Analysis of Trastuzumab (Tzb) Sensitivity in HER2+ PTEN-Deficient Breast Cancer Using Single Cell RNA-Seq

Abstract

HER2+ breast cancer is marked by the overexpression and/or amplification of the HER2 protein or HER2 gene, respectively. Current standard of care is trastuzumab-based therapy, but resistance remains a huge hurdle for metastatic HER2+ breast cancer patients. The loss of tumor suppressor PTEN has been regarded to contribute to trastuzumab resistance, but the exact role of PTEN status in HER2+ breast cancer and its prognostic value remains controversial. This dissertation aims to unravel the role of PTEN expression status in HER2+ breast cancer to gain insight on its contribution to trastuzumab sensitivity. We aim to understand how PTEN deficiency alters HER2+ breast cancer subpopulations and how changes induced by PTEN deficiency could result in an aggressive cancer phenotype and/or impact response to trastuzumab. We hypothesized that PTEN deficiency in HER2+ breast cancer increases the aggressive cancer cell subpopulations that are responsible for trastuzumab resistance. To test our hypothesis, we used an unbiased single cell RNA sequencing approach called Drop-seq to profile transcriptomes of cells constituting HER2+ breast cancer in vitro. By profiling four different HER2+ breast cancer cell line pairs containing a parental and shPTEN cell line with Drop-seq, we were able to dissect the functional consequences of PTEN deficiency in vitro. Also, we investigated both the intra- and intertumoral heterogeneity effects of PTEN deficiency in HER2+ breast cancer. Comparative analyses of the transcriptomes arising from parental and shPTEN cell lines provided information about the intratumoral consequences of PTEN deficiency. These studies revealed that PTEN deficiency in HER2+ breast cancer cell lines resulted in a global increase of quiescent features in the shPTEN cell line relative to the parental cell lines for HCC1954 and SKBR3 but not for BT474. Furthermore, PTEN deficiency resulted in a 84 fold, 120 fold, and a 2.4 fold increase in a quiescent, epithelial, early EMT subpopulation in HCC1954, SKBR3, and BT474, respectively. PTEN deficiency introduced intra-subpopulation heterogeneity by altering the expression of a subset of EMT, cytokine, cell cycle, and cell adhesion genes in HCC1954 and SKBR3 but not BT474. Comparative analyses of changes to the single cell transcriptomes resulting from PTEN deficiency between cell lines afforded insight on the intertumoral consequences of PTEN deficiency in HER2+ breast cancer. These analyses revealed that effects of PTEN deficiency were similar in HCC1954 and SKBR3 due to the similarities in magnitudes of subpopulation level changes and presence of intra-subpopulation level changes while hinted that BT474 represented a unique case for studying the consequences of PTEN deficiency. Altogether, these analyses captured the context-dependent effects that PTEN deficiency in HER2+ breast cancer. Additionally, we aimed to elucidate how transcriptomes shaped by a pre-existing PTEN deficiency could impact trastuzumab response by performing treatment studies with two HER2+ breast cancer cell line pairs (parental and shPTEN cell line). Treatment studies of BT474 and MDA-MB-361 showed minor changes in cancer subpopulations between treated and untreated cells, and these studies remain inconclusive at this point. Collectively, studies presented in this dissertation could have important clinical implications about the consequences of PTEN deficiency in HER2+ breast cancer as we highlighted the intra- and intertumoral consequences of PTEN deficiency. This insight could contribute to the identification of biomarkers that predicts patient response to trastuzumab and facilitate the discovery of alternative therapeutic strategies for patients who acquire resistance to trastuzumab.