Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Ran, Sophia

Abstract

Breast cancer (BC) is the most common cancer and ranks second as a cause of cancer death in women. Every year about one million women diagnosed with breast cancer some of which becomes metastatic leading to decreased patient survival. Paclitaxel (PXL) is a potent cytotoxic drug widely used as a first and second line drug-of-choice for the treatment of metastatic breast cancer. However, resistance develops frequently with evasion mechanisms remaining largely unclear. PXL elicits both cytotoxic and pro-survival responses in tumor cells. The tumor-promoting effect of PXL is currently unrecognized determinant for decreasing the apoptotic effect of PXL therapy. The likely mechanism for paclitaxel-dependent tumor-activating effects is the ability of PXL to activate Toll-like Receptor-4 (TLR4) pathway. TLR4 is activated by lipopolysaccharide (LPS) and other ligands including the widely used drug PXL. 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. Also, overexpression of Toll-like receptor-4 (TLR4) in human tumors often correlates with chemoresistance and metastasis. Despite these logical considerations implicating, TLR4 in chemoresistance and metastasis, limited evidence is available to support this claim experimentally. Most of the previous studies had used LPS, to show a tumor-promoting role of TLR4 in different cancers, although this bacterial component is unlikely to be found in the sterile breast cancer environment. Therefore, we formulated the following hypothesis i.e. (1) TLR4 activation by PXL in breast cancer cells promotes chemoresistance via activation of NF-kB pathway. (2) TLR4 activation by PXL in breast cancer cells promotes metastasis and tumor recurrence. To test our first hypothesis we analyzed the expression profile of TLR4 in a panel of breast carcinoma cells and clinical breast tumor specimens. TLR4 was found to be expressed in 60% of human breast cancer cell lines and majority of breast tumor specimens. Based on the analysis of TLR4 expression we selected two breast cancer cell line MDA-MB-231 (TLR4 positive) and HCC1806 (TLR4 negative) for further experiments. TLR4-positive (MDA-MB-231) and negative (HCC1806) lines were engineered to stably down-regulate and over-express TLR4, respectively. We correlated TLR4 expression with resistance to PXL in these 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 PXL 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 PXL in vitro and enhanced tumor resistance to paclitaxel therapy in vivo. 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 in MDA-MB-231 (TLR4 positive) and HCC1806 (TLR4 over-expressed) breast cancer cells. To test our second hypothesis, we determined the incidence of tumor metastasis and recurrence (as a measure of response to PXL therapy) in these 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 to and post-PXL treatment. We found that PXL treatment was very effective in inhibiting growth of TLR4-negative tumors, but highly increased the incidence and burden of pulmonary and lymphatic metastasis in tumors that expressed TLR4. Activation of TLR4 pathway substantially increases tumor local inflammation as demonstrated by the increase in protein level of inflammatory cytokines IL6 (250pg/ml) and IL8 (350pg/ml) in HCC1806 (TLR4 overexpressed group) on paclitaxel treatment. TLR4 activation by PXL also promoted systemic inflammation as demonstrated by the increased protein levels of IL6 and IL8 in blood samples of HCC1806 (TLR4 over-expressed) mice group. Generation of these local tumor and systemic inflammatory responses leads to increased generation of myeloid progenitors (i.e. Ly6C and Ly6G) that can promote metastasis by enhancing the formation of tumor blood and lymphatic vessels. There was also an enhancement of blood and lymphatic vessels in HCC1806 (TLR4 over-expressed) tumors on paclitaxel treatment as analyzed by IHC. PXL-dependent activation of TLR4-positive tumors induced de novo generation of deep-suited intratumoral lymphatic vessels that were highly permissive to invasion by malignant cells. These data show that PXL 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. Also, PXL therapy of patients with TLR4-expressing tumors may activate systemic inflammatory circuits that promote angiogenesis, lymphangiogenesis, and metastasis at both the site of local invasion and the premetastatic niche in distal organs. Therefore, it can be speculated that tumor expression of TLR4 may indicate poor prognosis and response to therapy, and that blocking the TLR4 pathway might improve current anti-cancer treatments. In Summary my research has revealed the important role of TLR4 in acquisition of chemoresistance and promotion of metastasis on paclitaxel (PXL) treatment in breast cancer cells that is both a stimulator of this receptor and a widely-used chemotherapeutic drug.

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