Svasti Haricharan

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

Mechanism and preclinical prevention of increased breast cancer risk caused by pregnancy

While a first pregnancy before age 22 lowers breast cancer risk, a pregnancy after age 35 significantly increases life-long breast cancer risk. Pregnancy causes several changes to the normal breast that raise barriers to transformation, but how pregnancy can also increase cancer risk remains unclear. We show in mice that pregnancy has different effects on the few early lesions that have already developed in the otherwise normal breast—it causes apoptosis evasion and accelerated progression to cancer. The apoptosis evasion is due to the normally tightly controlled STAT5 signaling going astray—these precancerous cells activate STAT5 in response to pregnancy/lactation hormones and maintain STAT5 activation even during involution, thus preventing the apoptosis normally initiated by oncoprotein and involution. Short-term anti-STAT5 treatment of lactation-completed mice bearing early lesions eliminates the increased risk after a pregnancy. This chemoprevention strategy has important implications for preventing increased human breast cancer risk caused by pregnancy. DOI: http://dx.doi.org/10.7554/eLife.00996.001

Contribution of an alveolar cell of origin to the high-grade malignant phenotype of pregnancy-associated breast cancer

Pregnancy-associated breast cancers (PABCs) are tumors diagnosed during pregnancy or up to 5 years following parturition, and are usually high-grade, connective tissue-rich, and estrogen receptor (ER)/progesterone receptor-negative. Little is known about the cellular origin of PABCs or the mechanisms by which PABCs are initiated. Using the RCAS retrovirus to deliver the ErbB2 oncogene into the mammary epithelium of our previously reported MMTV-tva transgenic mice, we detected high-grade, poorly differentiated, stroma-rich and ER-negative tumors during pregnancy and lactation. These high-grade and stroma-rich tumors were less frequent in involuted mice or in age-matched nulliparous mice. More importantly, by generating a WAP-tva transgenic line for expression of ErbB2 selectively in WAP+ mammary alveolar cells, we found that tumors had similar morphological phenotypes (high grade, poorly differentiated, stroma-rich and ER-negative), irrespective of the time since pregnancy and even in the absence of pregnancy. These data suggest that PABCs arise preferentially from an alveolar cell population that expands during pregnancy and lactation. This somatic mouse model may also be useful for preclinical testing of new prophylactic and therapeutic strategies against PABC.

Luminal epithelial cells within the mammary gland can produce basal cells upon oncogenic stress

In the normal mammary gland, the basal epithelium is known to be bipotent and can generate either basal or luminal cells, whereas the luminal epithelium has not been demonstrated to contribute to the basal compartment in an intact and normally developed mammary gland. It is not clear whether cellular heterogeneity within a breast tumor results from transformation of bipotent basal cells or from transformation and subsequent basal conversion of the more differentiated luminal cells. Here we used a retroviral vector to express an oncogene specifically in a small number of the mammary luminal epithelial cells and tested their potential to produce basal cells during tumorigenesis. This in-vivo lineage-tracing work demonstrates that luminal cells are capable of producing basal cells on activation of either polyoma middle T antigen or ErbB2 signaling. These findings reveal the plasticity of the luminal compartment during tumorigenesis and provide an explanation for cellular heterogeneity within a cancer.

Loss of MutL Disrupts CHK2-Dependent Cell-Cycle Control through CDK4/6 to Promote Intrinsic Endocrine Therapy Resistance in Primary Breast Cancer

Significant endocrine therapy-resistant tumor proliferation is present in ≥20% of estrogen receptor-positive (ER+) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair (MMR) complex (MLH1/3, PMS1/2), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER+ breast cancer abrogates CHK2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER+ breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit aromatase inhibitor-resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of patients with breast cancer who exhibit marked resistance to the current standard of care.Significance: MutL deficiency in a subset of ER+ primary tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy-resistant tumors. Therefore, markers of MutL dysregulation could guide CDK4/6 inhibitor use in the adjuvant setting, where the risk benefit ratio for untargeted therapeutic intervention is narrow. Cancer Discov; 7(10); 1168-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1047.

Comprehensive Profiling of DNA Repair Defects in Breast Cancer Identifies a Novel Class of Endocrine Therapy Resistance Drivers

Purpose: This study was undertaken to conduct a comprehensive investigation of the role of DNA damage repair (DDR) defects in poor outcome ER+ disease. Experimental Design: Expression and mutational status of DDR genes in ER+ breast tumors were correlated with proliferative response in neoadjuvant aromatase inhibitor therapy trials (discovery dataset), with outcomes in METABRIC, TCGA, and Loi datasets (validation datasets), and in patient-derived xenografts. A causal relationship between candidate DDR genes and endocrine treatment response, and the underlying mechanism, was then tested in ER+ breast cancer cell lines. Results: Correlations between loss of expression of three genes: CETN2 (P < 0.001) and ERCC1 (P = 0.01) from the nucleotide excision repair (NER) and NEIL2 (P = 0.04) from the base excision repair (BER) pathways were associated with endocrine treatment resistance in discovery dataset, and subsequently validated in independent patient cohorts. Complementary mutation analysis supported associations between mutations in NER and BER genes and reduced endocrine treatment response. A causal role for CETN2, NEIL2, and ERCC1 loss in intrinsic endocrine resistance was experimentally validated in ER+ breast cancer cell lines, and in ER+ patient-derived xenograft models. Loss of CETN2, NEIL2, or ERCC1 induced endocrine treatment resistance by dysregulating G1–S transition, and therefore, increased sensitivity to CDK4/6 inhibitors. A combined DDR signature score was developed that predicted poor outcome in multiple patient cohorts. Conclusions: This report identifies DDR defects as a new class of endocrine treatment resistance drivers and indicates new avenues for predicting efficacy of CDK4/6 inhibition in the adjuvant treatment setting. Clin Cancer Res; 24(19); 4887–99. ©2018 AACR.

Functional Annotation of ESR1 Gene Fusions in Estrogen Receptor-Positive Breast Cancer.

SUMMARY RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective.

Mammary Ductal Environment Is Necessary for Faithful Maintenance of Estrogen Signaling in ER+ Breast Cancer

In this issue of Cancer Cell, Sflomos et al. (2016) describe a robust preclinical animal model of ER⁺ breast cancer. The authors identify the critical role of the breast microenvironment in determining hormone response of ER⁺ breast cancer cells and in driving the luminal phenotype of breast cancer.

Abstract 489: Mismatch repair defects and endocrine therapy resistance in estrogen receptor positive breast cancer

Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic resistance occurs in ~1/3 of patients and acquired resistance in ~1/5 of the remainder. While many resistance mechanisms have been explored, therapeutic strategies to overcome resistance in the clinical setting have seen mixed outcomes, and appear most effective in the acquired resistance setting. Understanding mechanisms of resistance and finding therapeutic strategies to target them, therefore, remain important challenges facing breast cancer researchers. In this study we systematically examine the role of DNA damage repair defects in inducing endocrine therapy resistance, a relatively understudied question of recent interest. We use in silico analysis of clinical datasets, in vitro experiments evaluating endocrine therapy resistance in response to DDR dysregulation in multiple breast cancer celllines, and in vivo validation using cellline xenograft and patient-derived xenograft models. We also use gene expression microarrays and RPPA data from cell lines, patient-derived xenografts and primary ER+ breast tumors to uncover therapeutic options that are validated in vitro and in vivo and corroborated by clinical trial data. The results of this study uncover an intriguing link between mismatch repair (MMR) deficiency, specifically of the MutL complex (MLH1/3, PMS1/2), and poor prognosis in ER+ disease. We find a direct role for MutL loss in endocrine therapy resistance in vitro and in vivo by knocking down multiple MutL genes using CRISPR and stable shRNA approaches validated using standard rescue experiments. We identify the underlying mechanism: MutL deficiency in ER+ breast cancer abrogates Chk2-mediated feedback inhibition of CDK4/6 that appears necessary for endocrine therapy responsiveness. Consequently, pharmacological targeting of CDK4/6 in vitro and in vivo significantly inhibits growth of endocrine therapy resistant MutL-deficient ER+ breast cancer cells. These results are corroborated by data from a neoadjuvant clinical trial demonstrating that cell cycle regulation of MutL-mutant tumors tends to be estrogen-independent but sensitive to CDK4/6 inhibitors. The results of this study provide important biological and clinically relevant insights. 1) MMR deficiency is unexpectedly causal to intrinsic endocrine therapy resistance 2) This causal effect appears to be mediated by abrogation of cell cycle checkpoint activation in response to endocrine therapy 3) MMR deficiency in a subset of ER+ tumors explains why CDK4/6 inhibition is effective against some de novo endocrine therapy resistant tumors. While there are currently no biomarkers to guide the use of CDK4/6 inhibitors for ER+ breast cancer, markers of MMR dysregulation could identify patients in whom CDK4/6 inhibition should be used to prevent disease recurrence. Citation Format: Svasti Haricharan, Jacob Schmelz, Cheryl Schmidt, Purba Singh, Kimberly R. Holloway, Meenakshi Anurag, Shunqiang Li, Shyam M. Kavuri, Shixia Huang, Dean P. Edwards, Vera Suman, Kelly Hunt, John A. Olson, Jeremy Hoog, Cynthia X. Ma, Matthew N. Bainbridge, Matthew J. Ellis. Mismatch repair defects and endocrine therapy resistance in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 489. doi:10.1158/1538-7445.AM2017-489

Abstract PD2-06: Mismatch repair deficiency induces endocrine therapy resistance in breast cancer

Estrogen receptor positive (ER+) breast cancer accounts for the majority of breast cancers diagnosed worldwide but fortunately, outcomes are markedly improved by pharmacological interventions that interrupt ER function. Unfortunately, suppression of relapse risk from early stage disease with endocrine therapy (anti-estrogens or aromatase inhibitors) is only ∼50%, and for advanced disease, pan endocrine therapy resistance is almost inevitable. While many mechanisms for intrinsic and acquired endocrine resistance have been explored, links between defects in DNA repair, the fundamental drivers of cancer pathogenesis, and endocrine therapy resistance have been understudied. Here we link mismatch repair (MMR) deficiency to poor clinical outcomes in ER+ breast cancer using whole exome DNA sequencing data and mRNA expression analysis. We subsequently demonstrate that MMR deficiency bypasses ER dependent cell cycle regulation through disruption of Chk2/p21-mediated feedback inhibition of CDK4 in breast cancer cell lines and tumor samples, as well as through correlations with human clinical data. We also show that pharmacological targeting of CDK4 significantly inhibits growth of MMR-deficient ER+ breast cancer cells in vitro and in vivo. This mechanism provides a new explanation for why endocrine therapy resistant ER+ breast cancers can respond to CDK inhibition and suggests that primary tumors exhibiting MMR deficiency are good candidates for adjuvant CDK4 inhibitor treatment. Citation Format: Haricharan S, Schmelz J, Schmidt C, Singh P, Holloway K, Anurag M, Suman V, Olson JA, Hunt K, Bainbridge MN, Ellis MJ. Mismatch repair deficiency induces endocrine therapy resistance in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-06.