in M1-TAM phagocytosis occurred via suppression of IL6. Slit2 was also shown to diminish fibrosis in breast cancer mouse models by increasing the expression of matrix metalloproteinase 13 in M1-TAMs. Analysis of patient samples showed high Slit2 expression strongly associated with better patient survival and inversely correlated with the abundance of CD163+ TAMs. Overall, these studies define the role of Slit2 in inhibiting metastasis by activating M1-TAMs and depleting tumor fibrosis. Furthermore, these findings suggest that Slit2 can be a promising immunotherapeutic agent to redirect TAMs to serve as tumor killers for aggressive and metastatic breast cancers. In addition, Slit2 expression along with CD163+ TAMs could be used as an improved prognostic biomarker in patients with breast cancer.Significance:This study provides evidence that the antitumor effect of Slit2 in breast cancer occurs by activating the phagocytic activity of M1-like tumor-associated macrophages against tumor cells and diminishing fibrosis.

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RAGE/mS100a7a15 axis enhances M2 macrophage recruitment to lungs.

S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways. Cancer Res; 72(3); 604–15. ©2011 AACR.

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RAGE is a multifunctional receptor implicated in diverse processes including inflammation and cancer. In this study, we report that RAGE expression is upregulated widely in aggressive triple-negative breast cancer (TNBC) cells, both in primary tumors and in lymph node metastases. In evaluating the functional contributions of RAGE in breast cancer, we found that RAGE-deficient mice displayed a reduced propensity for breast tumor growth. In an established model of lung metastasis, systemic blockade by injection of a RAGE neutralizing antibody inhibited metastasis development. Mechanistic investigations revealed that RAGE bound to the proinflammatory ligand S100A7 and mediated its ability to activate ERK, NF-κB, and cell migration. In an S100A7 transgenic mouse model of breast cancer (mS100a7a15 mice), administration of either RAGE neutralizing antibody or soluble RAGE was sufficient to inhibit tumor progression and metastasis. In this model, we found that RAGE/S100A7 conditioned the tumor microenvironment by driving the recruitment of MMP9-positive tumor-associated macrophages. Overall, our results highlight RAGE as a candidate biomarker for TNBCs, and they reveal a functional role for RAGE/S100A7 signaling in linking inflammation to aggressive breast cancer development. Cancer Res; 75(6); 974–85. ©2015 AACR.

Background: Metastasis is the major cause of mortality in breast cancer; however, the molecular mechanisms remain elusive. In our previous study, we demonstrated that S100A7/RAGE mediates breast cancer growth and metastasis by recruitment of tumor-associated macrophages. However, the downstream S100A7-mediated inflammatory oncogenic signaling cascade that enhances breast tumor growth and metastasis by generating the immunosuppressive tumor microenvironment (iTME) has not been studied. In this present study, we aimed to investigate the S100A7 and cPLA2 cross-talk in enhancing tumor growth and metastasis through enhancing the iTME.Methods: Human breast cancer tissue and plasma samples were used to analyze the expression of S100A7, cPLA2, and PGE2 titer. S100A7-overexpressing or downregulated human metastatic breast cancer cells were used to evaluate the S100A7-mediated downstream signaling mechanisms. Bi-transgenic mS100a7a15 overexpression, TNBC C3(1)/Tag transgenic, and humanized patient-derived xenograft mouse models and cPLA2 inhibitor (AACOCF3) were used to investigate the role of S100A7/cPLA2/PGE2 signaling in tumor growth and metastasis. Additionally, CODEX, a highly advanced multiplexed imaging was employed to delineate the effect of S100A7/cPLA2 inhibition on the recruitment of various immune cells.Results: S100A7 and cPLA2 are highly expressed and positively correlated in malignant breast cancer patients. S100A7/RAGE upregulates cPLA2/PGE2 axis in aggressive breast cancer cells. Furthermore, S100A7 is positively correlated with PGE2 in breast cancer patients. Moreover, cPLA2 pharmacological inhibition suppressed S100A7-mediated tumor growth and metastasis in multiple pre-clinical models. Mechanistically, S100A7-mediated activation of cPLA2 enhances the recruitment of immunosuppressive myeloid cells by increasing PGE2 to fuel breast cancer growth and its secondary spread. We revealed that cPLA2 inhibitor mitigates S100A7-mediated breast tumorigenicity by suppressing the iTME. Furthermore, CODEX imaging data showed that cPLA2 inhibition increased the infiltration of CD4+/CD8+ T cells in the TME. Analysis of metastatic breast cancer samples revealed a positive correlation between S100A7/cPLA2 with CD163+ tumor-associated M2-macrophages.Conclusions: Our study shows that cross-talk between S100A7 and cPLA2 plays an important role in enhancing breast tumor growth and metastasis by generating an immunosuppressive tumor microenvironment and reducing infiltration of T cells. Furthermore, S100A7 could be used as a novel non-invasive prognostic marker and cPLA2 inhibitors as promising drugs against S100A7-overexpressing metastatic breast cancer.

The role of commensal bacterial microbiota in the pathogenesis of human malignancies has been a research field of incomparable progress in recent years. Although breast tissue is commonly assumed to be sterile, recent studies suggest that human breast tissue may contain a bacterial microbiota. In this study, we used an immune-competent orthotopic breast cancer mouse model to explore the existence of a unique and independent bacterial microbiota in breast tumors. We observed some similarities in breast cancer microbiota with skin; however, breast tumor microbiota was mainly enriched with Gram-negative bacteria, serving as a primary source of lipopolysaccharide (LPS). In addition, dextran sulfate sodium (DSS) treatment in late-stage tumor lesions increased LPS levels in the breast tissue environment. We also discovered an increased expression of S100A7 and low level of TLR4 in late-stage tumors with or without DSS as compared to early-stage tumor lesions. The treatment of breast cancer cells with LPS increased the expression of S100A7 in breast cancer cells in vitro. Furthermore, S100A7 overexpression downregulated TLR4 and upregulated RAGE expression in breast cancer cells. Analysis of human breast cancer samples also highlighted the inverse correlation between S100A7 and TLR4 expression. Overall, these findings suggest that the commensal microbiota of breast tissue may enhance breast tumor burden through a novel LPS/S100A7/TLR4/RAGE signaling axis.

Cancer-associated fibroblasts (CAFs) play an active role in remodeling the local tumor stroma to support tumor initiation, growth, invasion, metastasis, and therapeutic resistance. The CAF-secreted chemokine, CXCL12, has been directly implicated in the tumorigenic progression of carcinomas, including breast cancer. Using a 3-D in vitro microfluidic-based microtissue model, we demonstrate that stromal CXCL12 secreted by CAFs has a potent effect on increasing the vascular permeability of local blood microvessel analogues through paracrine signaling. Moreover, genetic deletion of fibroblast-specific CXCL12 significantly reduced vessel permeability compared to CXCL12 secreting CAFs within the recapitulated tumor microenvironment (TME). We suspected that fibroblast-mediated extracellular matrix (ECM) remodeling and contraction indirectly accounted for this change in vessel permeability. To this end, we investigated the autocrine effects of CXCL12 on fibroblast contractility and determined that antagonistic blocking of CXCL12 did not have a substantial effect on ECM contraction. Our findings indicate that fibroblast-secreted CXCL12 has a significant role in promoting a leakier endothelium hospitable to angiogenesis and tumor cell intravasation; however, autocrine CXCL12 is not the primary upstream trigger of CAF contractility.

Background: Induced electric fields (iEFs) control directional breast cancer cell migration. While the connection between migration and metabolism is appreciated in the context of cancer and metastasis, effects of iEFs on metabolic pathways especially as they relate to migration, remain unexplored. Materials and Methods: Quantitative cell migration data in the presence and absence of an epidermal growth factor (EGF) gradient in the microfluidic bidirectional microtrack assay was retrospectively analyzed for additional effects of iEFs on cell motility and directionality. Surrogate markers of oxidative phosphorylation (succinate dehydrogenase [SDH] activity) and glycolysis (lactate dehydrogenase activity) were assessed in MDA-MB-231 breast cancer cells and normal MCF10A mammary epithelial cells exposed to iEFs and EGF. Results: Retrospective analysis of migration results suggests that iEFs increase forward cell migration speeds while extending the time cells spend migrating slowly in the reverse direction or remaining stationary. Furthermore, in the presence of EGF, iEFs differentially altered flux through oxidative phosphorylation in MDA-MB-231 cells and glycolysis in MCF10A cells. Conclusions: iEFs interfere with MDA-MB-231 cell migration, potentially, by altering mitochondrial metabolism, observed as an inhibition of SDH activity in the presence of EGF. The energy intensive process of migration in these highly metastatic breast cancer cells may be hindered by iEFs, thus, through hampering of oxidative phosphorylation.

Background:Triple negative breast cancer (TNBC) represents about 10-15% of all breast cancers. Given the lack of targeted therapies, chemotherapy and immunotherapy are the only treatment options. The five-year survival rate of TNBC patients is approximately 15% less than other forms of breast cancer. Therefore, there is an urgent need to develop novel effective therapeutics for TNBC.Material and Methods:RNA isolated from human immortalized mammary epithelial MCF10A, and various TNBC cell lines was DNase treated and quantitative RT-PCR (qRT-PCR) performed using gene-specific primers spanning exon-exon junctions that include introns in the corresponding genomic sequence to avoid genomic DNA amplification. Gene expression was calculated by ΔΔCt method using GAPDH as an internal control. Immuno-blot analyses were performed on lysates prepared from cells in log phase of growth. Proteins were detected by Enhanced Chemi-Luminescence (ECL) method. Viability of cells treated with ERβ specific agonists was determined by using CellTiter-Glo® 2.0 assay. MB231 cells were treated with ERβ agonists following transfection with pcDNA3 (vector) or Flag-tagged ESR2 plasmids. Western blot analyses using specific antibodies and qRT-PCR were performed with the protein extracts and RNA isolated from the cells, respectively. Results:Our experiments demonstrate that ESR2 (ERβ) is differentially expressed and its level of expression is about 3 to 22-fold higher in TNBC cell lines tested than MCF10A. We have shown that viability of estrogen receptor (ER) positive breast cancer cell lines can be significantly inhibited by selective activation of ERβ. A highly selective ERβ agonist has been invented in the Drug Development Institute at the Ohio State University. Treatment of TNBC lines with these ERβ agonists exhibited a dramatic reduction of cell viability. While comparable IC50 values were observed with our in house and commercially available compounds, TNBC had 2-3-fold lesser IC50 values than ER-positive cell lines. Increased expression of FOXO3 and c-Myc1 and decreased expression of c-Myc2 isoform was noted in ERβ overexpressing MB231 cells upon treatment with ERβ agonists.Conclusion:Our results demonstrate that treatment with highly selective ERβ agonists could be an effective therapeutic strategy for TNBC patients. To improve and develop novel therapeutics for TNBC understanding the mechanism of action of these compounds will be crucial. Citation Format: Jharna Datta, Natalie Willingham, Jasmine Manouchehri, Joel David, Mirisha Sheth, Bhuvaneswari Ramaswamy, Ramesh Ganju, Mathew A Cherian. Estrogen receptor beta agonists for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-39.

Background: Epidemiological studies indicate that prolonged breast feeding reduces the risk of triple negative breast cancer (TNBC), which carries the worst prognosis. Prolonged breastfeeding allows gradual involution (GI) of the breast while lack of or short-term breast feeding leads to abrupt involution (AI). We developed a novel murine model mimicking AI and GI of breast, and found that GI offers better protection to mammary glands from tissue remodeling associated injuries. Our data showed that AI leads to the development of pro-tumorigenic microenvironment and ductal hyperplasia1. Tissue remodeling involves orchestrated cell death and repopulation, and is closely associated with metabolic reprogramming from mitochondrial oxidative phosphorylation (OXPHOS) to glycolysis. Such metabolic alterations can contribute to cellular changes aiding malignant transformation2. We used our murine model to evaluate whether AI affects cellular metabolism differentially when compared to GI. Methods: Wild-type mice of FVB background were used in all our experiments. Twelve to fourteen week old uniparous mice were allowed to nurse (6 pups/dam) for 7 days postpartum. All pups were removed on day7 postpartum from the dams in AI cohort and three each on day28 and day31 from dams in GI cohort. Whole mammary glands and sorted luminal progenitor (LP) cells harvested on postpartum day28 were subjected to Affymetrix microarray analysis. Gene Set Enrichment Analysis (GSEA) was used to compute pathway enrichments in AI vs. GI glands. Differentially expressed genes were validated using qRT-PCR. Mammary glands harvested on day28 postpartum were subjected to mass spectrometry based untargeted metabolic profiling using Agilent QTOF. Raw data were analyzed using XCMS to assess key metabolic networks altered in AI vs GI glands. Targeted analysis for lactate, pyruvate, succinate and palmitic acid were performed using C13 labelled internal standards to compare OXPHOS vs glycolysis reliance in the AI and GI glands. Results: We observed enrichment of mitochondrial OXPHOS pathway, fatty acid metabolism and Myc target genes in both whole mammary gland and LP cells of AI vs. GI mice. Adipogenesis and hypoxia related genes were enriched in AI-glands. We observed significant upregulation of genes involved in glucose transport and fatty acid synthesis in AI glands, namely, Glut-5, Cidea, Acss2, Acsm3, Acly, Atp6v0d2, Acot11, and Elvol3. Several factors indicating a higher reliance on OXPHOS vs glycolysis, such as, Ppar-γ, Pgc1α, Cpt-2, Srebp1c and Chrebp were upregulated in AI glands. Upregulation of Cpt-2 and Srebp1c in the AI glands indicate higher flux through fatty acid oxidation and reliance on cholesterol synthesis. Metabolomic profiling revealed significant alteration in L-carnitine, GMP and XMP in AI glands which reflect mitochondrial fatty acid transport and nucleotide biosynthesis via guanine-guanosine salvage pathway. Pyruvate and lactate associated with glycolysis were increased in GI vs. AI glands. Conclusion: We show for the first time that in the abruptly involuting (AI) mammary glands following short-term breast feeding, there is a significant shift in the metabolic pathways towards mitochondrial OXPHOS and fatty acid oxidation compared to GI glands. Studies are underway to determine the effect of this metabolic shift on cellular transformation and tumorigenesis and the potential to target these pathways to reverse the detrimental effects of AI. 1. Basree MM, Shinde N, Koivisto C, et al. Breast Cancer Res. 2019; 21(1):80. 2. Ward PS, Thompson CB. Cancer Cell. 2012; 21(3):297. Citation Format: Neelam Shinde, Kirti Kaul, Allen Zhang, Saba Mehra, Resham Mawalkar, Hee Kyung Kim, Ramesh Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Abrupt involution of lactating mammary gland induces metabolic reprogramming conducive to pro-tumorigenic changes [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-28.

Triple-negative breast cancer (TNBC), defined as loss of estrogen, progesterone, and Her2 receptors, is a subtype of highly aggressive breast cancer with worse prognosis and poor survival rate. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine aberrantly expressed in many solid tumors and known to promote tumor progression and metastasis. However, its role in TNBC progression and metastasis is unexplored. Here we have shown that in TNBC patients, MIF expression was significantly enriched in the tumor compared to adjacent normal tissue. Using publically available patient datasets, we showed that MIF overexpression correlates with worse survival in TNBC compared to other hormonal status. Orthotopic implantation of TNBC cells into MIF knockout mice showed reduced tumor growth compared to wild-type mice. In addition, we have shown that MIF downregulation inhibits TNBC growth and progression in a syngeneic mouse model. We further showed that CPSI-1306, a small-molecule MIF inhibitor, inhibits the growth of TNBC cells in vitro. Mechanistic studies revealed that CPSI-1306 induces intrinsic apoptosis by alteration in mitochondrial membrane potential, cytochrome c (Cyt c) release, and activation of different caspases. In addition, CPSI-1306 inhibits the activation of cell survival and proliferation-related molecules. CPSI-1306 treatment also reduced the tumor growth and metastasis in orthotopic mouse models of mammary carcinoma. CPSI-1306 treatment of tumor-bearing mice significantly inhibited TNBC growth and pulmonary metastasis in a dose-dependent manner. Histological analysis of xenograft tumors revealed a higher number of apoptotic cells in CPSI-1306-treated tumors compared to vehicle controls. Our studies, for the first time, show that MIF overexpression in TNBC enhances growth and metastasis. Taken together, our results indicate that using small molecular weight MIF inhibitors could be a promising strategy to inhibit TNBC progression and metastasis.

BackgroundAmong women, breast cancer is the leading cause of cancer-related death worldwide. Estrogen receptor α-positive (ERα+) breast cancer accounts for 70% of all breast cancer subtypes. Although ERα+ breast cancer initially responds to estrogen deprivation or blockade, the emergence of resistance compels the use of more aggressive therapies. While ERα is a driver in ERα+ breast cancer, ERβ plays an inhibitory role in several different cancer types. To date, the lack of highly selective ERβ agonists without ERα activity has limited the exploration of ERβ activation as a strategy for ERα+ breast cancer.MethodsWe measured the expression levels of ESR1 and ESR2 genes in immortalized mammary epithelial cells and different breast cancer cell lines. The viability of ERα+ breast cancer cell lines upon treatments with specific ERβ agonists, including OSU-ERb-12 and LY500307, was assessed. The specificity of the ERβ agonists, OSU-ERb-12 and LY500307, was confirmed by reporter assays. The effects of ERβ agonists on cell proliferation, cell cycle, apoptosis, colony formation, cell migration, and expression of tumor suppressor proteins were analyzed. The expression of ESR2 and genes containing ERE-AP1 composite response elements was examined in ERα+ human breast cancer samples to determine the correlation between ESR2 expression and overall survival and that of putative ESR2-regulated genes.ResultsIn this study, we demonstrate the efficacy of highly selective ERβ agonists in ERα+ breast cancer cell lines and drug-resistant derivatives. ERβ agonists blocked cell proliferation, migration, and colony formation and induced apoptosis and S and/or G2/M cell-cycle arrest of ERα+ breast cancer cell lines. Also, increases in the expression of the key tumor suppressors FOXO1 and FOXO3a were noted. Importantly, the strong synergy between ERβ agonists and ERα antagonists suggested that the efficacy of ERβ agonists is maximized by combination with ERα blockade. Lastly, ESR2 (ERβ gene) expression was negatively correlated with ESR1 (ERα gene) and CCND1 RNA expression in human metastatic ERα+/HER2- breast cancer samples.ConclusionOur results demonstrate that highly selective ERβ agonists attenuate the viability of ERα+ breast cancer cell lines in vitro and suggest that this therapeutic strategy merits further evaluation for ERα+ breast cancer.

Chemotherapy forms the backbone of current treatments for many patients with advanced non-small-cell lung cancer (NSCLC). However, the survival rate is low in these patients due to the development of drug resistance, including cisplatin resistance. In this study, we developed a novel strategy to combat the growth of cisplatin-resistant (CR) NSCLC cells. We have shown that treatment with the plant-derived, non-psychotropic small molecular weight molecule, cannabidiol (CBD), significantly induced apoptosis of CR NSCLC cells. In addition, CBD treatment significantly reduced tumor progression and metastasis in a mouse xenograft model and suppressed cancer stem cell properties. Further mechanistic studies demonstrated the ability of CBD to inhibit the growth of CR cell lines by reducing NRF-2 and enhancing the generation of reactive oxygen species (ROS). Moreover, we show that CBD acts through Transient Receptor Potential Vanilloid-2 (TRPV2) to induce apoptosis, where TRPV2 is expressed on human lung adenocarcinoma tumors. High expression of TRPV2 correlates with better overall survival of lung cancer patients. Our findings identify CBD as a novel therapeutic agent targeting TRPV2 to inhibit the growth and metastasis of this aggressive cisplatin-resistant phenotype in NSCLC.

IntroductionBreast cancer affects two million patients worldwide every year and is the most common cause of cancer-related death among women. The triple-negative breast cancer (TNBC) sub-type is associated with an especially poor prognosis because currently available therapies fail to induce long-lasting responses. Therefore, there is an urgent need to develop novel therapies that result in durable responses. One universal characteristic of the tumor microenvironment is a markedly elevated concentration of extracellular adenosine triphosphate (eATP). Chemotherapy exposure results in further increases in eATP through its release into the extracellular space of cancer cells via P2RX channels. eATP is degraded by eATPases. Given that eATP is toxic to cancer cells, we hypothesized that augmenting the release of eATP through P2RX channels and inhibiting extracellular ATPases would sensitize TNBC cells to chemotherapy.MethodsTNBC cell lines MDA-MB 231, Hs 578t and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells were treated with increasing concentrations the chemotherapeutic agent paclitaxel in the presence of eATPases or specific antagonists of P2RXs with cell viability and eATP content being measured. Additionally, the mRNA, protein and cell surface expressions of the purinergic receptors P2RX4 and P2RX7 were evaluated in all examined cell lines via qRT-PCR, western blot, and flow cytometry analyses, respectively.ResultsIn the present study, we observed dose-dependent declines of cell viability and increases in eATP of paclitaxel-treated TNBC cell lines in the presence of inhibitors of eATPases, but not of the MCF-10A cell line. These effects were reversed by specific antagonists of P2RXs. Similar results, as those observed with eATPase inhibitors, were seen with P2RX activators. All examined cell lines expressed both P2RX4 and P2RX7 at the mRNA, protein and cell surface levels.ConclusionThese results reveal that eATP modulates the chemotherapeutic response in TNBC cell lines, which could be exploited to enhance the efficacy of chemotherapy regimens for TNBC.

Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.

BACKGROUND: Atherosclerosis of the aorta and coronary arteries is still one of the major causes of death. We recently reported obesity paradox between body mass index (BMI) and atherosclerosis of the aortas (AA) in morbidly obese decedent patients. The cause of this obesity paradox is unknown. The aim of the present study was to carry out genomic microarray analysis in order to determine gene expression profiles in the aortas of morbidly obese decedents with either mild or severe atherosclerosis of the aorta. METHODS: Microarray studies using Affymetrix GeneChips Clariom D Human array chips were performed on the aortas obtained from six morbidly obese decedents, three of whom had minimal AA and three who had severe disease. RESULTS: Group 1 (severe AA) and group 2 (mild AA) included 3 patients each. The patients were matched by age and BMI. There were significant (p

The first evidence that cannabinoids may have in vitro and in vivo antineoplastic activity against tumor cell lines and animal tumor models was published in the Journal of the National Cancer Institute nearly 50 years ago. Cannabinoids appear to induce apoptosis in rodent brain tumors by way of direct interaction with the cannabinoid receptor. They may inhibit angiogenesis and tumor cell invasiveness. Despite preclinical findings, attempts to translate the benefits from bench to bedside have been limited. This session provides a review of the basic science supporting the use of cannabinoids in gliomas, paired with the first randomized clinical trial of a cannabis-based therapy for glioblastoma multiforme. Another preclinical presentation reports the effects of cannabinoids on triple-negative breast cancer cell lines and how cannabidiol may affect tumors. The session’s second human trial raises concerns about the use of botanical cannabis in patients with advanced cancer receiving immunotherapy suggesting inferior outcomes.

Recent studies revealed that ethnic differences in mechanistic target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK-1/2) signaling pathways might be associated with the development and progression of different human malignancies. The African American (AA) population has an increased rate of cancer incidence and mortality compared to the Caucasian American (CA) population. Although the socioeconomic differences across different ethnic groups contribute to the disparity in developing different cancers, recent scientific evidence indicates the association of molecular and genetic variations in racial disparities of different human malignancies. The mTOR and ERK-1/2 signaling pathways are one of the well-known oncogenic signaling mechanisms that regulate diverse molecular and phenotypic aspects of normal as well as cancer cells in response to different external or internal stimuli. To date, very few studies have been carried out to explore the significance of racial disparity with abnormal mTOR and ERK-1/2 kinase signaling pathways, which may contribute to the development of aggressive human cancers. In this review, we discuss the differential regulation of mTOR and ERK-1/2 kinase signaling pathways across different ethnic groups, especially between AA and CA populations. Notably, we observed that key signaling proteins associated with mTOR and ERK-1/2 pathway including transforming growth factor-beta (TGF-β), Akt, and VEGFR showed racially disparate expression in cancer patients. Overall, this review article encompasses the significance of racially disparate signaling molecules related to mTOR/ERK1/2 and their potential in developing tailor-made anti-cancer therapies.

Purpose: Epidemiological data links lack of breastfeeding with increased risk of breast cancer, particularly triple negative breast cancer (TNBC). The involution process is characterized by mammary tissue remodeling, including adipocyte repopulation and re-differentiation fueled by metabolic rewiring. Our mouse model mimicking short-term breast feeding that leads to abrupt involution (AI) revealed a chronic inflammatory state in the mammary gland (MG) (Basree, et al. Breast Cancer Research; 2019). As metabolic dysfunction is linked to increased BC risk, we sought to elucidate the effects of AI and gradual involution (GI) on MG energy metabolism and associated oxidative stress. In addition, we evaluated the impact of AI and GI on whole body glucose metabolism and insulin response/resistance. Methods: FVB/n mice (8week old) were paired for breeding. At partum, dams were randomized to AI or GI cohort and standardized to 6 pups per dam. AI mice had pups removed on postpartum day 7 (d7) to mimic short-term breastfeeding. GI mice had 3 pups removed on day 28 and 31 each to mimic gradual weaning. MGs were harvested on d28, 56, and 120 postpartum. Prior to harvest, mice underwent an echoMRI and insulin tolerance test (ITT). At harvest, mice were fasted for 4 hours, and blood was collected for serum insulin measurement using Ultra-Sensitive Insulin ELISA. Lipid peroxidation (MDA) and DNA adduct formation (8-OHdG), which represent chronic oxidative stress, were measured in MG by ELISA. Whole MG RNA were subjected to affymetrix followed by gene set enrichment analyses (GSEA) and qPCR for target validation. MGs were also subjected to untargeted metabolomics analysis and Mitofuel flex assay to assess substrate dependence by Seahorse Bioanalyzer. Results: GSEA and qPCR revealed enrichment of fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) pathways in AI MGs at d28, further validated by expression of genes (PGC-1α, Cpt-2, Srebp1c, and Chrebp). Metabolites associated with FAO were enriched in AI MGs at d28 and 56. Mitofuel flex assay indicated a significantly higher dependence on FAO in AI MGs. On d56, fasted blood glucose was significantly higher in AI mice with serum insulin and HOMA-IR trending to be higher than in GI mice. Level of 8-OHdG was elevated in AI MGs at d120. AI mice trended to be heavier, have greater body fat, and lower lean mass than GI Mice at d120. Body weight and percent body fat on d120 were positively correlated to MDA concentration in the mammary gland. Conclusion: Our mouse models of AI and GI revealed early MG specific metabolic shift towards increased FAO and OXPHOS that persists over time in AI glands. Similarly, increased oxidative stress and its association with adiposity at d120 indicates continued effect of AI. This change in adiposity, altered systemic glucose metabolism and metabolic shift seen in AI mice may contribute to the higher risk of breast cancer. [K.O. and K.K. are co-first authors.] Citation Format: Kate Ormiston, Kirti Kaul, Neelam Shinde, Allen Zhang, Morgan Bauer, Hee Kyung Kim, Ramesh K. Ganju, Sarmila Majumder, Bhuvaneswari Ramaswamy. Lack of breast feeding may contribute to increased breast cancer risk by altering metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5814.

The intricate interplay between malignant cells and host cellular and non-cellular components play crucial role in different stages of tumor development, progression, and metastases. Tumor and stromal cells communicate to each other through receptors such as integrins and secretion of signaling molecules like growth factors, cytokines, chemokines and inflammatory mediators. Chemokines mediated signaling pathways have emerged as major mechanisms underlying multifaceted roles played by host cells during tumor progression. In response to tumor stimuli, host cells-derived chemokines further activates signaling cascades that support the ability of tumor cells to invade surrounding basement membrane and extra-cellular matrix. The host-derived chemokines act on endothelial cells to increase their permeability and facilitate tumor cells intravasation and extravasation. The tumor cells-host neutrophils interaction within the vasculature initiates chemokines driven recruitment of inflammatory cells that protects circulatory tumor cells from immune attack. Chemokines secreted by tumor cells and stromal immune and non-immune cells within the tumor microenvironment enter the circulation and are responsible for formation of a “pre-metastatic niche” like a “soil” in distant organs whereby circulating tumor cells “seed’ and colonize, leading to formation of metastatic foci. Given the importance of host derived chemokines in cancer progression and metastases several drugs like Mogamulizumab, Plerixafor, Repertaxin among others are part of ongoing clinical trial which target chemokines and their receptors against cancer pathogenesis. In this review, we focus on recent advances in understanding the complexity of chemokines network in tumor microenvironment, with an emphasis on chemokines secreted from host cells. We especially summarize the role of host-derived chemokines in different stages of metastases, including invasion, dissemination, migration into the vasculature, and seeding into the pre-metastatic niche. We finally provide a brief description of prospective drugs that target chemokines in different clinical trials against cancer.

Tumor-associated macrophages (TAM) are heterogeneous in nature and comprise antitumor M1-like (M1-TAM) or pro-tumor M2-like (M2-TAM) TAMs. M2-TAMs are a major component of stroma in breast tumors and enhance metastasis by reducing their phagocytic ability and increasing tumor fibrosis. However, the molecular mechanisms that regulate phenotypic plasticity of TAMs are not well known. Here we report a novel tumor suppressor Slit2 in breast cancer by regulating TAMs in the tumor microenvironment. Slit2 reduced the in vivo growth and metastasis of spontaneous and syngeneic mammary tumor and xenograft breast tumor models. Slit2 increased recruitment of M1-TAMs to the tumor and enhanced the ability of M1-TAMs to phagocytose tumor cells in vitro and in vivo. This Slit2-mediated increase in M1-TAM phagocytosis occurred via suppression of IL6. Slit2 was also shown to diminish fibrosis in breast cancer mouse models by increasing the expression of matrix metalloproteinase 13 in M1-TAMs. Analysis of patient samples showed high Slit2 expression strongly associated with better patient survival and inversely correlated with the abundance of CD163+ TAMs. Overall, these studies define the role of Slit2 in inhibiting metastasis by activating M1-TAMs and depleting tumor fibrosis. Furthermore, these findings suggest that Slit2 can be a promising immunotherapeutic agent to redirect TAMs to serve as tumor killers for aggressive and metastatic breast cancers. In addition, Slit2 expression along with CD163+ TAMs could be used as an improved prognostic biomarker in patients with breast cancer. Significance: This study provides evidence that the antitumor effect of Slit2 in breast cancer occurs by activating the phagocytic activity of M1-like tumor-associated macrophages against tumor cells and diminishing fibrosis.

Recent studies have demonstrated that racial differences can influence breast cancer incidence and survival rate. African American (AA) women are at two to three fold higher risk for breast cancer than other ethnic groups. AA women with aggressive breast cancers show worse prognoses and higher mortality rates relative to Caucasian (CA) women. Over the last few years, effective treatment strategies have reduced mortality from breast cancer. Unfortunately, the breast cancer mortality rate among AA women remains higher compared to their CA counterparts. The focus of this review is to underscore the racial differences and differential regulation/expression of genetic signatures in CA and AA women with breast cancer. Moreover, immune cell infiltration significantly affects the clinical outcome of breast cancer. Here, we have reviewed recent findings on immune cell recruitment in the tumor microenvironment (TME) and documented its association with breast cancer racial disparity. In addition, we have extensively discussed the role of cytokines, chemokines, and other cell signaling molecules among AA and CA breast cancer patients. Furthermore, we have also reviewed the distinct genetic and epigenetic changes in AA and CA patients. Overall, this review article encompasses various molecular and cellular factors associated with breast cancer disparity that affects mortality and clinical outcome.

Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) belong to a superfamily of nuclear receptors called steroid hormone receptors, which, upon binding ligand, dimerize and translocate to the nucleus where they activate or repress the transcription of a large number of genes, thus modulating critical physiologic processes. ERβ has multiple isoforms that show differing association with prognosis. Expression levels of the full length ERβ1 isoform are often lower in aggressive cancers as compared to normal tissue. High ERβ1 expression is associated with improved overall survival in women with breast cancer. The promise of ERβ activation, as a potential targeted therapy, is based on concurrent activation of multiple tumor suppressor pathways with few side effects compared to chemotherapy. Thus, ERβ is a nuclear receptor with broad-spectrum tumor suppressor activity, which could serve as a potential treatment target in a variety of human cancers including breast cancer. Further development of highly selective agonists that lack ERα agonist activity, will be necessary to fully harness the potential of ERβ.

Breast cancer is one of the leading causes of death among women in the United States. Among all the breast cancer subtypes, triple-negative breast cancer (TNBC) is denoted as having the highest rate of mortality due to inefficacious therapy, drug resistance, and high recurrences rate. Despite many therapeutic advances over the past decades, novel therapeutic approaches are required to treat TNBC. Although lysosomes have recently been identified as novel targets in a variety of cancers, not much known about their role in breast cancer especially in TNBC. Due to the altered features of lysosomes in cancerous cells, its functional ability affects cell proliferation, metabolism, adaptation, and autophagy in tumor cells. Thus, the induction of lysosomal membrane permeabilization (LMP) may be used as a novel approach in controlling the aberrant cellular functions in TNBC. Cannabinoid receptor 2 (CB2) is expressed in breast cancer and its agonist JWH-015 has been shown to inhibit TNBC growth and metastasis. However, not much is known about the mechanisms by which JWH015 inhibit growth and metastasis. To investigate JWH015-mediated therapeutic mechanisms, we examined the lysosome-mediated cell death in TNBC cells. We found that JWH015 induces cell death in TNBC cells through lysosomal disruption mechanism. We also showed that JWH-015 treatment increases releases of lysosomal proteins, cathepsin D/B, and autophagy marker LC3B and that leads to activation of apoptotic cell death signaling in TNBC. These alterations result in the formation of autolysosomes to promote LMP. Our findings elucidate the mechanism by which JWH-015 promotes LMP induced autophagy and regulates TNBC cell proliferation. These studies elucidate novel lysosomal-mediated cell death mechanisms through activation of CB2 receptors in TNBC. Citation Format: Chatterjee N, Das S, Ahiwar D, Mishra S, Kaul K, Ganju R. Cannabinoid receptors agonist induces lysosome-mediated cell death in TNBC [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-24.

Discoidin domain receptors (DDR1 and DDR2) are receptor tyrosine kinases that signal in response to collagen. We had previously shown that collagen binding leads to clustering of DDR1b, a process partly mediated by its extracellular domain (ECD). In this study, we investigated (i) the impact of the oligomeric state of DDR2 ECD on collagen binding and fibrillogenesis, (ii) the effect of collagen binding on DDR2 clustering, and (iii) the spatial distribution and phosphorylation status of DDR1b and DDR2 after collagen stimulation. Studies were conducted using purified recombinant DDR2 ECD proteins in monomeric, dimeric or oligomeric state, and MC3T3-E1 cells expressing full-length DDR2-GFP or DDR1b-YFP. We show that the oligomeric form of DDR2 ECD displayed enhanced binding to collagen and inhibition of fibrillogenesis. Using atomic force and fluorescence microscopy, we demonstrate that unlike DDR1b, DDR2 ECD and DDR2-GFP do not undergo collagen-induced receptor clustering. However, after prolonged collagen stimulation, both DDR1b-YFP and DDR2-GFP formed filamentous structures consistent with spatial re-distribution of DDRs in cells. Immunocytochemistry revealed that while DDR1b clusters co-localized with non-fibrillar collagen, DDR1b/DDR2 filamentous structures associated with collagen fibrils. Antibodies against a tyrosine phosphorylation site in the intracellular juxtamembrane region of DDR1b displayed positive signals in both DDR1b clusters and filamentous structures. However, only the filamentous structures of both DDR1b and DDR2 co-localized with antibodies directed against tyrosine phosphorylation sites within the receptor kinase domain. Our results uncover key differences and similarities in the clustering abilities and spatial distribution of DDR1b and DDR2 and their impact on receptor phosphorylation.

The chemokine CXCL12 has been shown to regulate breast tumor growth, however, its mechanism in initiating distant metastasis is not well understood. Here, we generated a novel conditional allele of Cxcl12 in mice and used a fibroblast-specific Cre transgene along with various mammary tumor models to evaluate CXCL12 function in the breast cancer metastasis. Ablation of CXCL12 in stromal fibroblasts of mice significantly delayed the time to tumor onset and inhibited distant metastasis in different mouse models. Elucidation of mechanisms using in vitro and in vivo model systems revealed that CXCL12 enhances tumor cell intravasation by increasing vascular permeability and expansion of a leaky tumor vasculature. Furthermore, our studies revealed CXCL12 enhances permeability by recruiting endothelial precursor cells and decreasing endothelial tight junction and adherence junction proteins. High expression of stromal CXCL12 in large cohort of breast cancer patients was directly correlated to blood vessel density and inversely correlated to recurrence and overall patient survival. In addition, our analysis revealed that stromal CXCL12 levels in combination with number of CD31+ blood vessels confers poorer patient survival compared to individual protein level. However, no correlation was observed between epithelial CXCL12 and patient survival or blood vessel density. Our findings describe the novel interactions between fibroblasts-derived CXCL12 and endothelial cells in facilitating tumor cell intrvasation, leading to distant metastasis. Overall, our studies indicate that cross-talk between fibroblast-derived CXCL12 and endothelial cells could be used as novel biomarker and strategy for developing tumor microenvironment based therapies against aggressive and metastatic breast cancer.

S100A7 has been shown to be associated with breast cancer growth and metastasis. However, the molecular mechanism through which S100A7 promotes breast tumorigenesis is not completely understood. In this study, we revealed that S100A7 enhanced Stat3-mediated secretion of Serpin-E1 in triple negative breast cancer (TNBC) cell lines. Serpin-E1 has been shown to promote recruitment and polarization toward a pro-tumorigenic phenotype of tumor-associated macrophages through activating the Stat3 pathway. Importantly, we reported that the inhibition of Stat3 led to reduction of S100A7-mediated invasiness of TNBC cells and also significantly decreased the secretion of Serpin-E1 by cancer cells. We also revealed that Stat3 inhibition in TNBC cells or Serpin-E1 neutralization significantly reduced the migration of human monocytes/macrophages by transwell migration assay. We further demonstrate that murine ortholog of S100A7, mS100a7a15 enhanced mammary hyperplasia, tumor growth and metastasis through Stat3 activation. Previously, we reported that S100A7 mediated its tumorigenic effects through RAGE in breast cancer cells. Therefore, we also discovered that combinatorial treatment of Stat3 and RAGE inhibitors in mammary tumor bearing mS100a7a15 overexpression mouse model strongly decreased the tumor burden and enhanced the recruitment of tumor-suppressing M1-macrophages. We also found that combined inhibition of RAGE and Stat3 significantly increased the infiltration of CD4 T cells in the tumor microenvironment (TME). Collectively, these findings suggest S100A7/RAGE signaling pathway could be used as a novel therapeutic target to inhibit breast cancer growth and metastasis through modulating the TME. Citation Format: Ajeet K Verma, Tasha Wilkie, Sanjay Mishra, Manish Charan, Ramesh K Ganju. S100a7/rage signaling promotes breast tumorigenesis through modulating tumor-associated macrophages [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-13.

Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and lacks the expression of estrogen, progesterone, and Her2 receptors. TNBC is a highly aggressive subtype of breast cancer that has been associated with poorer prognosis and worse overall survival rate. Macrophage migration inhibitory factor (MIF) is a multipotent pro-inflammatory cytokine, highly expressed in various cancers and reported to enhance tumor growth and metastasis. However, its role in influencing the anti-tumor immunity is unexplored. We have previously shown that MIF promotes TNBC growth and metastasis. In addition, CPSI-1306 (MIF inhibitor), reduces its oncogenic effects. Here, we have shown that MIF downregulation in human TNBC xenografts correlate with reduced infiltration of myeloid derived suppressor cells (MDSCs). Furthermore, using a MIF knockout mouse model, we detected reduced MDSC population in the tumor microenvironment. In addition, we observed higher recruitment and increased proliferation of CD3+ T cells. Furthermore, we utilized a syngeneic mouse model to study the effect of MIF inhibition on infiltration of different immune cells in-vivo. CPSI-1306 treatment decreased the recruitment of MDSCs in both the tumor and spleen. In addition, CPSI-1306 treatment promotes the infiltration of CD8+ T cells in both the tumor and spleen. However, CPSI-1306 treatment did not influence the recruitment of CD4+ T cells. We further analyzed the expression of various cytokines and chemokines in blood sera of control and CPSI-1306 treated mice groups using a cytokine array kit. We observed that CPSI-1306 treatment group showed reduced levels of GCSF, GMCSF, IL-2 and IL-4 levels compared to vehicle control groups. Overall, our data strongly suggests that small molecular weight MIF inhibitors could be a potential strategy against TNBC. Citation Format: Manish Charan, Subhadip Das, Sanjay Mishra, Sanjay Varikuti, Abhay R Satoskar, Ramesh K Ganju. Macrophage migration inhibitory factor regulates triple-negative breast cancer progression by enhancing the recruitment of immune suppressive cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-04-12.

This abstract was withdrawn by the authors. Citation Format: Ahirwar DK, Nasser MW, Shukla RK, Shilo K, Ganju RK. Withdrawn [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-07-09.

Interactions between cells and their environment influence key physiologic processes such as their propensity to migrate. However, directed migration controlled by extrinsically applied electrical signals is poorly understood. Using a novel microfluidic platform, we found that metastatic breast cancer cells sense and respond to the net direction of weak (∼100 µV cm−1), asymmetric, non-contact induced Electric Fields (iEFs). iEFs inhibited EGFR (Epidermal Growth Factor Receptor) activation, prevented formation of actin-rich filopodia, and hindered the motility of EGF-treated breast cancer cells. The directional effects of iEFs were nullified by inhibition of Akt phosphorylation. Moreover, iEFs in combination with Akt inhibitor reduced EGF-promoted motility below the level of untreated controls. These results represent a step towards isolating the coupling mechanism between cell motility and iEFs, provide valuable insights into how iEFs target multiple diverging cancer cell signaling mechanisms, and demonstrate that electrical signals are a fundamental regulator of cancer cell migration., Ayush Arpit Garg et al. demonstrate that metastatic breast cancer cells respond to weak, non-contact induced Electric Fields, reducing their motility in an Akt-dependent manner. This study provides insight into how non-contact induced Electric Fields regulate cancer cell signaling to hinder their migration.

The family of chemical structures that interact with a cannabinoid receptor are broadly termed cannabinoids. Traditionally known for their psychotropic effects and their use as palliative medicine in cancer, cannabinoids are very versatile and are known to interact with several orphan receptors besides cannabinoid receptors (CBR) in the body. Recent studies have shown that several key pathways involved in cell growth, differentiation and, even metabolism and apoptosis crosstalk with cannabinoid signaling. Several of these pathways including AKT, EGFR, and mTOR are known to contribute to tumor development and metastasis, and cannabinoids may reverse their effects, thereby by inducing apoptosis, autophagy and modulating the immune system. In this book chapter, we explore how cannabinoids regulate diverse signaling mechanisms in cancer and immune cells within the tumor microenvironment and whether they impart a therapeutic effect. We also provide some important insight into the role of cannabinoids in cellular and whole body metabolism in the context of tumor inhibition. Finally, we highlight recent and ongoing clinical trials that include cannabinoids as a therapeutic strategy and several combinational approaches towards novel therapeutic opportunities in several invasive cancer conditions.

Background A large collaborative analysis of data from 47 epidemiological studies concluded that longer duration of breastfeeding reduces the risk of developing breast cancer. Despite the strong epidemiological evidence, the molecular mechanisms linking prolonged breastfeeding to decreased risk of breast cancer remain poorly understood. Methods We modeled two types of breastfeeding behaviors in wild type FVB/N mice: (1) normal or gradual involution of breast tissue following prolonged breastfeeding and (2) forced or abrupt involution following short-term breastfeeding. To accomplish this, pups were gradually weaned between 28 and 31 days (gradual involution) or abruptly at 7 days postpartum (abrupt involution). Mammary glands were examined for histological changes, proliferation, and inflammatory markers by immunohistochemistry. Fluorescence-activated cell sorting was used to quantify mammary epithelial subpopulations. Gene set enrichment analysis was used to analyze gene expression data from mouse mammary luminal progenitor cells. Similar analysis was done using gene expression data generated from human breast samples obtained from parous women enrolled on a tissue collection study, OSU-2011C0094, and were undergoing reduction mammoplasty without history of breast cancer. Results Mammary glands from mice that underwent abrupt involution exhibited denser stroma, altered collagen composition, higher inflammation and proliferation, increased estrogen receptor α and progesterone receptor expression compared to those that underwent gradual involution. Importantly, when aged to 4 months postpartum, mice that were in the abrupt involution cohort developed ductal hyperplasia and squamous metaplasia. Abrupt involution also resulted in a significant expansion of the luminal progenitor cell compartment associated with enrichment of Notch and estrogen signaling pathway genes. Breast tissues obtained from healthy women who breastfed for

Increased circulating catecholamines have been linked with cardiovascular anomalies as well as with peripheral vascular diseases. Although the roles of epinephrine and norepinephrine have received considerable attention, the role of the other catecholamine, dopamine, has been less studied. Since dopamine is a potent endogenous inhibitor of angiogenesis and as angiogenesis is essential for ischemic healing, we therefore studied the role played by dopamine during ischemic healing using dopamine D2 receptor knockout (KOD2) mice. Although concentration of dopamine and its rate-limiting enzyme, tyrosine hydroxylase, was considerably high in the muscle tissues of wild-type and KOD2 mice with unilateral hind limb ischemia (HLI), recovery was significantly faster in the KOD2 mice compared to the wild-type controls, thereby indicating that peripheral dopamine might have a role in this healing process. In addition, we observed significant differences in post-ischemic angiogenesis between these two groups. Our study further revealed that elevated dopamine independently suppressed activation of local tissue-based renin-angiotensin system (RAS), a critical growth factor system stimulating angiogenesis in ischemia. Angiotensin II (ATII) and its receptor, angiotensin receptor type 1 (AT1R), are the key players in RAS-mediated angiogenesis. Dopamine acting through its D2 receptors in endothelial cells inhibited ATII-mediated angiogenesis by suppressing the expression of AT1R in these cells. This study thus for the first time demonstrates the role played by dopamine in prolonging post-ischemic recovery. Therefore, pharmacological intervention inhibiting the action of dopamine holds promise as future therapeutic strategy for the treatment of HLI and other peripheral arterial diseases.

Breast cancer is the second leading cause of cancer deaths among women. Cannabinoid receptor 2 (CNR2 or CB2) is an integral part of the endocannabinoid system. Although CNR2 is highly expressed in the breast cancer tissues as well as breast cancer cell lines, its functional role in breast tumorigenesis is not well understood. We observed that estrogen receptor-α negative (ERα-) breast cancer cells highly express epidermal growth factor receptor (EGFR) as well as insulin-like growth factor-I receptor (IGF-IR). We also observed IGF-IR upregulation in ERα+ breast cancer cells. In addition, we found that higher CNR2 expression correlates with better recurrence free survival in ERα- and ERα+ breast cancer patients. Therefore, we analyzed the role of CNR2 specific agonist (JWH-015) on EGF and/or IGF-I-induced tumorigenic events in ERα- and ERα+ breast cancers. Our studies showed that CNR2 activation inhibited EGF and IGF-I-induced migration and invasion of ERα+ and ERα- breast cancer cells. At the molecular level, JWH-015 inhibited EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and matrix metalloproteinases (MMPs). In vivo studies showed that JWH-015 significantly reduced breast cancer growth in ERα+ and ERα- breast cancer mouse models. Furthermore, we found that the tumors derived from JWH-015-treated mice showed reduced activation of EGFR and IGF-IR and their downstream targets. In conclusion, we show that CNR2 activation suppresses breast cancer through novel mechanisms by inhibiting EGF/EGFR and IGF-I/IGF-IR signaling axes.

// Mohamad Elbaz 1, 2, 5 , Dinesh Ahirwar 1, 2 , Zhang Xiaoli 3 , Xinyu Zhou 6 , Maryam Lustberg 4 , Mohd W. Nasser 1, 2 , Konstantin Shilo 1, 2 , Ramesh K. Ganju 1, 2 1 Department of Pathology, Wexner Medical Center, Ohio State University (OSU), OH, USA 2 The Comprehensive Cancer Center, Ohio State University (OSU), Wexner Medical Center, OH, USA 3 Center for Biostatistics, Ohio State University (OSU), OH, USA 4 Department of Internal Medicine, Ohio State University (OSU), OH, USA 5 Department of Pharmacology, Pharmacy School, Helwan University, Helwan, Egypt 6 Department of surgery, Davis Heart and Lung Research Institute, Ohio State University, OH, USA Correspondence to: Ramesh K. Ganju, email: Ramesh.ganju@osumc.edu Keywords: TRPV2, chemotherapy, TNBC, uptake, doxorubicin Abbreviations: Transient Receptor Potential Vanilloid type 2 (TRPV2), Triple negative Breast cancer (TNBC), Doxorubicin (Dox), CBD (cannabidiol). Received: March 24, 2016 Accepted: April 27, 2016 Published: May 27, 2016 ABSTRACT Transient receptor potential vanilloid type-2 (TRPV2) is an ion channel that is triggered by agonists like cannabidiol (CBD). Triple negative breast cancer (TNBC) is an aggressive disease with limited therapeutic options. Chemotherapy is still the first line for the treatment of TNBC patients; however, TNBC usually gains rapid resistance and unresponsiveness to chemotherapeutic drugs. In this study, we found that TRPV2 protein is highly up-regulated in TNBC tissues compared to normal breast tissues. We also observed that TNBC and estrogen receptor alpha negative (ERα - ) patients with higher TRPV2 expression have significantly higher recurrence free survival compared to patients with lower TRPV2 expression especially those who were treated with chemotherapy. In addition, we showed that TRPV2 overexpression or activation by CBD significantly increased doxorubicin (DOX) uptake and apoptosis in TNBC cells. The induction of DOX uptake was abrogated by TRPV2 blocking or downregulation. In vivo mouse model studies showed that the TNBC tumors derived from CBD+DOX treated mice have significantly reduced weight and increased apoptosis compared to those treated with CBD or DOX alone. Overall, our studies for the first time revealed that TRPV2 might be a good prognostic marker for TNBC and ERα - breast cancer patient especially for those who are treated with chemotherapy. In addition, TRPV2 activation could be a novel therapeutic strategy to enhance the uptake and efficacy of chemotherapy in TNBC patients.