Sandeep Rajput

Paclitaxel Therapy Promotes Breast Cancer Metastasis in a TLR4-Dependent Manner

Emerging evidence suggests that cytotoxic therapy may actually promote drug resistance and metastasis while inhibiting the growth of primary tumors. Work in preclinical models of breast cancer has shown that acquired chemoresistance to the widely used drug paclitaxel can be mediated by activation of the Toll-like receptor TLR4 in cancer cells. In this study, we determined the prometastatic effects of tumor-expressed TLR4 and paclitaxel therapy and investigated the mechanisms mediating these effects. While paclitaxel treatment was largely efficacious in inhibiting TLR4-negative tumors, it significantly increased the incidence and burden of pulmonary and lymphatic metastasis by TLR4-positive tumors. TLR4 activation by paclitaxel strongly increased the expression of inflammatory mediators, not only locally in the primary tumor microenvironment but also systemically in the blood, lymph nodes, spleen, bone marrow, and lungs. These proinflammatory changes promoted the outgrowth of Ly6C(+) and Ly6G(+) myeloid progenitor cells and their mobilization to tumors, where they increased blood vessel formation but not invasion of these vessels. In contrast, paclitaxel-mediated activation of TLR4-positive tumors induced de novo generation of deep intratumoral lymphatic vessels that were highly permissive to invasion by malignant cells. These results suggest that paclitaxel therapy of patients with TLR4-expressing tumors may activate systemic inflammatory circuits that promote angiogenesis, lymphangiogenesis, and metastasis, both at local sites and premetastatic niches where invasion occurs in distal organs. Taken together, our findings suggest that efforts to target TLR4 on tumor cells may simultaneously quell local and systemic inflammatory pathways that promote malignant progression, with implications for how to prevent tumor recurrence and the establishment of metastatic lesions, either during chemotherapy or after it is completed.

TLR4 Is a Novel Determinant of the Response to Paclitaxel in Breast Cancer

Overexpression of Toll-like receptor-4 (TLR4) in human tumors often correlates with chemoresistance and metastasis. We found that TLR4 is overexpressed in the majority of clinical breast cancer samples and in 68% of the examined breast cancer lines. TLR4 is activated by lipopolysaccharide (LPS) and other ligands including the widely used drug paclitaxel. LPS is frequently used to show a tumor-promoting role of TLR4 although this bacterial component is unlikely to be found in the breast cancer environment. We reasoned that paclitaxel-dependent activation of TLR4 is more relevant to breast cancer chemoresistance that could be mediated by activation of the NF-κB pathway leading to upregulation of prosurvival genes. To test this hypothesis, we correlated TLR4 expression with resistance to paclitaxel in two modified breast cancer lines with either depleted or overexpressed TLR4 protein. Depletion of TLR4 in naturally overexpressing MDA-MB-231 cells downregulated prosurvival genes concomitant with 2- to 3-fold reduced IC50 to paclitaxel in vitro and a 6-fold decrease in recurrence rate in vivo. Conversely, TLR4 overexpression in a negative cell line HCC1806 significantly increased expression of inflammatory and prosurvival genes along with a 3-fold increase of IC50 to paclitaxel in vitro and enhanced tumor resistance to paclitaxel therapy in vivo. Importantly, both tumor models showed that many paclitaxel-upregulated inflammatory cytokines were coinduced with their receptors suggesting that this therapy induces autocrine tumor-promoting loops. Collectively, these results show that paclitaxel not only kills tumor cells but also enhances their survival by activating TLR4 pathway. These findings suggest that blocking TLR4 could significantly improve response to paclitaxel therapy. Mol Cancer Ther; 12(8); 1676–87. ©2013 AACR.

Murine aldo-keto reductase family 1 subfamily B: identification of AKR1B8 as an ortholog of human AKR1B10.

Abstract Aldo-keto reductase family 1 member B10 (AKR1B10), over-expressed in multiple human cancers, might be implicated in cancer development and progression via detoxifying cytotoxic carbonyls and regulating fatty acid synthesis. In the present study, we investigated the ortholog of AKR1B10 in mice, an ideal modeling organism greatly contributing to human disease investigations. In the mouse, there are three aldo-keto reductase family 1 subfamily B (AKR1B) members, i.e., AKR1B3, AKR1B7, and AKR1B8. Among them, AKR1B8 has the highest similarity to human AKR1B10 in terms of amino acid sequence, computer-modeled structures, substrate spectra and specificity, and tissue distribution. More importantly, similar to human AKR1B10, mouse AKR1B8 associates with murine acetyl-CoA carboxylase-α and mediates fatty acid synthesis in colon cancer cells. Taken together, our data suggest that murine AKR1B8 is the ortholog of human AKR1B10.

Heat shock protein 90-α mediates aldo-keto reductase 1B10 (AKR1B10) protein secretion through secretory lysosomes.

Background: Aldo-keto reductase 1B10 (AKR1B10) protein is a new tumor biomarker in humans. Results: Heat shock protein 90α (HSP90α) is a chaperone molecule that mediates transportation to lysosomes and secretion of AKR1B10. Helix 10 of AKR1B10 protein mediates its interaction with HSP90α. Conclusion: HSP90α mediates AKR1B10 secretion through binding to its helix 10 domain. Significance: This finding is significant in exploiting the use of AKR1B10 in cancer clinics. Aldo-keto reductase 1B10 (AKR1B10) protein is a new tumor biomarker in humans. Our previous studies have shown that AKR1B10 is secreted through a lysosome-mediated nonclassical pathway, leading to an increase in the serum of breast cancer patients. This study illuminates the regulatory mechanism of AKR1B10 secretion. The cytosolic AKR1B10 associates with and is translocated to lysosomes by heat shock protein 90α (HSP90α), a chaperone molecule. Ectopic expression of HSP90α significantly increased the secretion of endogenous AKR1B10 and exogenous GFP-AKR1B10 fusion protein when cotransfected. Geldanamycin, a HSP90α inhibitor, dissociated AKR1B10-HSP90α complexes and significantly reduced AKR1B10 secretion in a dose-dependent manner. We characterized the functional domain in AKR1B10 and found that helix 10 (amino acids 233–240), located at the C terminus, regulates AKR1B10 secretion. Targeted point mutations recognized that amino acids Lys-233, Glu-236, and Lys-240 in helix 10 mediate the interaction of AKR1B10 with HSP90α. Together, our data suggest that HSP90α mediates AKR1B10 secretion through binding to its helix 10 domain. This finding is significant in exploiting the use of AKR1B10 in cancer clinics.

Abstract 1700: TLR4 as a novel determinant of paclitaxel response in metastatic breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Paclitaxel elicits both cytotoxic and pro-survival responses in tumor cells. The tumor-promoting effect of paclitaxel is a currently unrecognized determinant for decreasing the apoptotic effect of paclitaxel therapy. The likely mechanism for paclitaxel-dependent tumor-activating effects is the ability of paclitaxel to activate Toll-like Receptor-4 (TLR4) pathway. TLR4 is often over-expressed in malignant epithelial cells in which its signaling hyper-activates NF-kB, MAPK and PI3K pathways providing a pro-survival benefit to the residual cancer cells. The goal of this project was to identify the TLR4 role in paclitaxel resistance by correlating the expression profile of TLR4 pathway in a panel of breast carcinoma cells with sensitivity to paclitaxel therapy. Methods: TLR4 mRNA levels were determined in 18 breast carcinoma lines by qRT-PCR. The functionality of TLR4 pathway was determined by measuring mRNA of downstream products (IL-6, IL-8, MCP-1 and TNFα) after stimulating cells with a natural TLR4 ligand, lipopolysaccharide (LPS), or paclitaxel. Protein levels were measured by ELISA. Cytotoxic assays were used to correlate the level of TLR4 expression and responsiveness to paclitaxel. TLR4-positive (MDA-MB-231) and negative (HCC1806) lines were engineered to stably down-regulate and over-express TLR4, respectively. The target expression in modified clones was determined by qRT-PCR and Western Blot. The modified lines were analyzed for functionality and altered responsiveness to paclitaxel using the aforementioned methods. Results: TLR4 was expressed in 60% of human breast cancer cell lines. TLR4 receptor in MDA-MB-231 line was functional as demonstrated by up-regulation of inflammatory cytokines. LPS increased IL-6 and MCP-1 by 8-10 fold whereas IL-8 and TNF-alpha were increased by 120-300 fold. Paclitaxel had 1/10 to 1/3 of the LPS activity. MDA-MB-231 line was ∼5 fold more resistant to paclitaxel than HCC1806 lacking TLR4. Paclitaxel treatment not only drastically increases secretion of NF-kB dependent cytokines but also up-regulated the expression levels of their receptors suggesting establishment of novel autocrine pro-survival and proliferative positive loops. Anti-TLR4 antibody inhibited paclitaxel stimulating effects by nearly 100%. Conclusions: These data show that paclitaxel up-regulates both inflammatory cytokines and their receptors in human breast carcinoma cells, likely through activation of the TLR4 pathway. Inflammatory pathway signaling increases survival and proliferation in TLR4-positive cells, suggesting that activation of this pathway in malignant cells maintain chronic inflammation and promote tumor growth and metastasis through both paracrine and autocrine loops. This study suggests that tumor resistance to paclitaxel might be determined by TLR4 expression, and that blocking TLR4 might significantly improve tumor response to paclitaxel therapy. 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 1700. doi:10.1158/1538-7445.AM2011-1700

Abstract 1078: Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models

Purpose: Triple negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer with limited therapeutic options. Development of molecularly targeted agents for TNBC is an unmet clinical need. The phosphoinositide 3-kinase (PI3K) pathway, which is a major cell growth and survival pathway, is frequently activated in TNBC as a result of genetic aberrations such as loss of the negative regulator PTEN or gain of function mutations in PIK3CA, therefore an attractive therapeutic target. However, single agent PI3K inhibitors have shown limited anti-tumor activity in both preclinical models and in clinical trials. One of the most important mechanisms of resistance to PI3K inhibitor is amplification of Myc. Dinaciclib, a potent inhibitor of cyclin-dependent kinases (CDKs)1, 2, 5 and 9, has shown to be particularly effective in Myc dependent tumors in preclinical studies and in TNBC. We therefore hypothesized that dinaciclib could overcome tumor cell resistance to PI3K inhibitors and improve the therapeutic efficacy in TNBC. In this study, we evaluated the anti-tumor and molecular effect of dinaciclib and BKM120 (Pan-PI3K inhibitor), either alone or in combination, in a panel of patient derived xenograft (PDX) models of TNBC. Methods: Four TNBC PDXs models were selected for the study for in-vivo and ex-vivo response to vehicle, BKM120, dinaciclib, or the combination of BKM120 and dinaciclib. Tumor volume changes over time in each group were documented to calculate the percentage of tumor growth inhibition by either agent alone or in combination. Tumor tissues harvested post treatment were examined by immunohistochemistry for cleaved PARP to determine the extent of apoptosis and phospho-Histone H3 for G2 to M phase cell cycle progression. Western blot analysis and reverse protein phase array (RPPA) were also performed to determine treatment effect on PI3K downstream targets and cell cycle molecules. Results: The combination of BKM120 and dinaciclib induced significantly greater growth inhibitory effect on tumor growth than either single agent alone in TNBC PDX models. This is accompanied by an enhanced apoptotic induction and reduced cell cycle progression. In addition, the combination of dinaciclib and BKM120 significantly reduced the level of cyclin B and the key anti-apoptotic protein survivin as well as significant downregulation of pAKT, and pS6. Conclusions: These data suggest that dinaciclib and BKM120 combination is an effective approach in treating TNBC. Additional mechanistic investigation for the efficacy of this combination is underway. Citation Format: Sandeep Rajput, Fang Guo, Li Shun, Cynthia Ma. Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models [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 1078. doi:10.1158/1538-7445.AM2017-1078

Abstract 771: TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis

Background: Paclitaxel (PXL) is a widely-used drug for breast cancer therapy. Resistance, however, occurs frequently and the evasion mechanisms remain unclear. PXL is known to activate Toll-like Receptor-4 (TLR4), a major signaling receptor expressed on immune cells responding to lipopolysaccharide (LPS). LPS activation of TLR4 in the immune cells substantially enhances their migratory, invasive, proliferative, and pro-survival functions as necessary for the body defense. Since PXL is a functional LPS mimetic, we hypothesized that acquisition of such phenotype by TLR4-expressing cancer cells promotes tumor growth, metastasis, and resistance to therapy. Methods: We tested this hypothesis in two breast cancer luciferase-tagged models genetically modified to either suppress TLR4 expression in a positive line, MDA-MB-231, or overexpress it in a negative line, HCC1806. Modified cell lines and their controls were orthotopically implanted in mice followed by measuring tumor growth prior and post-PXL treatment. Incidence and burden of lymph node (LN) and lung metastases were quantified by measuring luciferase activity in respective organs. TLR4-induced inflammation was assessed by measuring cytokines in blood, tumor lysates, and metastatic organs using RT-qPCR and ELISA. Cell composition of spleens and bone marrow from control and treated mice were analyzed by FACS. TLR4-induced changes in tumor vasculature were determined by immunostaining for blood and lymphatic vessel markers. Results: TLR4 expressed in tumor cells significantly increased rate of recurrence and metastasis, an event augmented by PXL treatment. Local inflammation was also enhanced by the PXL•TLR4 axis as illustrated by a 276-fold increase in IL-6 in lysates of TLR4+ tumors from treated mice as compared with samples of isogenic tumors with depleted TLR4. Activation of this pathway also increased cytokine levels in the blood and distant organs as indicted by 3-6 fold increase in IL-4 and IL-10 in LNs and lungs. These pro-inflammatory systemic changes promoted generation of myeloid progenitors in bone marrow and spleen evident by 4-5 fold increase in Ly6C+ cell population in TLR4-overexpressing tumors compared with those lacking TLR4. Importantly, activation of the PXL•TLR4 axis induced intratumoral formation of lymphatic vessels in HCC1806 tumors absent in all other experimental groups. Conclusion: These data imply that PXL therapy activates TLR4 often overexpressed in breast cancer. Activation of this pathway substantially increases local tumor and systemic inflammation leading to generation of myeloid progenitors that can promote metastasis by inducing angiogenesis and lymphatic vessel formation, particularly inside the tumor. These findings suggest that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway may improve current anti-cancer treatments. Citation Format: Sandeep Rajput, Lisa Volk-Draper, Kelly Hall, Sophia Ran. TLR4 activation by paclitaxel promotes breast cancer recurrence and metastasis. [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 771. doi:10.1158/1538-7445.AM2014-771

Abstract 12: Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature

Background: Lymph node (LN) metastasis, the strongest prognostic factor for breast cancer (BC) patient survival, directly correlates with increased density of lymphatic vessels. The formation of new lymphatic vessels (i.e. lymphangiogenesis) is strongly promoted by tumor-associated macrophages (TAMs) through mechanisms that are not fully understood. We recently discovered that inflammatory lymphangiogenesis is driven by bone marrow derived myeloid cells that transdifferentiate into lymphatic-like cells and structurally contribute to growing lymphatic vasculature. Clinical BC are characterized by an inflammatory environment and massive recruitment of macrophages. We, therefore, hypothesize that BC mobilized monocytic precursors may promote tumor lymphangiogenesis by induction of their transdifferentiation into Macrophage-derived Lymphatic Endothelial Cell Progenitors designated here as M-LECPs. Methodology: To test this hypothesis, we analyzed 75 clinical specimens of invasive breast carcinoma and 5 normal breast tissues for the presence of M-LECPs and incidence of their integration into lymphatic vessels. M-LECPs were identified by double or triple staining using multiple specific markers for myeloid (CD68, CD14, and CD11b) and lymphatic lineages (LYVE-1, podoplanin and VEGFR-3). Integrated M-LECPs were identified by double staining of LYVE-1 + vascular structures with antibodies against myeloid-specific markers as well as confocal microscopy using anti-VE-cadherin staining. Results: Out of 75 tumors, 58% had lymphatics, and 46% of these specimens showed integration of M-LECPs in the lymphatic vessels. In stark contrast, normal mammary tissues showed less than 5% of either double-stained macrophages or dual-identity vessels. The predominant M-LECP association with tumors was also evident in metastatic BC mouse models including MDA-MB-231 (human xenograft), R3L (transplantable syngeneic), and MMTV-PyMT (spontaneous syngeneic). All three models contained 40-50% of TAMs positive for lymphatic markers and up to 90% of lymphatic vessels positive for myeloid markers. In vitro studies showed that inflammatory triggers induce lymphatic proteins in myeloid cells but not myeloid markers in inflamed lymphatic endothelial cells. Conclusions/Significance: These findings show, for the first time, that majority of TAMs in clinical BC exhibit the lymphatic phenotype and integrate into lymphatic vessels. Reproduction of this phenomenon in three BC mouse models and cultured myeloid cells treated with inflammatory mediators suggests a strong self-autonomous role of TAMs into outgrowth of tumor lymphatics. Further investigation of this novel mechanism of TAM-dependent lymphangiogenesis may identify new targets for inhibiting lymphatic metastasis leading to improved survival of breast cancer patients. Citation Format: Kelly Hall, Lisa Volk-Draper, Sandeep Rajput, David DeNardo, Sophia Ran. Tumor macrophages in clinical breast cancers transdifferentiate into lymphatic-like cells and structurally contribute to lymphatic vasculature. [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 12. doi:10.1158/1538-7445.AM2014-12

Abstract 5585: Generation of novel cell lines for analyzing the role of TLR4 in breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Toll like receptor 4 (TLR4) is a transmembrane receptor expressed in macrophages that plays a major role in pathogen recognition and activation of innate immunity. TLR4 signaling has been linked to tumorigenesis and promotion of metastasis in ovarian, lung, prostate and head-neck cancers through up-regulation of the expression of inflammatory cytokines (e.g., IL-6, IL-8 and TNF-alpha). These factors increase cancer cell survival and resistance to chemotherapy through up-regulation of anti-apoptotic and down-regulation of apoptotic proteins, respectively. The goal of this study was to generate new isogenic cell lines that would allow analysis of the TLR4 role in paclitaxel resistance in breast cancer. To this end, we used TLR4-positive and negative lines (MDA-MB-231 and HCC1806, respectively) to generate stable TLR4 knockdown and over-expressing line derivatives. Methods: To generate a TLR4 knockdown line, MDA-MB-231 cells were transfected with pisiRNA-TLR4 plasmid and selected with zeocin (50μg/ml). To generate a TLR4 over-expressing line, HCC1806 cells were transfected with pKT2-TLR4-PURO plasmid and selected with puromycin (1μg/ml). Ten to 20 monoclonal colonies were selected from each line and characterized for TLR4 expression and TLR4-dependent cell activities by qRT-PCR and western blot. Results: TLR4 mRNA expression was reduced by >90% as compared with parental line in five out of 8 MDA-MB-231TLR4− clones examined. One clone had 60% reduction and two clones had no effect on TLR4 mRNA determined by qRT-PCR. TLR4 mRNA was up-regulated 1,000-12,000 fold in five (out of 11) clones derived from HCC1806 line. Two clones overexpressed TLR4 mRNA by 300-500 fold and four clones had increased TLR4 expression by 1-6 fold. All selected clones were analyzed for TLR4 functional signaling by exposure to LPS (1μg/ml), which was reduced by 50-70% in MDA-MB-231TLR4− clones and increased by 15-25 folds in HCC1806TLR4+ sub-lines, as compared with cells transfected with empty vectors. Conclusions: These data show that we successfully created sub-lines of MDA-MB-231 with stable knockdown of TLR4 and sub-lines of breast carcinoma HCC1806 line with stable over-expression of TLR4. Ectopically expressed human TLR4 as well as TLR4 downregulating sequence had significant functional effects on the TLR4 signaling as demonstrated by increase and decrease of LPS-activated targets (IL-6, IL-8 and TNF-alpha) in MDA-MB-231TLR4− and HCC1806TLR4+ sub-lines, respectively. These new sub-lines will serve as excellent tools for analyzing the role of the TLR4 pathway signaling in breast cancer progression, metastasis and resistance to therapy. * These authors contributed equally in this work. 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 5585. doi:10.1158/1538-7445.AM2011-5585