Janani Ravi

S100A7 enhances mammary tumorigenesis through upregulation of inflammatory pathways

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.

RAGE Mediates S100A7-Induced Breast Cancer Growth and Metastasis by Modulating the Tumor Microenvironment

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.

Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway: Novel anti‐tumor mechanisms of Cannabidiol in breast cancer

The anti‐tumor role and mechanisms of Cannabidiol (CBD), a non‐psychotropic cannabinoid compound, are not well studied especially in triple‐negative breast cancer (TNBC). In the present study, we analyzed CBDs anti‐tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here ‐for the first time‐that CBD significantly inhibits epidermal growth factor (EGF)‐induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF‐induced activation of EGFR, ERK, AKT and NF‐kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor‐associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD‐treated cancer cells. The conditioned medium of CBD‐treated cancer cells also showed lower levels of GM‐CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows ‐for the first time‐that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.

Vitamin D receptor as a therapeutic target for benign prostatic hyperplasia

The bioactive form of vitamin D, 1α, 25-dihydroxyvitamin D3 (1α, 25(OH)2D3), is a secosteroid hormone that binds to the vitamin D receptor (VDR), a member of the nuclear receptor super-family expressed in many cell types, and modulates a variety of biological functions. 1α, 25(OH)2D3 is essential for bone and mineral homeostasis, but also regulates growth and differentiation of multiple cell types, and displays immunoregulatory and anti-inflammatory activities. The antiproliferative, prodifferentiative, antibacterial, immunomodulatory and anti-inflammatory properties of synthetic VDR agonists could be exploited to treat a variety of chronic inflammatory and autoimmune diseases, including benign prostatic hyperplasia (BPH). It has been hypothesized that VDR may influence both the risk of a variety of diseases and their occurrence and prognosis. However, earlier studies investigating the associations between specific VDR polymorphisms and various diseases often show controversial results. We performed a systematic review of the current literature on vitamin D and BPH using the PubMed and Web of Knowledge databases. The aim of this review is to summarize the current knowledge on the utility of the VDR gene regarding prostate growth as well as the pathogenesis and treatment of BPH, a complex syndrome characterized by a static component related to prostate overgrowth, a dynamic component responsible for urinary storage symptoms, and an inflammatory component. Despite the massive advances in recent decades, further research is needed to fully characterize the exact underlying mechanisms of VDR action on BPH and to comprehend how these cellular changes translate into clinical development in physical concert.

Cannabinoid receptor-2 agonist inhibits macrophage induced EMT in non-small cell lung cancer by downregulation of EGFR pathway.

JWH‐015, a cannabinoid receptor 2 (CB2) agonist has tumor regressive property in various cancer types. However, the underlying mechanism by which it acts in lung cancer is still unknown. Tumor associated macrophage (TAM) intensity has positive correlation with tumor progression. Also, macrophages recruited at the tumor site promote tumor growth by enhancing epithelial to mesenchymal (EMT) progression. In this study, we analyzed the role of JWH‐015 on EMT and macrophage infiltration by regulation of EGFR signaling. JWH‐015 inhibited EMT in NSCLC cells A549 and also reversed the mesenchymal nature of CALU‐1 cells by downregulation of EGFR signaling targets like ERK and STAT3. Also, in vitro co‐culture experiments of A549 with M2 polarized macrophages provided evidence that JWH‐015 decreased migratory and invasive abilities which was proved by reduced expression of FAK, VCAM1, and MMP2. Furthermore, it decreased macrophage induced EMT in A549 by attenuating the mesenchymal character by downregulating EGFR and its targets. These results were confirmed in an in vivo subcutaneous syngenic mouse model where JWH‐015 blocks tumor growth and also inhibits macrophage recruitment and EMT at the tumor site which was regulated by EGFR pathway. Finally, JWH‐015 reduced lung tumor lesions in an in vivo tumorigenicity mouse model. These data confer the impact of this cannabinoid on anti‐proliferative and anti‐tumorigenic effects, thus enhancing our understanding of its therapeutic efficacy in NSCLC. Our findings open new avenues for cannabinoid receptor CB2 agonist‐JWH‐015 as a novel and potential therapeutic target based on EGFR downregulation mechanisms in NSCLC.

Novel role of cannabinoid receptor 2 in inhibiting EGF/EGFR and IGF-I/IGF-IR pathways in breast cancer.

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.

Abstract 729: Novel role of CB2R in tuning breast tumor microenvironment, EGF/EGFR and IGF-I/IGF-IR pathways

Cannabinoid receptor-2 (CB2R) is an integral part of the endocannabinoid system. It is upregulated in the primary breast cancer lesions and in different types of immune cells however; its functional role in breast tumorigenesis is not well understood. The present study was aimed at evaluating the mechanistic anti-tumor role of CB2R activation on breast cancer cells and immune cells within the breast tumor microenvironment. First, we analyzed the anti-tumorigenic mechanisms of CB2R activation in ERa- and ERa+ breast cancer cells. Our studies showed that CB2R specific agonist (JWH-015) inhibits EGF and IGF-I-induced migration and invasion of ERa+ and ERa- breast cancer cells. At the molecular level, JWH-015 inhibits EGFR and IGF-IR activation and their downstream targets STAT3, AKT, ERK, NF-kB and MMP-9/MMP-2. Interestingly, We found that JWH-015 significantly reduces breast cancer growth in vivo and the tumors that were derived from CB2R agonist treatment showed reduced activation of EGFR and IGF-IR and their downstream targets compared to control group. Since CB2R is overexpressed in immune cells, we assessed for the role of CB2R activation on modulation of immune cells present in tumor stroma. We observed increased tumor weight, more myeloid derived suppressor cells (MDSCs) (CD11b+/Gr-1+) and less CD3+/CD8+ cells in orthotopically injected CB2R knock out mice compared to wild type mice. Furthermore, we found that JWH-015-treated wild type mice have reduced tumor growth and metastasis, more CD3+/CD8+ cells and less MDSCs within the tumor stroma. For the first time, we show that CB2R activation might suppress breast tumor growth and metastasis through novel mechanisms of inhibiting EGFR and IGF-IR signaling axes on tumor cells and modulating the immune cells’ compositions within the breast tumor microenvironment. Citation Format: Mohamad Elbaz, Mohd Nasser, Janani Ravi, Dinesh Ahirwar, Ramesh Ganju. Novel role of CB2R in tuning breast tumor microenvironment, EGF/EGFR and IGF-I/IGF-IR pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 729.

Abstract 3363: RAGE/S100A7/Stat3-axis enhances breast cancer growth and metastasis via modulating tumor microenvironment

The Receptor for Advanced Glycation Endproducts (RAGE), a multifunctional, multi-ligand receptor, has been shown to play an important role in inflammation. However, not much is known about its role in breast cancer growth and metastasis. In this report, we observed that RAGE expression is upregulated in triple negative breast cancer (TNBC) cell lines, primary tumors and lymph-node metastasis samples. RAGE-/- mice show reduced breast cancer growth. Additionally, blocking RAGE with neutralizing-antibody inhibited lung metastasis in an intracardiac mouse model. Further elucidation of RAGE-mediated mechanisms revealed that RAGE binds to S100A7 and mediates S100A7-induced cell migration, Stat3 and NF-ĸB activation. Our results also indicate that S100A7/RAGE axis-modulates invasion/migration through Stat3 dependent MMP9 activation. In addition, RAGE neutralizing antibody and soluble RAGE inhibited breast cancer progression and metastasis in the inducible mS100a7a15 mouse model system. We demonstrated that RAGE/S100A7 enhanced mammary hyperplasia, tumor growth and metastasis through Stat3 activation. Notably, our studies revealed that RAGE/mS100a7a15 modulates the breast tumor microenvironment through recruitment of phospho-Stat3/MMP9-positive tumor-associated macrophages. Our studies suggest that RAGE expression could be used as a novel biomarker for aggressive/invasive breast cancer, especially TNBC. Collectively, these findings suggest that RAGE/S100A7/Stat3-axis has a novel role in linking inflammation to the development of invasive/aggressive breast cancer including TNBC. Citation Format: Mohd W. Nasser, Nissar A. Wani, Janani Ravi, Grace A. Amponsah, Dinesh K. Ahirwar, Catherine A. Powell, Mohamad Elbaz, Helong Zhao, Konstantin Shilo, Ramesh K. Ganju. RAGE/S100A7/Stat3-axis enhances breast cancer growth and metastasis via modulating tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3363. doi:10.1158/1538-7445.AM2015-3363

Abstract 3324: The endocannabinoid system inhibits non-small cell lung cancer tumorigenesis by modulating the EGF/EGFR pathway

Non-small cell lung cancer (NSCLC) is a leading form of cancer with limited treatment regimens and is a major cause of cancer related mortality in the United States. The endocannabinoid system composed of two cannabinoid receptors- CB1 and CB2, their endogenous ligands and the enzymes for their metabolism has been implicated in several pathophysiological conditions. They have been extensively studied in various cancers as inhibitors of tumor cell growth by modulating key cell survival pathways. Anandamide (AEA), a well characterized neurotransmitter is an endogenous cannabinoid and agonist for the cannabinoid receptor CB1. One way to enhance the effect of endocannabinoid signaling is to inhibit the enzyme fatty acid amide hydrolase (FAAH), which metabolizes AEA by using the selective FAAH inhibitor URB597. To understand the physiological role of the endocannabinoid system, the NSCLC cell lines A549 and H460 were treated with either Met-F-AEA, URB597 or in combination and subjected to further analysis. Cells which were incubated in combination with Met-F-AEA and URB597 showed reduced proliferative and chemotactic activities in vitro when compared to AEA alone, which were confirmed by reduced MMP2 secretion and stress fiber formation. Also, we have shown that the combination treatment modulates activation of epidermal growth factor receptor (EGFR) and its downstream targets. In response to EGFR pathway activation, cells treated with Met-F-AEA in combination with URB597 underwent GO/G1 cell cycle arrest, ultimately leading to apoptosis via activation of caspase-9 and PARP. Furthermore, these results were validated in vivo in a xenograft nude mouse model system. Mouse xenografts treated with both Met-F-AEA and URB597 underwent reduction in tumor growth when compared to Met-F-AEA or URB597 alone. In summary, our results presented here show that Met-F-AEA in combination with URB597 inhibits the EGFR pathway, resulting in cell cycle arrest and apoptosis. Our findings suggest a novel perspective for the antitumorigenic activity of the endocannabinoid Met-F-AEA when used in combination with URB597 in NSCLC. Citation Format: Janani Ravi, Amita Sneh, Konstantin Shilo, Mohd Nasser, Ramesh Ganju. The endocannabinoid system inhibits non-small cell lung cancer tumorigenesis by modulating the EGF/EGFR pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3324. doi:10.1158/1538-7445.AM2014-3324

Abstract 388: mS100a7a15 enhances mammary tumor growth and metastasis by recruiting tumor associated macrophages

mS100a7a15 is the murine ortholog of human S100A7 and S100A15 proteins. Both S100A7 and S100A15 have been shown to play an important role in breast cancer. S100A7 has been shown to be highly expressed in high grade ductal carcinoma in situ and invasive breast cancers. Its expression is also related to poor prognosis and associated with increased inflammatory infiltrates and various inflammatory disorders. However, the exact mechanism by which S100A7 or S100A15 enhances breast cancer growth is not known. In the present study, we determined the molecular mechanisms by which mS100a7a15 enhances growth by overexpressing mS100a7a15 in MDA-MB-231 cells. We showed that mS100a7a15 enhances expression of proinflammatory molecules CXCL1 and CXCL8. In addition, we observed that supernatants obtained from mS100a7a15 overexpressing cells enhanced chemotaxis of murine RAW264.7 macrophages. Further elucidation revealed that mS100a7/a15 mediates its effects by binding to receptor for advanced glycation end products (RAGE). We further analyzed the role of mS100a7a15 on modulation of tumor growth and inflammatory pathways in breast cancer by generating inducible bi-transgenic MMTV-rtTA; mS100a7a15 mice (MMTV-mS100a7a15). These mice showed enhanced mS100a7a15 protein expression upon doxycycline treatment. Mammary glands isolated from these mice showed enhanced hyperplasia upon doxycycline treatment for 3 months compared to uninduced mice. Further studies revealed enhanced recruitment of macrophages in mammary glands and activation of STAT3 in induced mice. Orthotopic implantation of MVT-1 breast tumor cells (derived from MMTV-c-Myc; MMTV-VEGF mice) into the mammary glands of these mice showed enhanced tumor growth and metastasis in doxycycline treated mice compared to the control. Tumors and lung tissues obtained from these mice showed enhanced pro-metastatic gene expression and recruitment of F4/80 and CD206 positive M2 tumor-associated macrophages (TAM). However, no difference was observed in CD3 + and CD4 + T lymphocytes. Further elucidation of the role of macrophages by in vivo depletion of macrophages using clodronate liposomes revealed that mS100a7a15-mediated recruitment of TAM is important for tumor growth, angiogenesis and metastasis. Furthermore, mice treated with STAT3 inhibitors showed reduced mS100a7a15-induced hyperplasia. STAT3 has been shown to regulate expression of various inflammatory molecules. These studies using a novel mS100a7a15 bi-transgenic model system demonstrate that mS100a7a15 enhances breast tumor growth and metastasis by enhancing inflammatory signals that result in enhanced recruitment of TAM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 388. doi:1538-7445.AM2012-388