Signaling from estrogen receptor alpha (ERα) and its ligand estradiol (E2) is critical for growth of ≈70% of breast cancers. Therefore, several drugs that inhibit ERα functions have been in clinical use for decades and new classes of anti-estrogens are continuously being developed. Although a significant number of ERα+ breast cancers respond to anti-estrogen therapy, ≈30% of these breast cancers recur, sometimes even after 20 years of initial diagnosis. Mechanism of resistance to anti-estrogens is one of the intensely studied disciplines in breast cancer. Several mechanisms have been proposed including mutations in ESR1, crosstalk between growth factor and ERα signaling, and interplay between cell cycle machinery and ERα signaling. ESR1 mutations as well as crosstalk with other signaling networks lead to ligand independent activation of ERα thus rendering anti-estrogens ineffective, particularly when treatment involved anti-estrogens that do not degrade ERα. As a result of these studies, several therapies that combine anti-estrogens that degrade ERα with PI3K/AKT/mTOR inhibitors targeting growth factor signaling or CDK4/6 inhibitors targeting cell cycle machinery are used clinically to treat recurrent ERα+ breast cancers. In this review, we discuss the nexus between ERα-PI3K/AKT/mTOR pathways and how understanding of this nexus has helped to develop combination therapies.

Signaling from estrogen receptor alpha (ER) and its ligand estradiol (E2) is critical for growth of ~70% of breast cancers. Therefore, several drugs that inhibit ER functions are in clinical use for decades and new classes of anti-estrogens are continuously being developed. Although a significant number of ER+ breast cancers respond to anti-estrogen therapy, ~30% of these breast cancers recur, sometimes even after 20 years of initial diagnosis. Mechanism of resistance to anti-estrogens is one of the intensely studied disciplines in breast cancer. Several mechanisms have been proposed including mutations in ESR1, crosstalk between growth factor and ER signaling, and interplay between cell cycle machinery and ER signaling. ESR1 mutations as well as crosstalk with other signaling networks lead to ligand independent activation of ER thus rendering anti-estrogens ineffective, particularly when treatment involved anti-estrogens that do not degrade ERa. As a result of these studies, several therapies that combine anti-estrogens that degrade ER with PI3K/AKT/mTOR inhibitors targeting growth factor signaling or CDK4/6 inhibitors targeting cell cycle machinery are used clinically to treat recurrent ER+ breast cancers. In this review, we discuss nexus between ER-PI3K/AKT/mTOR pathways and how understanding of this nexus has helped to develop combination therapies.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: The Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR.

Genetic approaches in Drosophila have successfully identified many genes involved in regulation of growth control as well as genetic interactions relevant to the initiation and progression of cancer in vivo. Here, we report on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. We also report on an independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. We also find a number of novel connections to Yki and EGFR driven tissue growth, mostly unique to one of the two. Thus, resources provided here would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use.

Study of genomic aberrations leading to immortalization of epithelial cells has been technically challenging due to the lack of isogenic models. To address this, we used healthy primary breast luminal epithelial cells of different genetic ancestry and their hTERT-immortalized counterparts to identify transcriptomic changes associated with immortalization. Elevated expression of TONSL (Tonsoku-like, DNA repair protein) was identified as one of the earliest events during immortalization. TONSL, which is located on chromosome 8q24.3, was found to be amplified in approximately 20% of breast cancers. TONSL alone immortalized primary breast epithelial cells and increased telomerase activity, but overexpression was insufficient for neoplastic transformation. However, TONSL-immortalized primary cells overexpressing defined oncogenes generated estrogen receptor–positive adenocarcinomas in mice. Analysis of a breast tumor microarray with approximately 600 tumors revealed poor overall and progression-free survival of patients with TONSL-overexpressing tumors. TONSL increased chromatin accessibility to pro-oncogenic transcription factors, including NF-κB and limited access to the tumor-suppressor p53. TONSL overexpression resulted in significant changes in the expression of genes associated with DNA repair hubs, including upregulation of several genes in the homologous recombination (HR) and Fanconi anemia pathways. Consistent with these results, TONSL-overexpressing primary cells exhibited upregulated DNA repair via HR. Moreover, TONSL was essential for growth of TONSL-amplified breast cancer cell lines in vivo, and these cells were sensitive to TONSL–FACT complex inhibitor CBL0137. Together, these findings identify TONSL as a regulator of epithelial cell immortalization to facilitate cancer initiation and as a target for breast cancer therapy. Significance: The chr.8q24.3 amplicon-resident gene TONSL is upregulated during the initial steps of tumorigenesis to support neoplastic transformation by increasing DNA repair and represents a potential therapeutic target for treating breast cancer.

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Background: Inheritance of BRCA1 and BRCA2 mutations is associated with increased risk of breast and ovarian cancers. Previous studies with low-throughput flow cytometry-based assays suggested elevated number of luminal progenitor cells in the breast tissues of BRCA1 mutation carriers compared to breast tissues of non-carriers. However, breast epithelial cell-specific transcriptome differences between BRCA1, BRCA2 mutation carriers, and non-carriers and how these differences alter susceptibility to transformation are yet to be elucidated. Methods: We generated a single cell transcriptome atlas of breast tissues from BRCA1 (six samples, 17,220 cells), BRCA2 (four samples, 25,046 cells) mutation carriers and non-carriers (11 samples, >50,000 cells). Using previously described markers, epithelial cells were sub-clustered into basal, luminal progenitor, and mature luminal cells. Genes differentially expressed in epithelial cells of BRCA1 and BRCA2 mutation carriers compared to those in non-carrier donors were subjected to Ingenuity Pathway Analysis to determine signaling pathways uniquely active in BRCA1 and BRCA2 mutant epithelial cells. Breast epithelial cells derived from three donor types were immortalized using hTERT and then transformed with PIK3CAH1047R mutant or H-RasG12V ± SV40-T/t antigens, and tumorigenicity was determined in vivo. Results: BRCA1 but not BRCA2 mutations altered the ratio between basal, luminal progenitor and mature luminal cells in breast tissues compared to breast tissues in non-carriers. A unique cluster of cells within luminal progenitors was underrepresented in case of BRCA2 mutation carriers compared to non-carriers or BRCA1 mutation carriers. BRCA1 or BRCA2 mutations specifically altered transcriptomes which are an integral part of mTOR and MYCN signaling, and the translational machinery. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included YAP1, BRD4, SMARCA4, and TGFβ1 signaling. BRCA2 mutations were associated with upregulation of IL-6, FOXO3, and TNFSF11 signaling. Breast epithelial cells from BRCA2 mutation carriers but not BRCA1 mutation carriers or non-carriers modified to overexpress hTERT + PIK3CAH1047R generated tumors in NSG mice. These tumors displayed large cystic structures with basilar epithelial cells lining the rim of cysts with focal areas of cellular hyperplasia and neoplastic cells that extended into the lumen. However, BRCA1/2 mutation status did not influence tumorigenicity by hTERT+ H-RASG12V +SV40-T/t antigens. Conclusions: Our studies provide a high resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers, which also reveal potentially targetable signaling networks uniquely deregulated in these cells. BRCA2 mutations are associated with distinct susceptibility to PIK3CA mutation-driven transformation. Since PIK3CA mutations are observed in clinically normal breast tissues, screening for such mutations in BRCA2 mutation carriers may help to detect pre-neoplastic or early stage breast cancer. Citation Format: Harikrishna Nakshatri, Poornima Bhat-Nakshatri, Duojiao Chen, Katie Chen, Henry Mang, Christopher A Herodotou, Aditi S Khatpe, Patrick C McGuire, Xiaoling Xuei, Yunlong Liu, George Sandusky, Anna Maria Storniolo. Single cell transcriptomic analysis reveals the effects of BRCA1 and BRCA2 mutations on distinct signaling networks and cancer susceptibility [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-07-09.

Single cell transcriptomics studies have begun to identify breast epithelial cell and stromal cell specific transcriptome differences between BRCA1/2 mutation carriers and non-carriers. We generated a single cell transcriptome atlas of breast tissues from BRCA1, BRCA2 mutation carriers and compared this single cell atlas of mutation carriers with our previously described single cell breast atlas of healthy non-carriers. We observed that BRCA1 but not BRCA2 mutations altered the ratio between basal, luminal progenitor and mature luminal cells in breast tissues. A unique subcluster of cells within luminal progenitors is underrepresented in case of BRCA1 and BRCA2 mutation carriers compared to non-carriers. Both BRCA1 and BRCA2 mutations specifically altered transcriptomes in epithelial cells which are an integral part of NF-kappaB, LARP1, and MYC signaling. Reduction of MYC signaling and translational machinery in BRCA1/2 mutant epithelial cells is reminiscent of embryonic diapause-like adaptation that occurs in drug tolerant populations of cells. Signaling pathway alterations in epithelial cells unique to BRCA1 mutations included STAT3, BRD4, SMARCA4, HIF2A/EPAS1, and Inhibin-A signaling. BRCA2 mutations were associated with upregulation of IL-6, PDK1, FOXO3, and TNFSF11 signaling. These signaling pathway alterations are sufficient to alter sensitivity of BRCA1/BRCA2-mutant breast epithelial cells to transformation as epithelial cells from BRCA1 mutation carriers overexpressing hTERT + PIK3CAH1047R generated adenocarcinomas, whereas similarly modified mutant BRCA2 cells generated basal carcinomas in NSG mice. Thus, our studies provide a high-resolution transcriptome atlas of breast epithelial cells of BRCA1 and BRCA2 mutation carriers and reveal their susceptibility to PIK3CA mutation-driven transformation.