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Breast cancer is one of the most common cancer types that happens to global females. The major challenges for breast cancer include therapy resistance and metastasis. Various factors and cancer-related immune cells are involved in tumor chemotherapy resistance process, including pro-inflammatory cytokine, interleukin (IL) 17, CXCR2 ligands (pro-inflammatory chemokines) and neutrophils. They are the key players for promoting cancer progression, and numerous reports indicated them as a potential therapeutic target for various cancer types including breast cancer. However, the detailed mechanism(s) remains unclear. In the present study, we investigate the role of neutrophils in chemotherapy resistance and metastasis in breast cancer. In our previous study, we have observed increased levels of CXCR2 ligands in chemotherapy-resistant cells comparing with Cl66 parent cells. In this study, we observed upregulated IL17, IL17 receptor (IL-17R), granulocyte colony stimulating factor (GCSF) by qRT- PCR in resistant cells compared with parent cells. We further evaluated the protein levels of CXCR2 and its ligands, IL17 and its receptor, frequencies of neutrophils and T-helper 17 (Th17) cells in primary and metastatic tumors through using immunohistochemistry method. There were higher levels of IL17R, CXCR2, and CXCR2 ligands in metastatic tumor sites in comparison to the primary tumor sites. Moreover, we observed increased levels of IL17R, CXCR2, CXCR2 ligands together with the higher frequencies of neutrophils and Th17 cells in resistant tumors than Cl66 tumors. At the same time, tumor cells treated with IL-17 increased its proliferation rate and its expression of CXCL1 & CXCL5; and the increase was all in a concentration-dependent manner. In addition, the increase of CXCR2 ligands by IL17 was inhibited when we treated the tumor cells with ERK and NF-KB signaling inhibitors, indicating both pathways are involved in the regulation of IL17-induced CXCR2 ligands secretion. Together, our data demonstrate an IL17-CXCR2 ligands axis plays protumorigenic inflammation, facilitating therapy resistance and metastasis through recruited neutrophils. Citation Format: Lingyun Wu, Sugandha Saxena, Mohammad Awaji, Bhawna Sharma, Rakesh Singh. IL-17-CXCR2 axis promotes breast cancer metastasis and therapy resistance through facilitating recruitment of neutrophils [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4554.

The major challenges for breast cancer includes therapy resistance and metastasis to distant organs. Chronic inflammation has been intimately linked with these processes. We have observed increased level of CXCR2 ligands, inflammatory immunosuppressive microenvironment, and increased metastasis in chemotherapy resistant cells. CXCR2 and its ligands have been shown to be the primary mechanism for neutrophils recruitment to the sites of primary tumors and metastases. However, their precise roles in therapy resistance and metastasis remains unclear. The aim of this project is to investigate the role of neutrophils in chemotherapy resistance and metastasis in breast cancer. In this report, we investigated the mechanisms and putative role of neutrophils in chemotherapy resistance and metastasis. We evaluated the expression level of the factors critical for neutrophil recruitments, such as interleukin (IL)-17 and its receptors (IL-17R), granulocyte colony stimulating factor (GCSF), CXCR2 and its ligands in primary tumors and metastases established from parents and chemotherapy resistant cells. The frequency of neutrophils and T-helper 17 (Th17) cells were analyzed using immunohistochemistry. Chemotherapy-resistant cell lines express higher levels of GCSF, IL-17, and IL-17R compared to Cl66 parent cells. We observed higher expression of IL-17R, CXCR2, and CXCR2 ligands in metastatic lesions compared to primary tumors. Furthermore, there were more recruitments of neutrophils and Th17 cells in resistant tumors than parent tumors. In addition, treatment of tumor cells with IL-17 significantly increased cellular proliferation together with CXCL1 and CXCL5 expression in a concentration-dependent manner. Moreover, we observed higher expression of TGFβ 2 and Th17 cell priming factors, IL-6 and IL-23, in neutrophils co-cultured with therapy resistant cells compared to the control suggesting a feed forward loop. In addition, we observed that IL17 treatment of tumor cells did not increase their tolerance to chemotherapy drug, indicating its role in establishing immune suppressive microenvironments. Together our data demonstrate an IL17-CXCR2 ligands axis induces protumorigenic inflammation, facilitating therapy resistance and metastasis through recruited neutrophils. Citation Format: Lingyun Wu, Bhawna Sharma, Rakesh K. Singh. Role of IL-17-CXCR2 axis in neutrophil recruitment facilitating breast cancer metastasis and therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 132.

Breast cancer is a deadly disease which accounts for more than 40,000 deaths each year in United States. With the availability of various treatment options, side effects and resistance associated with them is also becoming a major concern for the treatment of breast cancer. How breast cancer cells evade chemotherapy still remains a vast area for research. In this study, we proposed that mammary tumor cells resist cell death via VEGFR3 signaling pathway. To test our hypothesis, we used doxorubicin and paclitaxel resistant murine mammary tumor cells (Cl66-Dox and Cl66-Pac) which have been developed in our laboratory. When implanted in female Balb/c mice they form more aggressive tumor than the parent cells (Cl66) from which they have been derived. We observed that these resistant cells have higher expression of VEGFR3 receptor than parent cells at mRNA level. Treatment with VEGFR3 kinase inhibitor, MAZ51, Cl-66Dox and Cl66-Pac cells showed 50% growth inhibition at 10uM dose, suggesting VEGFR3 receptor activation as one of the mechanism for resistance. Moreover, when implanted in mice, tumor formed by resistant cells responded well to MAZ51 treatment with slower tumor growth in comparison to control group consistent with our in vitro data. However, when we treated mice bearing resistant tumors first with chemotherapeutic drug, docetaxel followed by MAZ51, we observed a significant decrease in tumor growth and tumor weight, examined upon endpoint analysis. These results suggest that combination treatment is more effective in inhibiting drug-resistant tumors growth in comparison to drug and inhibitor alone as determined by reduced tumor volume and weight in combination treatment group. Overall, our in vivo data showed that blocking VEGFR3 signaling pathway makes drug-resistant mammary tumor cells susceptible to chemotherapy suggesting the use of inhibitors for this signaling pathway to be beneficial for drug-resistant mammary tumors. Citation Format: Bhawna Sharma, Michelle L. Varney, Rakesh K. Singh. VEGFR3 signaling facilitates mammary tumor cells to resist chemotherapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-45. doi:10.1158/1538-7445.AM2014-LB-45

Chemotherapy has been the mainstay of cancer treatment for so many years and so do the increasing resistance to it. Recent studies emphasize the importance of cytokines in breast cancer therapy resistance and how targeting cytokines or their receptor may prove to be a better therapeutic regimen. This study focuses on the role of receptor activator of NFkB ligand (RANKL), recently been observed to provide survival benefits to cancer cells and helps in mammary stem cell maintenance. To the best of our knowledge the modulations of RANK and RANKL in response to chemotherapy has not been evaluated before. Our preliminary studies with drug-resistant cells showed higher expression of RANK and RANKL both in vitro in cells as well as in vivo in tumors formed by these cells, suggesting a role of this RANKL mediated signaling in breast cancer therapy resistance. Given that RANK and RANKL are expressed in aggressive breast tumors and this signaling provides survival benefits to cancer cells, we hypothesized that chemotherapy up-regulates RANK-RANKL signaling in breast cancer cells which eventually makes breast cancer cells resistant to further chemotherapy providing evasion to therapy. To investigate this, we monitored the expression of RANKL in murine 4T1, CL66 and MDA MB-231, MCF-7; human breast cancer cells lines after treatment with two commonly used drugs doxorubicin and paclitaxel for breast cancer treatment. We observed that both drugs up-regulate RANKL expression in these cells. To examine if the expression of RANK also changes after drug treatment in these cells, we treated 4T1 and Cl66 cells with paclitaxel for 4, 8 and 12 hours. We observed an increase in the expression of RANK in these cells in a dose- and duration-dependent manner. 4T1 and Cl66 along with MDA MB-231 and MCF-7 were also treated with both paclitaxel and doxorubicin to evaluate if the change in receptor and ligand expression was drug-dependent. Our data showed that both the drugs up-regulate RANKL at mRNA (qRT-PCR) and protein levels (Immunofluorescence) in Cl66 and 4T1 cells. This up-regulation was as early as 4hrs (at mRNA level) after drugs treatment and the expression decreases after 8 hrs, suggesting an early transcriptional up-regulation. Our data suggest that increased expression of both RANK and RANKL in the breast cancer cells following chemotherapy may contribute to the activation of RANK-RANKL signaling in breast cancer and targeting this signaling might provide a novel adjuvant therapeutic regimen. Citation Format: Bhawna Sharma, Michelle L. Varney, Rakesh K. Singh. Chemotherapy up-regulates RANK-RANKL signaling in breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4968. doi:10.1158/1538-7445.AM2013-4968

Resistance to therapy and disease relapse is major concerns in the field of breast cancer management. Accumulating evidence suggests the existence of breast cancer stem cells and it has been proposed that their induction after chemotherapy could be contributing to therapy resistance and relapse. Our preliminary data demonstrate that expression of CXCL1 and CXCL8 (CXCR1/2 ligands) increases after chemotherapy. We also observed that expression of RANKL (ligand of RANK) goes up in a dose dependent manner in both Cl66 and 4T1 cells after treatment with paclitaxel and doxorubicin. Recently, the involvement of RANK/RANKL signaling in mammary tumorigenesis by maintaining the stem cell pool has been elucidated. Given that RANK-RANKL signaling and CXCR1/2 signaling share the common mediator, NFkB, our working hypothesis is that chemotherapy-induced expression of CXCR1/2 ligands and the RANK/RANKL signaling axis plays an important role in selecting a subset of mammary tumor cells with stem cell-like characteristics. We selected Cl66, mammary tumor cells against doxorubicin and paclitaxel. We observed that cells selected against these drugs express higher levels of CXCR1/2 ligands at both the mRNA and protein level. Moreover, 4T1 (6-thioguanine resistant cells) and Cl66 Dox 500 (cells resistant to doxorubicin) express RANK whereas Cl66-parent and Cl66 Pac 400 (cells resistant to paclitaxel) do not. This suggests that doxorubicin specifically selects RANK expressing cancer cells and 4T1 inherently express this receptor. Cl66 resistant cells injected in BALB/c mice grew slower than parent cells. Furthermore, the extent of neovascularization in doxorubicin resistant Cl66 tumors was significantly higher as compared to parent Cl66 tumors and paclitaxel resistant Cl66 tumors. As a result of their slow growth and difference in neovascularization, we analyzed the expression of stem cell markers in these cells. We observed that both Cl66-parent and paclitaxel resistant cells express ALDH1, but the expression was higher in paclitaxel resistant cells. Moreover, resistant cells showed enhanced expression of the mesenchymal marker, vimentin in comparison to parent cells. Overall, our results suggest that use of a particular chemotherapeutic agent may influence the selection of different sub-type of cancer cells with stem cell-like properties. This data also proposes the importance of a chemotherapy-induced cytokine-chemokine axis in maintaining the cancer stem cell pool. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3484. doi:1538-7445.AM2012-3484

Breast cancer is the second leading cause of cancer deaths among women after lung cancer, accounting for more than 40,000 deaths each year. Although there has been advancement in the treatment and diagnosis of breast cancer, therapy resistance and relapse are still big hurdles in the way. Recent reports and our preliminary studies suggest that malignant cells that survive the initial chemo- and radiation therapy express higher levels of CXCR2 ligands which might provide a survival benefit to malignant tumors leading to therapy resistance. The specific objective for this study involves targeting CXCR2 receptor and its ligands mediated signaling for therapy resistance in mammary tumor cells. Our hypothesis is that CXCR2 along with its ligands plays an important role in therapy resistance and that targeting this signaling pathway will provide an effective strategy to overcome the problem. We used mammary tumor cells expressing different levels of CXCR2 (Cl66-wt and Cl66-shCXCR2) and examined their response to Paclitaxel and Doxorubicin. We observed significant enhancement of paclitaxel and doxorubicin-mediated toxicity at lower concentrations in Cl66-shCXCR2 cells as compared to Cl66-wt cells. Moreover, we observed an increase in the expression of CXCL1, a CXCR2 ligand, with increasing doses of drugs. However, paclitaxel and doxorubicin induced CXCL-1 levels was significantly lower in Cl66-shCXCR2 cells as compared to Cl66-wt cells. To further validate our findings we selected Cl66 against paclitaxel and doxorubicin resistant cells by treating with increasing doses of these drugs. Cells were maintained in 500nM doxorubicin and 400nM paclitaxel. We analyzed the sensitivity of these cells to doxorubicin and paclitaxel in comparison to wild type parent cells. We observed that resistant cells survive even at higher concentrations (higher than at which they are maintained) of drugs. Although, paclitaxel resistant cells appears to be sensitive at higher doses of paclitaxel when compared to doxorubicin resistant cells in the same set of experiments. Further studies showed an increase in the expression of CXCL1 in the supernatant of these cells after drug treatment. The expression of CXCL1 was higher even at the basal level in the resistant cells in comparison to the parent cells. As it has been shown that expression of CXCR2 ligands increase after chemotherapy, we examined the expression profile of various CXCR2 ligands in these cells at the mRNA level. We observed that these cells express higher levels of CXCL5, CXCL3 and CXCL7, in comparison to parent Cl66 cells. Together, these results demonstrate that i) CXCR2 and its ligand-mediated signaling plays an important role in the drug resistance of mammary tumors cells; ii) that paclitaxel seems to be more effective against mammary tumor cells in comparison to doxorubicin; and iii) targeting CXCR2 expression enhanced chemotherapeutic responses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 693. doi:10.1158/1538-7445.AM2011-693

Breast cancer is the second most common cause of cancer death in women in the United States after lung cancer and is responsible for more than 40,000 deaths each year. Many challenges exist in the current management of advanced stage breast cancer as there are fewer recognized therapeutic strategies, often due to therapy resistance. Recent reports suggest that malignant cells that survive initial chemo- and radiation therapy often express inflammatory cytokines, which provides survival benefit making tumors resistant to further chemotherapy. The specific objective of the study is to develop strategy to manipulate chemokine-chemokine receptor network for effective therapy with limited toxicity against drug-resistant breast cancer. We investigated whether modulation of CXCR2-dependent signaling in malignant mammary tumor enhances therapeutic efficacy of chemotherapy drugs. We used mammary tumor cells expressing different levels of CXCR2 (Cl66-wt and Cl66-shCXCR2) and examined their response to Paclitaxel and Doxorubicin. We observed significant enhancement of paclitaxel and doxorubicin-mediated toxicity at concentrations 5 and 10nM in Cl66-shCXCR2 cells as compared to Cl66-wt cells. Moreover, the expression of CXCL1, a CXCR2 ligand, was increased in paclitaxel and doxorubicin treated mammary tumor cells. Paclitaxel and doxorubicin induced CXCL-1 levels was significantly lower in Cl66-shCXCR2 cells as compared to Cl66-wt cells. In vivo studies using wild type and knockdown cells showed no significant difference in the tumor growth. However, we observed a difference in the tumor growth between wild type and knockdown tumors in mice treated with 10mg/kg concentration of paclitaxel for 3 weeks after starting the treatment. When these studies were further extended there wasn't any difference between the two groups. Furthermore, we extended these studies using human breast cancer cell lines (MDA-MB231, MDA-MET, and SKBR3). MDA-MB231 and MDA-MET are triple negative and considered to be more metastatic and drug resistant than SKBR3 which is HER2-over expressing cell line. We observed differential expression of CXCR2 and its ligands in these cells. Our observation also showed significant difference in the paclitaxel and doxorubicin-mediated inhibitory response in MDA-MB231 and MDA-MET cells as compared to SKBR3 cells. Together these studies demonstrate that manipulating CXCR2-dependent activity in malignant breast cancer cells significantly enhanced therapeutic efficacy of paclitaxel and doxorubicin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5462.