Mohamad Elbaz

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.

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.

Conditioning solid tumor microenvironment through inflammatory chemokines and S100 family proteins.

Recently, there has been growing attention to the role of the tumor microenvironment (TME) in cancer growth, metastasis and emergence of chemotherapy resistance. Stromal and tumor cells make up the TME and interact with each other through a complex cross-talk manner. This interaction is facilitated by a variety of growth factors, cytokines, chemokines and S100 proteins. In this review, we focus on chemokines and their cognate receptors in regulating the tumorigenic process. Chemokines are cytokines that have chemotactic potential. Chemokine receptors are expressed on tumor cells and stromal cells. Chemokines and their cognate receptors modulate tumor growth and metastasis in a paracrine and autocrine manner. They play a major role in the modulation of stromal cell recruitment, angiogenic potential, cancer cell proliferation, survival, adhesion, invasion and metastasis to distant sites. In addition, a new class of calcium binding family S100 proteins has been getting attention as they play significant roles in tumor progression and metastasis by modulating TME. Here, we highlight recent developments regarding the inflammatory chemokine/S100 protein systems in the TME. We also focus on how chemokines/S100 proteins, through their role in the TME, modulate cancer cell ability to grow, proliferate, invade and metastasize to different organs. This review highlights the possibility of using the chemokine/chemokine receptor axis as a promising strategy in cancer therapy, the current difficulties in achieving this goal, and how it could be overcome for successful future therapeutic intervention.

Fibroblast-derived CXCL12 promotes breast cancer metastasis by facilitating tumor cell intravasation

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.

TRPV2 is a novel biomarker and therapeutic target in triple negative breast cancer

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 (ERβ-) 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 ERβ- 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.

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.

STAT1 gene deficient mice develop accelerated breast cancer growth and metastasis which is reduced by IL-17 blockade

ABSTRACT Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling which activates the expression of various genes related to apoptosis, inflammation, cell cycle and angiogenesis. Several experimental and clinical studies have investigated the role of STAT1 in primary tumor growth in breast cancer; however, its role in tumor metastasis remains to be determined. To determine the role of STAT1 in breast cancer metastasis, we analyzed growth and metastasis in WT or STAT1−/− mice orthotopically implanted with metastatic 4T1.2 cells. Primary tumor development was faster in STAT1−/− mice and these mice developed significantly bigger primary tumors and displayed more lung metastasis compared with WT counterparts. STAT1−/− mice showed elevated Ly6G+CD11b+ granulocytic MDSC infiltration in their primary tumors and spleens with concomitant upregulation of Mmp9 and Cxcl1 expression in tumors compared with WT counterparts. Blockade of IL-17A in primary tumor-bearing STAT1−/− mice suppressed accumulation of Ly6G+CD11b+ cells and markedly reduced lung metastasis. These data show that STAT1 is an important suppressor of primary breast tumor growth and metastasis. Importantly, we found anti-IL-17 treatment can rescue STAT1 deficient animals from developing exacerbated metastasis to the lungs which could be important for immunotherapies for immunocompromised breast cancer patients.

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 916: Slit2 inhibits breast cancer growth and metastasis by modulating tumor microenvironment

Breast cancer (BC) continues to be a major health issue particularly in developed countries. The Slit2 gene is hyper-methylated in BC and acts through Roundabout Homolog1 (Robo1) receptor. Slit2/Robo1 signaling has been shown to inhibit the migration of a variety of cells including BC cells. Recently, it has also been reported to improve chemotherapy outcome in intestinal cancer. However, Slit2/Robo1-mediated mechanisms that regulate tumor growth and metastasis are not well known. Using syngeneic mouse model, we report that the over-expression of Slit2 using Adeno-Slit2 transduction successfully restrains tumor growth and also restrict the metastasis to lung, compared to Adeno-null control. To explore the Slit2 mediated cellular events, we analyzed the stromal cells recruitment to the tumor and observed reduced number of tumor associated macrophages (TAMs) in the Adeno-Slit2 group compared to Adeno-null group. We further analyzed the underlying molecular mechanisms and found that the soluble Slit2 inhibits TGF-β1 mediated alternative activation of macrophages to TAMs. Treatment with TGF-β1 significantly enhances the markers of TAMs by upregulating β-catenin levels. Interestingly, we observed that Soluble Slit2 inhibits TGF-β1 mediated activation of TAMs by inhibiting downstream signaling molecules Smad2 and β-catenin. We also analyzed Slit2 expression in human breast cancer TMA and an inverse correlation of Slit2 expression was observed with the incidence of breast cancer, metastasis and macrophage recruitment. This study highlights the ability of Slit2 to prevent macrophage alternative activation to TAMs, thereby restrict tumor growth and lung metastasis. Anti-cancer activity of Adeno-Slit2 suggests therapeutic potential of Slit2 to treat advanced breast cancer. Citation Format: Dinesh K. Ahirwar, Mohd W. Nasser, Mohamad Elbaz, Kontestine Shilo, Ramesh Ganju. Slit2 inhibits breast cancer growth and metastasis by modulating tumor microenvironment. [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 916.