Regulation and Action of Skp2 and Rhoa in Cell and Tumor Models: Investigation into the Molecular Mechanisms Responsible for the Aggressive Phenotype of Triplenegative Breast Cancer
Date of Award
Open Access Dissertation
Doctor of Philosophy (PhD)
Molecular and Cellular Biology
Kathleen F. Arcaro
Sallie Smith Schneider
Barbara A. Osborne
Breast cancer tops the list of new cancer cases and is predicted to be the second leading cause of cancer deaths in women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth and metastasis of triple-negative and basal-like breast cancers. To study increased growth and invasive behavior in triple-negative and basal-like breast cancers we utilize both an interesting and relevant cell culture model and examination of human tissue.
In this study, we use the Tamoxifen-selected, MCF-7 derivative, TMX2-28 breast cancer cell line. TMX2-28 cells are triple-negative in that they lack expression of the estrogen receptor alpha (ERα), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). They also have acquired a mixed basal/luminal cytokeratin profile, suggestive of a more basal-like phenotype. TMX2-28 cells are highly proliferative and invasive.
In addition to our cell culture model, we also examine human tissue. Thirty frozen breast carcinoma samples were evaluated for mRNA expression. Additionally, I analyzed protein expression, using immunohistochemistry (IHC), of 50 benign reduction mammoplasty and 188 breast tumors (formalin-fixed paraffin embedded). Of the 188 breast tumors, 93 were ERα-positive and 95 were ERα-negative. Of the 95 ERα-negative samples, 24 were further classified as non-triple negative (either PR or HER2 positive), 49 were classified as triple-negative, and 22 were not further classified due to unavailability of HER2 status and were used only in analyses of ERα-negative tumors. Thirty-seven of the 188 tumor samples were ductal carcinoma in situ, 138 were invasive ductal carcinomas, and 13 were classified as other. Lastly, 23 of the 188 tumors were grade 1, 48 were grade 2, 105 were grade 3, and 12 did not have grade data available.
S-phase kinase-associated protein 2 (SKP2) plays an important role in cell cycle regulation by targeting p27 for degradation. The cyclin-dependent kinase (CDK) inhibitor p27 regulates G1/S transition by binding cyclin/CDK complexes and abrogating its activity. By targeting p27 for degradation, SKP2 frees the complexes needed to progress into the S phase of the cell cycle. Evaluation of SKP2 expression in TMX2-28 revealed significantly higher levels than in other breast cancer cell lines. Despite the high levels of SKP2 expression, p27 protein was not reduced. However, levels of the Serine 10 phosphorylated form of p27 (pSer10p27), which has been associated with increased proliferation rates, was found to be increased. Furthermore, suppression of SKP2 completely eliminated the pSer10p27 and slowed cycle progression confirming the role of SKP2 in the aggressive growth of TMX2-28 cells.
Assessment of mRNA from 30 frozen human breast cancers demonstrated that SKP2 is more highly expressed in ERα-negative and basal-like breast cancers. Immunohistochemical analysis of 188 breast cancers and 50 benign reduction mammoplasty tissues confirmed that SKP2 is more highly expressed in ERα-negative breast cancers and for the first time demonstrated that triple-negative breast cancers are more likely to overexpress SKP2 than are non-triple-negative, but still ERα-negative, tumors. In contrast to some previous reports, we did not observe an inverse relationship between SKP2 and p27 expression. Only 11% of tumors expressed high SKP2 and low p27, while 32% of tumors had high SKP2 and high p27. Although no significant relationship between SKP2 and p27 expression was observed in human breast cancers, a significant positive relationship was discovered between SKP2 and pSer10p27. Furthermore, high levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple negative breast tumors than in ERα-positive breast cancers. Based on these results and those of the cell culture experiments showing complete elimination of pSer10p27 after suppression of SKP2 it appears that levels of pSer10p27 may be a better indicator of SKP2-dependent p27 degradation than are levels of p27. Therefore, that inhibiting SKP2 in triple-negative breast cancers expressing high levels of both SKP2 and pSer10p27 regardless of p27 levels may be a valid therapeutic approach.
A foremost threat to patients is tumor invasion and metastasis, with the greatest risk to patients diagnosed with triple-negative and basal-like breast cancers. Two distinct morphological/functional mechanisms are known for single cell migration in tissues: mesenchymal and amoeboid invasion. Mesenchymal movement involves the use of proteases that cause cellular lysis in tissues, thereby creating a path through which cells can invade. Amoeboid movement is protease-independent; cells find paths through the ECM by pushing and squeezing through regions of adequate size. Despite their invasive phenotype, TMX2-28 retains morphology similar to non-aggressive MCF-7 cells, suggesting that their invasion may be proteolytic-independent.
We determined that TMX2-28 lack MMP-1 mRNA, and MMP-2/MMP-9 protein expression; each of which is important in protease-dependent invasion. Furthermore, TMX2-28 cells have low expression of other genes key to protease-dependent invasion including Slug, Zeb 1, Zeb 2, Vimentin, Fibronectin and N-cadherin. RhoA is a member of the Rho superfamily of GTPases that acts as a molecular switch to control signal transduction and is critical to the amoeboid invasion mechanism. TMX2-28 cells have high expression of protease-independent invasion genes such as RhoA, ROCK 1, ROCK 2, and E-cadherin. Finally, treating TMX2-28 cells with a RhoA pathway inhibitor or an shRNA targeting RhoA significantly reduces their invasiveness. These data suggest that TMX2-28 cells use a RhoA-dependent, proteolytic-independent invasion mechanism. Collectively, the data presented here demonstrate the roles of SKP2 and RhoA in triple-negative and basal-like breast cancers, making both genes, as well as their pathways, desirable therapeutic targets.
Fagan-Solis, Katrina D., "Regulation and Action of Skp2 and Rhoa in Cell and Tumor Models: Investigation into the Molecular Mechanisms Responsible for the Aggressive Phenotype of Triplenegative Breast Cancer" (2013). Open Access Dissertations. 683.