Dinesh K. Ahirwar

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.

C-X-C motif chemokine 12/C-X-C chemokine receptor type 7 signaling regulates breast cancer growth and metastasis by modulating the tumor microenvironment.

IntroductionAlthough C-X-C motif chemokine 12 (CXCL12) has been shown to bind to C-X-C chemokine receptor type 7 (CXCR7), the exact molecular mechanism regulations by CXCL12/CXCR7 axis in breast tumor growth and metastasis are not well understood. CXCR7 expression has been shown to be upregulated during pathological processes such as inflammation and cancer.MethodsBreast cancer cell lines were genetically silenced or pharmacologically inhibited for CXCR7 and/or its downstream target signal transducer and activator of transcription 3 (STAT3). 4T1 or 4T1 downregulated for CXCR7 and 4T1.2 breast cancer cell lines were injected in mammary gland of BALB/c mice to form tumors, and the molecular pathways regulating tumor growth and metastasis were assessed.ResultsIn this study, we observed that CXCL12 enhances CXCR7-mediated breast cancer migration. Furthermore, genetic silencing or pharmacologic inhibition of CXCR7 reduced breast tumor growth and metastasis. Further elucidation of mechanisms revealed that CXCR7 mediates tumor growth and metastasis by activating proinflammatory STAT3 signaling and angiogenic markers. Furthermore, enhanced breast tumorigenicity and invasiveness were associated with macrophage infiltration. CXCR7 recruits tumor-promoting macrophages (M2) to the tumor site through regulation of the macrophage colony-stimulating factor (M-CSF)/macrophage colony-stimulating factor receptor (MCSF-R) signaling pathway. In addition, CXCR7 regulated breast cancer metastasis by enhancing expression of metalloproteinases (MMP-9, MMP-2) and vascular cell-adhesion molecule-1 (VCAM-1). We also observed that CXCR7 is highly expressed in invasive ductal carcinoma (IDC) and metastatic breast tissue in human patient samples. In addition, high CXCR7 expression in tumors correlates with worse prognosis for both overall survival and lung metastasis-free survival in IDC patients.ConclusionThese observations reveal that CXCR7 enhances breast cancer growth and metastasis via a novel pathway by modulating the tumor microenvironment. These findings identify CXCR7-mediated STAT3 activation and modulation of the tumor microenvironment as novel regulation of breast cancer growth and metastasis. These studies indicate that new strategies using CXCR7 inhibitors could be developed for antimetastatic therapy.

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.

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.

Non-contact method for directing electrotaxis

We present a method to induce electric fields and drive electrotaxis (galvanotaxis) without the need for electrodes to be in contact with the media containing the cell cultures. We report experimental results using a modification of the transmembrane assay, demonstrating the hindrance of migration of breast cancer cells (SCP2) when an induced a.c. electric field is present in the appropriate direction (i.e. in the direction of migration). Of significance is that migration of these cells is hindered at electric field strengths many orders of magnitude (5 to 6) below those previously reported for d.c. electrotaxis, and even in the presence of a chemokine (SDF-1α) or a growth factor (EGF). Induced a.c. electric fields applied in the direction of migration are also shown to hinder motility of non-transformed human mammary epithelial cells (MCF10A) in the presence of the growth factor EGF. In addition, we also show how our method can be applied to other cell migration assays (scratch assay), and by changing the coil design and holder, that it is also compatible with commercially available multi-well culture plates.

Endothelial Robo4 suppresses breast cancer growth and metastasis through regulation of tumor angiogenesis

Targeting tumor angiogenesis is a promising alternative strategy for improvement of breast cancer therapy. Robo4 (roundabout homolog 4) signaling has been shown to protect endothelial integrity during sepsis shock and arthritis, and inhibit Vascular Endothelial Growth Factor (VEGF) signaling during pathological angiogenesis of retinopathy, which indicates that Robo4 might be a potential target for angiogenesis in breast cancer. In this study, we used immune competent Robo4 knockout mouse model to show that endothelial Robo4 is important for suppressing breast cancer growth and metastasis. And this effect does not involve the function of Robo4 on hematopoietic stem cells. Robo4 inhibits breast cancer growth and metastasis by regulating tumor angiogenesis, endothelial leakage and tight junction protein zonula occludens protein‐1 (ZO‐1) downregulation. Treatment with SecinH3, a small molecule drug which deactivates ARF6 downstream of Robo4, can enhance Robo4 signaling and thus inhibit breast cancer growth and metastasis. SecinH3 mediated its effect by reducing tumor angiogenesis rather than directly affecting cancer cell proliferation. In conclusion, endothelial Robo4 signaling is important for suppressing breast cancer growth and metastasis, and it can be targeted (enhanced) by administrating a small molecular drug.

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.

Psoriasin (S100A7): a novel mediator of angiogenesis.

expression in melanoma cell lines and dermal fibroblasts indicated that all cells expressed cytoplasmic CXCL12 [detected by immunofluorescence (IF)], this cytoplasmic expression pattern did not correlate with the ability of the cells to secrete detectable levels of CXCL12 – a potential complication worth noting when interpreting CXCL12 IHC and IF expression studies. Nevertheless, elevated expression of CXCL12 in the tumour microenvironment (epidermis) correlated with an increase in the time taken to metastasize, the implication being that the high CXCL12 gradient in the epidermis would restrict deeper melanoma invasion and promote radial rather than vertical melanoma growth through the dermis. CXCL12 expression was downregulated in BRAF/NRAS-mutant tumours compared with wild-type tumours revealing a potential mode of regulation of CXCL12 expression by MAPK activation. It would be of interest now to evaluate the extent of the causal relationship between loss of CXCL12 expression and the mutant RAS/RAF melanoma phenotype. Little expression of the CXCR7 receptor was evident in primary melanoma tumour tissue, but CXCR7 expression was detected in tumour-associated endothelial cells, suggesting further potential tumour/microenvironment interactions and adding to the potential complexity of chemokine biology in melanoma. Indeed, expression of CXCR7 on tumour-associated endothelial cells in breast cancer restricts metastasis emphasizing the importance of the microenvironment in disease progression. In Desnoyer et al., expression of the CXCL12–CXCR4– CXCR7 trio is analysed in KS. KS has a complex aetiology involving a skin microenvironment loaded with growth factors and cytokines, immune suppression and human herpesvirus-8 infection. The authors report that CXCL12, CXCR4 and CXCR7 are all upregulated in both acquired immune deficiency syndrome-associated (n = 12) and classic (n = 12) KS when compared with benign angioma tissue (n = 10). This research builds on previous in vitro data implicating the trio in KS pathobiology but it extends the number of patient samples analysed for chemokine and chemokine receptor expression, and importantly provides analysis of clinical correlates of KS-type and lesion stage (macules, papules or nodules). The proportions of CXCL12, CXCR4 and CXCR7 positive cells were significantly higher in nodules (70%) compared with the less severe macules and papules. Expression of the trio therefore increases with lesion severity. Unfortunately, plasma CXCL12 levels did not correlate with the levels of detection of CXCL12 in lesions, so would not be useful clinically as a blood-borne biomarker for disease progression, severity or response to treatment. Both studies highlight the potential for CXCL12–CXCR4– CXCR7 as biomarkers in skin neoplasias. Further study is warranted with larger patient cohorts, coupled with the integration of these findings with expression patterns on lymphocytes and investigation of the significance and role of cytoplasmic ligand and cytoplasmic and nuclear receptor expression in tumour cell biology. This should help determine when the CXCL12–CXCR4–CXCR4 axis acts to promote disease progression and thus when it might be usefully targeted. In the future, specific reagents will be needed that can detect the six different CXCL12 isoforms (a, b, c, d, e and φ). These will be invaluable in deciphering the potentially different roles of the isoforms in cancer development.