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Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2. CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation.Implications:This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells.

Supplementary Data from Transforming Growth Factor-β Signaling–Deficient Fibroblasts Enhance Hepatocyte Growth Factor Signaling in Mammary Carcinoma Cells to Promote Scattering and Invasion

Supplementary Figure 1. Molecular characterization of fibroblasts. Supplemental Figure 2. Fibroblasts do not significantly affect metastasis of MCF10CA1d breast tumor xenografts. Supplemental Figure 3. CCR2 knockdown in 4T1 mammary carcinoma cells inhibits fibroblast mediated tumor growth. Supplementary Figure 4. CCL2 not affect AKT, SMAD3 or ERK1/2 phosphorylation in MCF10CA1d breast cancer cells. Supplemental Figure 5. Heatmap analysis of cell growth and cell cycle related proteins in CCR2 deficient breast cancer cells.

Supplemental Figures S1-S7

Fibroblasts are major cellular components of the tumor microenvironment, regulating tumor cell behavior in part through secretion of extracellular matrix proteins, growth factors, and angiogenic factors. In previous studies, conditional deletion of the type II transforming growth factor-β (TGF-β) receptor in fibroblasts (Tgfbr2FspKO) was shown to promote mammary tumor metastasis in fibroblast-epithelial cell cotransplantation studies in mice, correlating with increased expression of hepatocyte growth factor (HGF). Here, we advance our findings to show that Tgfbr2FspKO fibroblasts enhance HGF/c-Met and HGF/Ron signaling to promote scattering and invasion of mammary carcinoma cells. Blockade of c-Met and Ron by small interfering RNA silencing and pharmacologic inhibitors significantly reduced mammary carcinoma cell scattering and invasion caused by Tgfbr2FspKO fibroblasts. Moreover, neutralizing antibodies to c-Met and Ron significantly inhibited HGF-induced cell scattering and invasion, correlating with reduced Stat3 and p42/44MAPK phosphorylation. Investigation of the signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways by pharmacologic inhibition and small interfering RNA silencing revealed a cooperative interaction between the two pathways to regulate HGF-induced invasion, scattering, and motility of mammary tumor cells. Furthermore, whereas c-Met was found to regulate both the Stat3 and MAPK signaling pathways, Ron was found to regulate Stat3 but not MAPK signaling in mammary carcinoma cells. These studies show a tumor-suppressive role for TGF-β signaling in fibroblasts, in part by suppressing HGF signaling between mammary fibroblasts and epithelial cells. These studies characterize complex functional roles for HGF and TGF-β signaling in mediating tumor-stromal interactions during mammary tumor cell scattering and invasion, with important implications in the metastatic process. (Mol Cancer Res 2008;6(10):1521–33)

Description of additional details of materials and methodologies used in this study.

Supplemental Figure S2 Braf conditioned media does not induce TGFβ activation in CAFs.

Contributions of the tumor microenvironment (TME) to progression in thyroid cancer are largely unexplored and may illuminate a basis for understanding rarer aggressive cases of this disease. In this study, we investigated the relationship between the TME and thyroid cancer progression in a mouse model where thyroid-specific expression of oncogenic BRAF and loss of Pten (BrafV600E/Pten−/−/TPO-Cre) leads to papillary thyroid cancers (PTC) that rapidly progress to poorly differentiated thyroid cancer (PDTC). We found that fibroblasts were recruited to the TME of BrafV600E/Pten−/−/TPO-Cre thyroid tumors. Conditioned media from cell lines established from these tumors, but not tumors driven by mutant H-ras, induced fibroblast migration and proliferation in vitro. Notably, the extracellular matrix of BrafV600E/Pten−/−/TPO-Cre tumors was enriched with stromal-derived fibrillar collagen, compared with wild-type or Hras-driven tumors. Further, type I collagen enhanced the motility of BrafV600E/Pten−/−/TPO-Cre tumor cells in vitro. In clinical specimens, we found COL1A1 and LOX to be upregulated in PTC and expressed at highest levels in PDTC and anaplastic thyroid cancer. Additionally, increased expression levels of COL1A1 and LOX were associated with decreased survival in thyroid cancer patients. Overall, our results identified fibroblast recruitment and remodeling of the extracellular matrix as pivotal features of the TME in promoting thyroid cancer progression, illuminating candidate therapeutic targets and biomarkers in advanced forms of this malignancy. Cancer Res; 76(7); 1804–13. ©2016 AACR.

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Supplementary Tables 1-2 from Enhanced Hepatocyte Growth Factor Signaling by Type II Transforming Growth Factor-β Receptor Knockout Fibroblasts Promotes Mammary Tumorigenesis

Transforming growth factor-β (TGF-β) plays complex dual roles as an inhibitor and promoter of tumor progression. Although the influence of the stromal microenvironment on tumor progression is well recognized, little is known about the functions of TGF-β signaling in the stroma during tumor progression. Using cre-lox technology, expression of the type II TGF-β receptor was selectively knocked out in fibroblasts (Tgfbr2FspKO). In a co-xenograft model, we show that Tgfbr2FspKO fibroblasts enhance mammary carcinoma growth and metastasis in mice while increasing hepatocyte growth factor (HGF) expression and c-Met signaling downstream pathways including signal transducers and activators of transcription 3 (Stat3) and p42/44 mitogen-activated protein kinase (MAPK). Treatment of tumor-bearing mice with a pharmacologic inhibitor (EXEL-7592) of c-Met blocks tumor progression and reduces levels of phospho-Stat3 and phospho-p42/44 MAPK. Similarly, small interfering RNA knockdown of c-Met expression in mammary tumor cells reduces metastasis and c-Met signaling caused by Tgfbr2FspKO fibroblasts. The results show that TGF-β signaling in fibroblasts suppresses tumor metastasis by antagonizing HGF/c-Met signaling within tumor epithelial cells. Furthermore, this co-xenograft model represents a unique context to study stromal TGF-β and HGF signaling in mammary tumorigenesis. [Cancer Res 2007;67(10):4869–77]

Supplementary Methods from Enhanced Hepatocyte Growth Factor Signaling by Type II Transforming Growth Factor-β Receptor Knockout Fibroblasts Promotes Mammary Tumorigenesis

Objective: One of the leading causes of death following traumatic injury is exsanguination. Biological material-based hemostatic agents such as fibrin, thrombin, and albumin have a high risk for causing infection. Synthetic peptide-based hemostatic agents offer an attractive alternative. The objective of this study is to explore the potential of h9e peptide as an effective hemostatic agent in both in vitro and in vivo models. Approach: In vitro blood coagulation kinetics in the presence of h9e peptide was determined as a function of gelation time using a dynamic rheometer. In vivo hemostatic effects were studied using the Wistar rat model. Results were compared to those of the commercial hemostatic product Celox™, a chitosan-based product. Adhesion of h9e peptide was evaluated using the platelet adhesion test. Biocompatibility of h9e peptide was studied in vivo using a mouse model. Results: After h9e peptide solution was mixed with blood, gelation started immediately, increased rapidly with time, and reached more than 100 Pa within 3 s. Blood coagulation strength increased as h9e peptide wt% concentration increased. In the rat model, h9e peptide solution at 5% weight concentration significantly reduced both bleeding time and blood loss, outperforming Celox. Preliminary pathological studies indicate that h9e peptide solution is biocompatible and did not have negative effects when injected subcutaneously in a mouse model. Innovation: For the first time, h9e peptide was found to have highly efficient hemostatic effects by forming nanoweb-like structures, which act as a preliminary thrombus and a surface to arrest bleeding 82% faster compared to the commercial hemostatic agent Celox. Conclusion: This study demonstrates that h9e peptide is a promising hemostatic biomaterial, not only because of its greater hemostatic effect than commercial product Celox but also because of its excellent biocompatibility based on the in vivo mouse model study.

Up to 45% of breast cancer patients experience a condition called Skeletal Muscle Wasting (SMW). SMW causes significant progressive muscle loss, weakness, and fatigue. SMW in patients adversely affects quality of life during and results in a severely reduced ability of patients to tolerate chemotherapeutic drugs. Chemotherapy itself also exacerbates SMW in patients. There are currently no effective therapies for SMW. This condition is rarely reported in patient records and is often overlooked in patients with a high body mass index. Although this condition is associated with significantly lower survival rates compared to patients with relatively normal muscle mass, we do not yet understand the molecular mechanism by which it occurs in breast cancer patients. In this study, we investigate the role that CCL2 plays in breast cancer and chemotherapy induced SMW. CCL2 is a chemokine that regulates recruitment of immune cells to sites of injury and inflammation. CCL2 is overexpressed in breast cancer and SMW has been linked to elevated levels of CCL2. CCL2 overexpression is also associated with high mortality rates in breast cancer patients. To investigate the role of CCL2 in muscle wasting we treated C2C12 mouse muscle cells with increasing concentrations of CCL2. This led to increased expression of markers of muscle degradation, MuRF-1 and Atrogin-1, with reduced muscle cell proliferation and muscle cell size. This indicates that high levels of CCL2 cause muscle degradation. Using an MDA-MB-231 model of breast cancer, we show that CCL2 knockdown in the primary breast tumor decreases the expression of muscle degradation markers. Further experiments using the 4T1 mammary tumor model show that mice in which CCL2 was knocked down exhibited greater grip strength than those mice in which CCL2 levels remained unaltered. Therefore, CCL2 knockdown reduces muscle degradation and increases grip strength in a mouse model of breast cancer. We also investigated the effect of chemotherapy on SMW. Preliminary in vitro data shows increased expression of CCL2 in response to chemotherapy treatment, while in vivo experiments using the 4T1 breast tumor model indicate that chemotherapeutic treatment also significantly reduces grip strength in mice. Our findings suggest that we can improve patient survival rates and quality of life by developing therapies to combat muscle loss during and after treatment. Understanding the mechanism of muscle wasting is necessary to provide insight into novel, targeted therapeutics to reduce SMW and enhance effectiveness of anti-cancer drugs. Citation Format: Nadia Alissa, Wei Bin Fang, Gage Brummer, Marcela Mitchell, John Thyfault, Paige Geiger, Nikki Cheng. The chemokine C-C motif ligand 2 (CCL2) plays an important role in skeletal muscle wasting associated with breast cancer progression [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 3852.

With improved screening methods, the numbers of abnormal breast lesions diagnosed in women have been increasing over time. However, it remains unclear whether these breast lesions will develop into invasive cancers. To more effectively predict the outcome of breast lesions and determine a more appropriate course of treatment, it is important to understand the underlying mechanisms that regulate progression of non-invasive lesions to invasive breast cancers. A hallmark of invasive breast cancers is the accumulation of fibroblasts. Fibroblast proliferation and activation in the mammary gland is in part regulated by the Transforming Growth Factor beta1 pathway (TGF-β). In animal models, TGF-β suppression of CCL2 and CXCL1 chemokine expression is associated with metastatic progression of mammary carcinomas. Here, we show that transgenic overexpression of the Polyoma middle T viral antigen in the mouse mammary gland of C57BL/6 mice results in slow growing non-invasive lesions that progress to invasive carcinomas in a stage dependent manner. Invasive carcinomas are associated with accumulation of fibroblasts that show decreased TGF-β expression and high levels of CXCL1, but not CCL2. Using co-transplant models, we show that decreased TGF-β signaling in fibroblasts contribute to mammary carcinoma progression through enhancement of CXCL1/CXCR2 dependent mechanisms. Using cell culture models, we show that CXCL1 mediated mammary carcinoma cell invasion through NF-κB, AKT, Stat3 and p42/44MAPK dependent mechanisms. These studies provide novel mechanistic insight into the progression of pre-invasive lesions and identify new stromal biomarkers, with important prognostic implications.

Head and neck squamous cell carcinoma (HNSCC) is associated with low survival, and the current aggressive therapies result in high morbidity. Nutraceuticals are dietary compounds with few side effects. However, limited antitumor efficacy has restricted their application for cancer therapy. Here, we examine combining nutraceuticals, establishing a combination therapy that is more potent than any singular component, and delineate the mechanism of action. Three formulations were tested: GZ17-S (combined plant extracts from Arum palaestinum, Peganum harmala and Curcuma longa); GZ17-05.00 (16 synthetic components of GZ17-S); and GZ17-6.02 (3 synthetic components of GZ17S; curcumin, harmine and isovanillin). We tested the formulations on HNSCC proliferation, migration, invasion, angiogenesis, macrophage viability and infiltration into the tumor and tumor apoptosis. GZ17-6.02, the most effective formulation, significantly reduced in vitro assessments of HNSCC progression. When combined with cisplatin, GZ17-6.02 enhanced anti-proliferative effects. Molecular signaling cascades inhibited by GZ17-6.02 include EGFR, ERK1/2, and AKT, and molecular docking analyses demonstrate GZ17-6.02 components bind at distinct binding sites. GZ17-6.02 significantly inhibited growth of HNSCC cell line, patient-derived xenografts, and murine syngeneic tumors in vivo (P

Ductal carcinoma in situ (DCIS) is the most common form of breast cancer, with 50,000 cases diagnosed every year in the United States. Overtreatment and undertreatment remain significant clinical challenges in patient care. Identifying key mechanisms associated with DCIS progression could uncover new biomarkers to better predict patient prognosis and improve guided treatment. Chemokines are small soluble molecules that regulate cellular homing through molecular gradients. CCL2-mediated recruitment of CCR2+ macrophages are a well-established mechanism for metastatic progression. Although the CCL2/CCR2 pathway is a therapeutic target of interest, little is known about the role of CCR2 expression in breast cancer. Here, using a mammary intraductal injection (MIND) model to mimic DCIS formation, the role of CCR2 was explored in minimally invasive SUM225 and highly invasive DCIS.com breast cancer cells. CCR2 overexpression increased SUM225 breast cancer survival and invasion associated with accumulation of CCL2 expressing fibroblasts. CCR2-deficient DCIS.com breast cancer cells formed fewer invasive lesions with fewer CCL2+ fibroblasts. Cografting CCL2-deficient fibroblasts with DCIS.com breast cancer cells in the subrenal capsule model inhibited tumor invasion and survival associated with decreased expression of aldehyde dehydrogenase (ALDH1), a proinvasive factor, and decreased expression of HTRA2, a proapoptotic serine protease. Through data mining analysis, high expression of CCR2 and ALDH1 and low HTRA2 expression were correlated with poor prognosis of breast cancer patients. Implications: This study demonstrates that CCR2 overexpression in breast cancer drives early-stage breast cancer progression through stromal-dependent expression of CCL2 with important insight into prognosis and treatment of DCIS. Mol Cancer Res; 16(2); 296–308. ©2017 AACR.

Chemokines mediate immune cell trafficking during tissue development, wound healing and infection. The chemokine CCL2 is best known to regulate macrophage recruitment during wound healing, infection and inflammatory diseases. While the importance of CCL2/CCR2 signaling in macrophages during cancer progression is well documented, we recently showed that CCL2-mediated breast cancer progression depends on CCR2 expression in carcinoma cells. Using 3D Matrigel: Collagen cultures of SUM225 and DCIS.com breast cancer cells, this study characterized the mechanisms of CCL2/CCR2 signaling in cell growth and invasion. SUM225 cells, which expressed lower levels of CCR2 than DCIS.com cells, formed symmetrical spheroids in Matrigel: Collagen, and were not responsive to CCL2 treatment. DCIS.com cells formed asymmetric cell clusters in Matrigel: Collagen. CCL2 treatment increased growth, decreased expression of E-cadherin and increased TWIST1 expression. CCR2 overexpression in SUM225 cells increased responsiveness to CCL2 treatment, enhancing growth and invasion. These phenotypes corresponded to increased expression of Aldehyde Dehydrogenase 1A1 (ALDH1A1) and decreased expression of the mitochondrial serine protease HTRA2. CCR2 deficiency in DCIS.com cells inhibited CCL2-mediated growth and invasion, corresponding to decreased ALDH1A1 expression and increased HTRA2 expression. ALDH1A1 and HTRA2 expression were modulated in CCR2-deficient and CCR2-overexpressing cell lines. We found that ALDH1A1 and HTRA2 regulates CCR2-mediated breast cancer cell growth and cellular invasion in a CCL2/CCR2 context-dependent manner. These data provide novel insight on the mechanisms of chemokine signaling in breast cancer cell growth and invasion, with important implications on targeted therapeutics for anti-cancer treatment. This article has an associated First Person interview with the first author of the paper., Summary: Chemokines are known to regulate immune cell recruitment during inflammation. This report characterizes novel molecular mechanisms through which CCL2/CCR2 chemokine signaling in breast cancer cells regulates growth and invasion.

Breast cancer is the second leading cause of cancer related deaths for women, due mainly to metastatic disease. Invasive tumors exhibit aberrations in recruitment and activity of immune cells, including decreased cytotoxic T cells. Restoring the levels and activity of cytotoxic T cells is a promising anti-cancer strategy; but its success is tumor type-dependent. The mechanisms that coordinate recruitment and activity of immune cells and other stromal cells in breast cancer remain poorly understood. Using the MMTV-PyVmT/FVB mammary tumor model, we demonstrate a novel role for CCL2/CCR2 chemokine signaling in tumor progression by altering the microenvironment. Selective targeting of CCR2 in the PyVmT mammary epithelium inhibited tumor growth and invasion, elevated CD8+ T cells, decreased M2 macrophages and decreased angiogenesis. Co-culture models demonstrated these stromal cell responses were mediated by tumor derived CCL2 and CCR2-mediated suppression of the T cell activating cytokine, CD154. Co-culture analysis indicated that CCR2-induced stromal reactivity was important for tumor cell proliferation and invasion. In breast tumor tissues, CD154 expression inversely correlated with CCR2 expression and correlated with relapse free survival. Targeting the CCL2/CCR2 signaling pathway may reprogram the immune angiogenic and microenvironments and enhance effectiveness of targeted and immuno-therapies.

Despite advances in treatment, up to 30% of breast cancer patients experience disease recurrence accompanied by more aggressive disease and poorer prognosis. Treatment of breast cancer is complicated by the presence of multiple breast cancer subtypes, including: luminal, Her2 overexpressing, and aggressive basal-like breast cancers. Identifying new biomarkers specific to breast cancer subtypes could enhance the prediction of patient prognosis and contribute to improved treatment strategies. The microenvironment influences breast cancer progression through expression of growth factors, angiogenic factors and other soluble proteins. In particular, chemokine C-C ligand 2 (CCL2) regulates macrophage recruitment to primary tumors and signals to cancer cells to promote breast tumor progression. Here we employed a software-based approach to evaluate the prognostic significance of CCL2 protein expression in breast cancer subtypes in relation to its expression in the epithelium or stroma or in relation to fibroblast-specific protein 1 (Fsp1), a mesenchymal marker. Immunohistochemistry analysis of tissue microarrays revealed that CCL2 significantly correlated with Fsp1 expression in the stroma and tumor epithelium of invasive ductal carcinoma. In the overall cohort of invasive ductal carcinomas (n=427), CCL2 and Fsp1 expression in whole tissues, stroma and epithelium were inversely associated with cancer stage and tumor size. When factoring in molecular subtype, stromal CCL2 was observed to be most highly expressed in basal-like breast cancers. By Cox regression modeling, stromal CCL2, but not epithelial CCL2, expression was significantly associated with decreased recurrence-free survival. Furthermore, stromal CCL2 (HR=7.51 P=0.007) was associated with a greater hazard than cancer stage (HR=2.45, P=0.048) in multivariate analysis. These studies indicate that stromal CCL2 is associated with decreased recurrence-free survival in patients with basal-like breast cancer, with important implications on the use of stromal markers for predicting patient prognosis.

Contributions of the tumor microenvironment (TME) to progression in thyroid cancer are largely unexplored and may illuminate a basis for understanding rarer aggressive cases of this disease. In this study, we investigated the relationship between the TME and thyroid cancer progression in a mouse model where thyroid-specific expression of oncogenic BRAF and loss of Pten (BrafV600E/Pten−/−/TPO-Cre) leads to papillary thyroid cancers (PTC) that rapidly progress to poorly differentiated thyroid cancer (PDTC). We found that fibroblasts were recruited to the TME of BrafV600E/Pten−/−/TPO-Cre thyroid tumors. Conditioned media from cell lines established from these tumors, but not tumors driven by mutant H-ras, induced fibroblast migration and proliferation in vitro. Notably, the extracellular matrix of BrafV600E/Pten−/−/TPO-Cre tumors was enriched with stromal-derived fibrillar collagen, compared with wild-type or Hras-driven tumors. Further, type I collagen enhanced the motility of BrafV600E/Pten−/−/TPO-Cre tumor cells in vitro. In clinical specimens, we found COL1A1 and LOX to be upregulated in PTC and expressed at highest levels in PDTC and anaplastic thyroid cancer. Additionally, increased expression levels of COL1A1 and LOX were associated with decreased survival in thyroid cancer patients. Overall, our results identified fibroblast recruitment and remodeling of the extracellular matrix as pivotal features of the TME in promoting thyroid cancer progression, illuminating candidate therapeutic targets and biomarkers in advanced forms of this malignancy. Cancer Res; 76(7); 1804–13. ©2016 AACR.

The primary chemotherapeutic agents for epithelial ovarian cancer are platinum-based drugs, which are commonly used in combination with a taxane regimen. These treatments are generally effective at achieving remission, but the remission is often followed by a relapse and acquired resistance to chemotherapy. In order to overcome these barriers of drug resistance, it is important to understand the underlying mechanisms regulating the development of drug-resistant tumors. Tumors evolve through interactions with the surrounding microenvironment, which are comprised of a complex mixture of cells including fibroblasts and immune cells. In ovarian cancer, fibroblasts can make up a significant component of the primary tumor. While fibroblasts are known to influence the behavior of cancer cells directly through secretion of growth factors, and extracellular matrix (ECM) proteins, the interactions between fibroblasts and immune cells are less understood. In a recently published study from Cell, Wang and colleagues present intriguing work characterizing the role of fibroblast and T cells in modulating platinum resistance in ovarian cancer. Here, we briefly summarize and comment on their findings in relation to the tumor microenvironment and chemoresistance.

Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2. CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation. Implications: This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells.

Conventional and targeted chemotherapies remain integral strategies to treat solid tumors. Despite the large number of anti-cancer drugs available, chemotherapy does not completely eradicate disease. Disease recurrence and the growth of drug resistant tumors remain significant problems in anti-cancer treatment. To develop more effective treatment strategies, it is important to understand the underlying cellular and molecular mechanisms of drug resistance. It is generally accepted that cancer cells do not function alone, but evolve through interactions with the surrounding tumor microenvironment. As key cellular components of the tumor microenvironment, fibroblasts regulate the growth and progression of many solid tumors. Emerging studies demonstrate that fibroblasts secrete a multitude of factors that enable cancer cells to become drug resistant. This review will explore how fibroblast secretion of soluble factors act on cancer cells to enhance cancer cell survival and cancer stem cell renewal, contributing to the development of drug resistant cancer.

Increased cell motility and survival are important hallmarks of metastatic tumor cells. However, the mechanisms that regulate the interplay between these cellular processes remain poorly understood. In these studies, we demonstrate that CCL2, a chemokine well known for regulating immune cell migration, plays an important role in signaling to breast cancer cells. We report that in a panel of mouse and human breast cancer cell lines CCL2 enhanced cell migration and survival associated with increased phosphorylation of Smad3 and p42/44MAPK proteins. The G protein-coupled receptor CCR2 was found to be elevated in breast cancers, correlating with CCL2 expression. RNA interference of CCR2 expression in breast cancer cells significantly inhibited CCL2-induced migration, survival, and phosphorylation of Smad3 and p42/44MAPK proteins. Disruption of Smad3 expression in mammary carcinoma cells blocked CCL2-induced cell survival and migration and partially reduced p42/44MAPK phosphorylation. Ablation of MAPK phosphorylation in Smad3-deficient cells with the MEK inhibitor U0126 further reduced cell survival but not migration. These data indicate that Smad3 signaling through MEK-p42/44MAPK regulates CCL2-induced cell motility and survival, whereas CCL2 induction of MEK-p42/44MAPK signaling independent of Smad3 functions as an alternative mechanism for cell survival. Furthermore, we show that CCL2-induced Smad3 signaling through MEK-p42/44MAPK regulates expression and activity of Rho GTPase to mediate CCL2-induced breast cancer cell motility and survival. With these studies, we characterize an important role for CCL2/CCR2 chemokine signaling in regulating the intrinsic relationships between breast cancer cell motility and survival with implications on the metastatic process.

Fibroblasts are major cellular components of the tumor microenvironment, regulating tumor cell behavior in part through secretion of extracellular matrix proteins, growth factors, and angiogenic factors. In previous studies, conditional deletion of the type II transforming growth factor-β (TGF-β) receptor in fibroblasts (Tgfbr2FspKO) was shown to promote mammary tumor metastasis in fibroblast-epithelial cell cotransplantation studies in mice, correlating with increased expression of hepatocyte growth factor (HGF). Here, we advance our findings to show that Tgfbr2FspKO fibroblasts enhance HGF/c-Met and HGF/Ron signaling to promote scattering and invasion of mammary carcinoma cells. Blockade of c-Met and Ron by small interfering RNA silencing and pharmacologic inhibitors significantly reduced mammary carcinoma cell scattering and invasion caused by Tgfbr2FspKO fibroblasts. Moreover, neutralizing antibodies to c-Met and Ron significantly inhibited HGF-induced cell scattering and invasion, correlating with reduced Stat3 and p42/44MAPK phosphorylation. Investigation of the signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways by pharmacologic inhibition and small interfering RNA silencing revealed a cooperative interaction between the two pathways to regulate HGF-induced invasion, scattering, and motility of mammary tumor cells. Furthermore, whereas c-Met was found to regulate both the Stat3 and MAPK signaling pathways, Ron was found to regulate Stat3 but not MAPK signaling in mammary carcinoma cells. These studies show a tumor-suppressive role for TGF-β signaling in fibroblasts, in part by suppressing HGF signaling between mammary fibroblasts and epithelial cells. These studies characterize complex functional roles for HGF and TGF-β signaling in mediating tumor-stromal interactions during mammary tumor cell scattering and invasion, with important implications in the metastatic process. (Mol Cancer Res 2008;6(10):1521–33)

The human body combats infection and promotes wound healing through the remarkable process of inflammation. Inflammation is characterized by the recruitment of stromal cell activity including recruitment of immune cells and induction of angiogenesis. These cellular processes are regulated by a class of soluble molecules called cytokines. Based on function, cell target, and structure, cytokines are subdivided into several classes including: interleukins, chemokines, and lymphokines. While cytokines regulate normal physiological processes, chronic deregulation of cytokine expression and activity contributes to cancer in many ways. Gene polymorphisms of all types of cytokines are associated with risk of disease development. Deregulation RNA and protein expression of interleukins, chemokines, and lymphokines have been detected in many solid tumors and hematopoetic malignancies, correlating with poor patient prognosis. The current body of literature suggests that in some tumor types, interleukins and chemokines work against the human body by signaling to cancer cells and remodeling the local microenvironment to support the growth, survival, and invasion of primary tumors and enhance metastatic colonization. Some lymphokines are downregulated to suppress tumor progression by enhancing cytotoxic T cell activity and inhibiting tumor cell survival. In this review, we will describe the structure/function of several cytokine families and review our current understanding on the roles and mechanisms of cytokines in tumor progression. In addition, we will also discuss strategies for exploiting the expression and activity of cytokines in therapeutic intervention.

Breast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-Met action in breast cancer progression has been difficult as c-Met communicates with a number of secondary receptors that can lead to various pathological outcomes. Understanding how these secondary receptors facilitate HGF/c-Met cellular responses will aid in the development of better therapeutic treatment options for breast cancer patients with elevated HGF signaling. In the present study it was shown that the epidermal growth factor receptor (EGFR) plays a significant role in HGF/c-Met mediated biological activities indicative of advanced tumor pathology, including enhanced proliferation and invasion. The clinically relevant EGFR inhibitor gefitinib was used to determine the role of EGFR in HGF-induced proliferation and motility in several mammary carcinoma cells including PyVmT, MDA-MB-231 and 4T1. Our analyses indicated that EGFR inhibition significantly blocked HGF activation of c-Met and EGFR and that inhibition of these pathways mitigated HGF induced proliferation and motility. The data indicate that this inhibition was not through a direct effect of gefitinib on c-Met, but that EGFR is necessary for c-Met activation in the assays performed. These results provide a novel mechanism of action for EGFR as a mediator of HGF signaling thereby linking EGFR to the oncogenic potential of c-Met in mammary carcinomas cells.

Luminal A and B breast cancers are the most prevalent forms of breast cancer diagnosed in women. Compared to luminal A breast cancer patients, patients with luminal B breast cancers experience increased disease recurrence and lower overall survival. The mechanisms that regulate the luminal B subtype remain poorly understood. The chemokine CCL2 is overexpressed in breast cancer, correlating with poor patient prognosis. The purpose of this study was to determine the role of CCL2 expression in luminal B breast cancer cells. Breast tissues, MMTV-PyVmT and MMTV-Neu transgenic mammary tumors forming luminal B-like lesions, were immunostained for CCL2 expression. To determine the role of CCL2 in breast cancer cells, CCL2 gene expression was silenced in mammary tumor tissues and cells using TAT cell-penetrating peptides non-covalently cross linked to siRNAs (Ca-TAT/siRNA). CCL2 expression was examined by ELISA and flow cytometry. Cell growth and survival were analyzed by flow cytometry, immunocytochemistry, and fluorescence microscopy. CCL2 expression was significantly increased in luminal B breast tumors, MMTV- PyVmT and MMTV-Neu mammary tumors, compared or normal breast tissue or luminal A breast tumors. Ca-TAT delivery of CCL2 siRNAs significantly reduced CCL2 expression in PyVmT mammary tumors, and decreased cell proliferation and survival. CCL2 gene silencing in PyVmT carcinoma cells or BT474 luminal B breast cancer cells decreased cell growth and viability associated with increased necrosis and autophagy. CCL2 expression is overexpressed in luminal B breast cancer cells and is important for regulating cell growth and survival by inhibiting necrosis and autophagy.

Eph receptor tyrosine kinases (RTKs) and their ligands, known as ephrins, play an important role in vascular remodeling during embryogenesis, but their functions in adult angiogenesis are just beginning to be investigated. In this report, we investigated the effect of blocking EphA receptor activation on VEGF-induced angiogenic responses of cultured retinal endothelial cells and on retinal neovascularization in a rodent model of retinopathy of prematurity (ROP). Soluble EphA2-Fc receptors inhibited ephrin-A1 ligand or VEGF-induced BRMEC migration and tube formation without affecting proliferation in vitro. Since EphA2-Fc receptors can inhibit activation of multiple EphA receptors, the specific role of EphA2 receptor in angiogenesis was further investigated in EphA2-deficient endothelial cells. Loss of EphA2 in endothelial cells leads to defective cell migration and assembly in response to either ephrin-A1 or VEGF. Finally, a significant reduction in the severity of abnormal retinal neovascularization was observed in the eyes treated with soluble EphA2-Fc receptors, yet the normal total retinal vascular area was not significantly changed. Because soluble Eph receptor significantly inhibited pathologic retinal angiogenesis without affecting normal intraretinal vessels, it may be a promising agent for treatment of retinal angiogenesis in a number of human ocular diseases.

Transforming growth factor-beta (TGF-beta) is the prototype of a large family of signaling molecules. TGF-beta signaling profoundly influences tumor development as demonstrated in several engineered mouse models. The present study was designed to identify differences by cDNA microarray and MALDI-TOF MS analyses in mammary carcinomas with and without TGF-beta signaling. The results demonstrate a significant potential for combination of profiling technologies to further understand the molecular mechanisms of breast cancer.

Stromal fibroblasts regulate epithelial cell behavior through direct and indirect cell-cell interactions. To clarify the role of TGF-beta signaling in stromal fibroblasts during mammary development and tumorigenesis, we conditionally knocked out the TGF-beta type II receptor gene in mouse mammary fibroblasts (Tgfbr2(fspKO)). Tgfbr2(fspKO) mice exhibit defective mammary ductal development, characterized in part by increased ductal epithelial cell turnover associated with an increase in stromal fibroblast abundance. Tgfbr2(fspKO) mammary fibroblasts transplanted with mammary carcinoma cells promote growth and invasion, which is associated with increased activating phosphorylation of the receptors: erbB1, erbB2, RON, and c-Met. Furthermore, the increased receptor phosphorylation correlates with increased secretion of the cognate ligands by Tgfbr2(fspKO) fibroblasts. Treatment of tumor cells with fibroblast-conditioned medium leads to increased tumor cell proliferation and motility, which are blocked by addition of pharmacologic inhibitors of TGF-alpha signaling or neutralizing antibodies to macrophage-stimulating protein (MSP), HGF, or c-Met. These studies characterize a significant role for stromal TGF-beta signaling in mammary tissue homeostasis and mammary tumor progression via regulation of TGF-alpha, MSP, and HGF signaling pathways.

Elevated expression of Eph receptors has long been correlated with the growth of solid tumors. However, the functional role of this family of receptor tyrosine kinases in carcinogenesis and tumor angiogenesis has not been well characterized. Here we report that soluble EphA receptors inhibit tumor angiogenesis and tumor progression in vivo in the RIP-Tag transgenic model of vascular endothelial growth factor (VEGF)-dependent multistage pancreatic islet cell carcinoma. Soluble EphA receptors delivered either by a transgene or an osmotic minipump inhibited the formation of angiogenic islet, a premalignant lesion, and reduced tumor volume of solid islet cell carcinoma. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor volume but increased tumor and endothelial cell apoptosis in vivo. In addition, soluble EphA receptors inhibited VEGF and betaTC tumor cell-conditioned medium-induced endothelial cell migration in vitro and VEGF-induced cornea angiogenesis in vivo. A dominant negative EphA2 mutant inhibited--whereas a gain-of-function EphA2 mutant enhanced--tumor cell-induced endothelial cell migration, suggesting that EphA2 receptor activation is required for tumor cell-endothelial cell interaction. These data provide functional evidence for EphA class receptor regulation of VEGF-dependent tumor angiogenesis, suggesting that the EphA signaling pathway may represent an attractive novel target for antiangiogenic therapy in cancer.

Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell–cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.

Cancer associated fibroblasts are the most abundant stromal cells in breast cancer, but their function in cancer progression has not been fully understood. We previously identified that the chemokine CCL2 was highly expressed in breast cancer associated fibroblasts, and high stromal CCl2 expression predicted poor outcome in basal like breast cancer. CCL2 is known to recruit monocyte/macrophage and promotes cancer progression. We previously found that recombinant CCL2 can directly signal to breast cancer cells and promote cell survival and invasion in vitro. In this study, we aimed to determine the functional importance of CCL2 signaling mediated fibroblast-cancer cell interactions in breast cancer progression. We used a fibroblast and cancer cell co-graft mouse model as the main functional assay. We generated primary fibroblasts from mouse mammary tumor and human breast cancer, and confirmed most of they expressed high level of CCL2. When co-grafted with the human basal breast cancer cell line MCF10A-CA1D into nude mice, fibroblasts enhanced xenograft growth. Stable knockdown of CCL2 expression from fibroblasts significantly reduced its ability in tumor growth promotion, while knockdown CCL2 from cancer cells did not. Decreased CCL2 production from fibroblasts resulted in increased apoptosis and autophagy in tumor samples. To determine the importance of direct CCL2 signaling to cancer cell, we generated the CCL2 receptor CCR2 mutant CA1D cancer cell lines by CRISPR-Cas9 targeting technology. Mutation of CCR2 in cancer cell significantly reduced tumor growth when co-grafted with CCL2 secreting fibroblasts. Lastly, we tested continuous delivery of CCL2 neutralizing antibody in the co-graft tumor model, but observed minimal therapeutic effect. Further examination of blood CCL2 level revealed an increased production of CCL2 from fibroblasts and mouse host after antibody treatment, which may contribute to the lack of therapeutic effect. In summary, our studies demonstrated the importance of CCL2/CCR2 signaling mediated fibroblasts-cancer cell interaction in basal like breast cancer progression. The CCL2 signaling pathway can be potentially served as therapeutic target, but requires development of efficient targeting strategy. Citation Format: Min Yao, Wei Bin Fang, Fang Fan, Nehemiah Alvarez, Patrick E. Fields, Nikki Cheng. The chemokine CCL2/CCR2 signaling mediated fibroblasts-cancer cells crosstalk promotes basal like breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2984. doi:10.1158/1538-7445.AM2017-2984

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that induces a broad spectrum of responses including angiogenesis. Angiogenesis promoted by TNF-alpha is mediated, at least in part, by ephrin A1, a member of the ligand family for Eph receptor tyrosine kinases. Although TNF-alpha induces ephrin A1 expression in endothelial cells, the signaling pathways mediating ephrin A1 induction remain unknown. In this study, we investigated the signaling mechanisms of TNF-alpha-dependent induction of ephrin A1 in endothelial cells. Both TNFR1 and TNFR2 appear to be involved in regulating ephrin A1 expression in endothelial cells, because neutralizing antibodies to either TNFR1 or TNFR2 inhibited TNF-alpha-induced ephrin A1 expression. Inhibition of nuclear factor-kappaB (NF-kappaB) activation by a trans-dominant inhibitory isoform of mutant IkappaBalpha did not affect ephrin A1 induction, suggesting that NF-kappaB proteins are not major regulators of ephrin A1 expression. In contrast, ephrin A1 induction was blocked by inhibition of p38 mitogen-activated protein kinase (MAPK) or SAPK/JNK, but not p42/44 MAPK, using either selective chemical inhibitors or dominant-negative forms of p38 MAPK or TNF receptor-associated factor 2. These findings indicate that TNF-alpha-induced ephrin A1 expression is mediated through JNK and p38 MAPK signaling pathways. Taken together, the results of our study demonstrated that induction of ephrin A1 in endothelial cells by TNF-alpha is mediated through both p38 MAPK and SAPK/JNK, but not p42/44 MAPK or NF-kappaB, pathways.

Chimeric oligonucleotides consisting of RNA and DNA residues have been shown to catalyze site-directed genetic alteration in mammalian cells both in vitro and in vivo. Since the frequency of these events appears to be logs higher than the rates of gene targeting, a process involving homologous recombination, we developed a system to study the mechanisms of chimera-directed gene conversion. Using a mammalian cell-free extract and a genetic readout in Escherichia coli, we find that point mutations and single base deletions can be corrected at frequencies of approximately 0.1% and 0.005%, respectively. The reaction depends on an accurately designed chimera and the presence of functional hMSH2 protein. The results of genetic and biochemical studies reported herein suggest that the process of mismatch repair functions in site-directed gene correction.

Ductal carcinoma in situ (DCIS) is the most common type of non-invasive breast cancer diagnosed in women and an immediate precursor to invasive ductal carcinoma (IDC). It is largely unclear why some cases of DCIS progress to IDC, while others remain non-invasive. Current cyto- and histopathological approaches do not accurately predict disease progression, resulting in patients being under-treated or over-treated for DCIS. Our long-term goals are to identify key factors that lead to IDC that will enable the development of a molecular based approach to predict the risk of IDC, and a more tailored approach to treat DCIS. The CCL2/CCR2 chemokine pathway is best known for regulating macrophage recruitment to late stage breast tumors. In recent studies, we found that CCR2 and its binding ligand CCL2 were overexpressed in breast ductal carcinomas, correlating with tumor grade and poor patient prognosis. To further investigate the significance of CCR2 and CCL2, we performed flow cytometry analysis of breast epithelial cell lines, and found that increased CCR2 overexpression but not CCL2, was associated with invasive potential of the cell lines. CCL2 was found to be increased in stromal cells, particularly in cancer associated fibroblasts. Using a mammary intra-ductal injection model, we further examined for expression patterns of CCR2 and CCL2 in DCIS lesions derived from DCIS.com and Sum225 cells. Increased CCR2 was detected in DCIS.com lesions, which progressed to IDC within 10 weeks of injection, while CCR2 expression was found to be lowly expressed in non-invasive Sum225 lesions. CCL2 was detected in the stroma of both DCIS.com and Sum225 lesions. Overexpression of CCR2 in cultured non-invasive 67NR mammary carcinoma cells enhanced cell survival and increased wound closure. CCL2 treatment further enhanced cell survival and wound closure of CCR2 overexpressing 67NR cells. Knockdown of CCR2 in DCIS.com cells in 3D Matrigel: collagen cultures inhibited cellular invasion into the matrix. These studies indicate that CCR2 overexpression in ductal carcinoma cells, enhances intracellular signaling, and contributes to development of IDC. These data suggest that targeting the CCR2 pathway may be promising strategy for prevention or treatment of IDC. Citation Format: Benford Mafuvadze, Wei Bin Fang, Nikki Cheng. Overexpression of the chemokine receptor CCR2 in the breast epithelium is associated with progression of DCIS [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-02.

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

Accumulation of fibroblasts is a phenomenon that significantly correlates with formation of aggressive cancers. While studies have shown that the TGF-β signaling pathway is an important regulator of fibroblast activation, the functional contribution of TGF-β signaling in fibroblasts during multi-step tumor progression remains largely unclear. In previous studies, we used a sub-renal capsule transplantation model to demonstrate that homozygous knockout of the Tgfbr2 gene (Tgbr2(FspKO)) enhanced mammary tumor growth and metastasis. Here, we show for the first time a significant role for loss of one Tgfbr2 allele during multi-step mammary tumor progression. Heterozygous deletion of Tgfbr2 in stromal cells in MMTV-PyVmT transgenic mice (PyVmT/Tgfbr2(hetFspKO) mice) resulted in earlier tumor formation and increased stromal cell accumulation. In contrast to previous studies of Tgbr2(FspKO) fibroblasts, Tgfbr2(hetFspKO) fibroblasts did not significantly increase tumor growth, but enhanced lung metastasis in PyVmT transgenic mice and in co-transplantation studies with PyVmT mammary carcinoma cells. Furthermore, Tgfbr2(hetFspKO) fibroblasts enhanced mammary carcinoma cell invasiveness associated with expression of inflammatory cytokines including CXCL12 and CCL2. Analyses of Tgbr2(FspKO) and Tgfbr2(hetFspKO) fibroblasts revealed differences in the expression of factors associated with metastatic spread, indicating potential differences in the mechanism of action between homozygous and heterozygous deletion of Tgfbr2 in stromal cells. In summary, these studies demonstrate for the first time that loss of one Tgfbr2 allele in fibroblasts enhances mammary metastases in a multi-step model of tumor progression, and demonstrate the importance of clarifying the functional contribution of genetic alterations in stromal cells in breast cancer progression.

Chemokines are soluble factors shown to play important roles in regulating immune cell recruitment during inflammatory responses and defense against foreign pathogens. De-regulated expression and activity of several chemokine signaling pathways have been implicated in cancer progression, including: CCL2, CCL5, CXCL1 and CXCL12. While studies in the past have focused the role of these chemokine signaling pathways in regulating immune responses, emerging studies show that these molecules regulate diverse cellular processes including angiogenesis, and regulation of epithelial cell growth and survival. New evidence indicates that chemokines are critical for cancer progression and indicate complex and diverse functions in the tumor microenvironment. This review will focus on the contributions of chemokine signaling in regulating cancer microvironment and discuss the utility of targeting or delivering chemokines in cancer therapeutics.