Nearly 300,000 women were predicted to be diagnosed with breast cancer in 2011, thus making breast cancer one of the most common cancers in women and a disease of high priority. Breast cancer is considered to be a heterogeneous disease that is categorized based upon biomarker expression and drug sensitivity. However, a greater understanding of the basic cellular processes that are exploited by breast cancer cells may lead to the development of novel therapeutics, which can be used to overcome disease heterogeneity. Poor prognosis has been correlated to tumor metastasis and drug resistance. Therefore, there is a need to build on the present understanding of the mechanisms underlying cell migration and invasion, and to elucidate the actions and consequences of broad-targeting chemotherapies, such as taxol. The role of the endoplasmic reticulum (ER) in breast cancer and in cancer-relevant cellular processes is unclear. Recent discoveries identifying novel proteins involved in regulating ER structure and ER dynamics now permit investigators to test hypotheses relating ER structure and function. Additionally, ER dynamics along microtubules (MTs) have been characterized as tip- attachment complex (TAC) dynamics and ER sliding. Moreover, recent studies have reported that integrin-?3 and protein phosphatase 1B (PTP1B) trafficking to focal adhesions was dependent on ER tubule extension along microtubules. Thus, this proposal implicates a relationship between ER structure/ER dynamics and cell motility, and taxol resistance;and will elucidate the mechanisms underlying the relationship between the ER and focal adhesions in breast cancer cells.
The first aim of this proposal consists of foundational high-resolution studies that will characterize ER structure in non-invasive (MCF-7) and invasive (MDA-MB-231) breast cancer cells using live- cell and fixed-cell fluorescence microscopy. Furthermore, ER structure will be directly modulated in cells by overexpression or knockdown of ER structural proteins. The performance of ER-modulated breast cancer cells will be measured in matrigel migration, transendothelial-matrigel invasion and tracking assays. Finally, the distribution of ER-organellar contacts within the leading edge of migrating breast cancer cells will be elucidated by electron microscopy.
The second aim will test the hypothesis that taxols mediate breast cancer cell killing by disrupting ER dynamics along MTs, thus disrupting ER-dependent delivery of focal adhesion proteins to focal adhesions. ER dynamics along microtubules will be modulated by altering TAC dynamics via STIM1 expression, or ER sliding via HDAC6 expression in breast cancer cells, then testing the sensitivity of these cells to taxol in cell killing assays. Furthermore, co-transfection of breast cancer cells with GFP-tagged focal adhesion proteins (integrin-?3 or PTP1B) and RFP-KDEL (ER marker) will be used to visualize ER tubule-associated delivery of these proteins to focal adhesions, and to determine whether this delivery process is dependent on TAC ER dynamics or ER sliding. Thus, these studies aim to fill a void within the current understanding of the subcellular events underlying cell migration, and taxol treatment by investigating the role of the ER in the mechanisms of these processes.
Gaining a greater understanding of the basic cellular processes that are exploited by breast cancer cells may lead to the development of novel therapeutics, which can be used to treat patients of multiple breast cancer sub-types, and to prevent/overcome drug resistance. We propose to fill a void within the present understanding of the subcellular events underlying cell migration and invasion, and to elucidate the actions and consequences of broad-targeting therapies, such as taxol, by investigating the role of a major cellular compartment, named the endoplasmic reticulum, in breast cancer and in cancer-relevant cellular processes.
|Westrate, L M; Lee, J E; Prinz, W A et al. (2015) Form follows function: the importance of endoplasmic reticulum shape. Annu Rev Biochem 84:791-811|