Kelly LaPara

MicroRNAs Link Estrogen Receptor Alpha Status and Dicer Levels in Breast Cancer

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1− cells (∼100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3′ UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1− cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3′ UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα− cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1− cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα− breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.

The DNA cytosine deaminase APOBEC3B promotes tamoxifen resistance in ER-positive breast cancer

An antiviral enzyme promotes drug resistance in breast cancer. Breast tumors often display extreme genetic heterogeneity characterized by hundreds of gross chromosomal aberrations and tens of thousands of somatic mutations. Tumor evolution is thought to be ongoing and driven by multiple mutagenic processes. A major outstanding question is whether primary tumors have preexisting mutations for therapy resistance or whether additional DNA damage and mutagenesis are necessary. Drug resistance is a key measure of tumor evolvability. If a resistance mutation preexists at the time of primary tumor presentation, then the intended therapy is likely to fail. However, if resistance does not preexist, then ongoing mutational processes still have the potential to undermine therapeutic efficacy. The antiviral enzyme APOBEC3B (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3B) preferentially deaminates DNA C-to-U, which results in signature C-to-T and C-to-G mutations commonly observed in breast tumors. We use clinical data and xenograft experiments to ask whether APOBEC3B contributes to ongoing breast tumor evolution and resistance to the selective estrogen receptor modulator, tamoxifen. First, APOBEC3B levels in primary estrogen receptor–positive (ER+) breast tumors inversely correlate with the clinical benefit of tamoxifen in the treatment of metastatic ER+ disease. Second, APOBEC3B depletion in an ER+ breast cancer cell line results in prolonged tamoxifen responses in murine xenograft experiments. Third, APOBEC3B overexpression accelerates the development of tamoxifen resistance in murine xenograft experiments by a mechanism that requires the enzyme’s catalytic activity. These studies combine to indicate that APOBEC3B promotes drug resistance in breast cancer and that inhibiting APOBEC3B-dependent tumor evolvability may be an effective strategy to improve efficacies of targeted cancer therapies.

Disruption of insulin receptor function inhibits proliferation in endocrine-resistant breast cancer cells.

The insulin-like growth factor (IGF) system is a well-studied growth regulatory pathway implicated in breast cancer biology. Clinical trials testing monoclonal antibodies directed against the type I IGF receptor (IGF1R) in combination with estrogen receptor-α (ER) targeting have been completed, but failed to show benefits in patients with endocrine-resistant tumors compared to ER targeting alone. We have previously shown that the closely related insulin receptor (InsR) is expressed in tamoxifen-resistant (TamR) breast cancer cells. Here we examined if inhibition of InsR affected TamR breast cancer cells. InsR function was inhibited by three different mechanisms: InsR short hairpin RNA, a small InsR-blocking peptide, S961 and an InsR monoclonal antibody (mAb). Suppression of InsR function by these methods in TamR cells successfully blocked insulin-mediated signaling, monolayer proliferation, cell cycle progression and anchorage-independent growth. This strategy was not effective in parental cells likely because of the presence of IGFR /InsR hybrid receptors. Downregulation of IGF1R in conjunction with InsR inhibition was more effective in blocking IGF- and insulin-mediated signaling and growth in parental cells compared with single-receptor targeting alone. Our findings show TamR cells were stimulated by InsR and were not sensitive to IGF1R inhibition, whereas in tamoxifen-sensitive parental cancer cells, the presence of both receptors, especially hybrid receptors, allowed cross-reactivity of ligand-mediated activation and growth. To suppress the IGF system, targeting of both IGF1R and InsR is optimal in endocrine-sensitive and -resistant breast cancer.

MCF-7 cells selected for acquired resistance to an anti IGF-IR antibody remain sensitive to fulvestrant and to an IGFIR tyrosine kinase inhibitor (TKI).

Abstract #72 Monoclonal antibodies and tyrosine kinase inhibitors (TKI) targeting the type-1 Insulin-like growth factor receptor (IGFIR) are currently in clinical trials. Resistance to several targeted therapies has been observed and is not well understood. Estrogen receptor (ER) has been shown to interact with IGFIR signaling to promote cell growth. To examine acquired resistance to IGF1R monoclonal antibodies, we selected ER positive MCF7L cells in increasing concentrations of an IGFIR inhibitory antibody (scFvFc, treated over 2 years). These cells (MCF7L-scFvFc) had downregulated IGFIR levels, increased basal phosphorylation of IRS-1 with constitutive activation of Akt. MCF7L-scFvFc cells had enhanced basal growth and were no longer responsive to IGF-I and estradiol (E2). To examine the relationship between IGF1R and ER function, we used the pure steroidal anti-estrogen fulvestrant to examine effects on growth of the parent and antibody-resistance cells. In parent MCF7L cells, fulvestrant inhibited growth responses to IGF-I and E2 treatment when each ligand was given individually, but when both ligands were given together, the growth inhibition was reversed. In MCF7L-scFvFc, fulvestrant inhibited growth response to IGF-I, but cells still responded to E2 even in the presence of fulvestrant. Fulvestrant inhibition of MCF7L-scFvFc basal growth was associated with downregulation of ER and IGFIR system components. Fulvestrant treatment induced PARP cleavage in MCF7L, but not in MCF7L-scFvFc cells. IGF-I partially protected cells against fulvestrant induced PARP cleavage, and E2 treatment has full protective effects on these cells. These data suggest that MCF7L-scFvFc cells were more resistant to cell apoptosis induced by fulvestrant. Other monoclonal antibodies directed against IGF1R were ineffective in reversing the resistance in MCF7L-scFvFc cells. To determine if activated IGF1R was responsible for the behavior of MCF7L-scFvFc cells, we used the IGF1R TKI AEW541. AEW541 inhibited growth of these antibody resistant cells and a combination of fulvestrant and AEW541 completely inhibited cell growth and IGF signaling in both MCF7L and MCF7L-scFvFc cells. Thus, activated IGFIR signaling was still required for the growth of MCF7L-scFvFc cells. In conclusion, MCF7L-scFvFc had low levels of IGF1R but retained activated IGF1R signaling that was inhibited by TKI (AEW541). In addition, the ER antagonist fulvestrant also inhibited growth of these cells. In these ER-positive cells, combined blockade of ER and IGF1R could provide a more prolonged inhibition of cell growth. Moreover, cells selected for resistance to an IGF1R antibody remain sensitive to IGF1R TKIs. Thus, concurrent or sequential targeting of IGF1R and ER function should be examined in breast cancer clinical trials of ER positive breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 72.

Abstract S4-07: Tamoxifen resistance driven by the DNA cytosine deaminase APOBEC3B in recurrent estrogen receptor positive breast cancer

Recent studies have implicated the DNA cytosine deaminase APOBEC3B as a major source of mutation in breast cancer. APOBEC3B explains a large proportion of both dispersed and clustered cytosine mutations, the latter of which are also called kataegis. APOBEC3B expression levels correlate with poor outcomes for patients with estrogen receptor positive breast cancer. While targeted therapies, such as tamoxifen, are available to treat these tumors, secondary drug resistance often develops. Here we suppressed endogenous APOBEC3B in the estrogen receptor positive breast cell line MCF-7L with shRNA. Lowered levels of APOBEC3B did not affect in vitro growth or sensitivity to estradiol. In a xenograft model of tamoxifen therapy, suppression of APOBEC3B associated with prolonged responses to tamoxifen (p Citation Format: Harris R, Law E, Sieuwerts A, LaPara K, Leonard B, Starrett G, Temiz NA, Sweep F, Span P, Foekens J, Martens J, Yee D. Tamoxifen resistance driven by the DNA cytosine deaminase APOBEC3B in recurrent estrogen receptor positive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-07.

Abstract 295: The cytosine deaminase APOBEC3B affects responses to therapy in estrogen receptor positive breast cancer cells

Endocrine therapy, chemotherapy, or both represent the mainstay of therapy for estrogen receptor positive (ER+) breast cancer. However, acquired (secondary) drug resistance is a major clinical problem. We identified APOBEC3B (A3B), a DNA cytosine deaminase, as a source of DNA mutagenesis driving tumor evolution and contributing to poor clinical outcomes in several cancers by catalyzing genetic changes required for drug resistance and metastasis. A3B accounts for up to half of the mutational load in breast carcinomas expressing this enzyme. High levels of A3B correlate with poor clinical outcomes in tamoxifen treated patients. To evaluate the role for A3B in resistance to therapy, we used the ER+ MCF-7L breast cancer cell line. These cells have modestly elevated levels of endogenous A3B and we suppressed A3B levels by shRNA and enhanced levels by transduction. In vitro, alterations in A3B levels do not affect monolayer growth or cell doubling times. However, in vitro response to tamoxifen was prolonged in MCF-7L cells with suppressed A3B. In a xenograft model, MCF-7 cells with suppressed A3B remained responsive to tamoxifen for a longer period of time compared to wild type cells. Cells with overexpression of A3B had a very short period of tamoxifen suppression compared to wild-type cells. These data suggest high A3B levels are associated with a shorter period of response to tamoxifen. To evaluate the role for A3B in chemotherapy resistance, we treated cells with 5-fluorouracil. In contrast to tamoxifen therapy, cells with elevated A3B levels remained responsive to fluorouracil for longer periods of time compared to wild-type cells. Since A3B converts cytosine to uracil (C-to-U), we speculate that A3B enzymatic activity might enhance sensitivity to a fluoropyrimidine. Thus, the ability of A3B to affect therapeutic resistance in breast cancer may be dependent on the type and mechanism of drug treatment. Citation Format: Kelly S. LaPara, Emily Law, Reuben Harris, Douglas Yee. The cytosine deaminase APOBEC3B affects responses to therapy in estrogen receptor positive breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 295.

Abstract P4-06-03: Targeting the insulin receptor with a small peptide (S961) in cancer

The insulin-like growth factor (IGF)/insulin receptors are members of the superfamily of growth factor receptor tyrosine kinases. The insulin and type I IGF-I receptors (IGF-1R) have very similar structure and share high homology (∼84%) in the kinase domain. Their dimeric structure allows for the formation of either holo or hybrid receptors. Thus, targeting of any individual receptor has been a challenge. We have previously shown that the insulin receptor is expressed in tamoxifen resistant breast cancer cell lines. Therefore, specific targeting might be a useful strategy to block this pathway. The antagonist S961 is a single chain peptide that binds insulin receptor and has been reported to have partial antagonist activity. The affinity of S961 for the insulin receptor is comparable to that of insulin and the selectivity of the insulin receptor versus IGF1 is higher than that of insulin itself (Sturis et al). To determine if S961 has activity in breast cancer cells, we tested its ability to disrupt insulin and IGF-I signaling and growth in a panel of cancer cell lines (MCF-7L, MCF-7L TamR, MDA-231, and HepG2 cells). MCF-7L cells express high levels of IGF1, while HepG2 express mostly insulin receptor. Pre-incubation with S961 significantly suppressed p-Akt and p-MAPK after insulin stimulation in the HepG2, but not in MCF-7L. In contrast, insulin receptor stimulation in MCF-7L TamR cells, which do not express IGF1R, was inhibited by S961. Even a 1000-fold increase in S961 was unable to suppress insulin receptor activation in cells expressing both insulin receptor and IGF1R. S961 inhibits insulin-stimulate cell cycle progression in MCF7L TamR cells. In these TamR cells, S961 also suppresses colony formation in anchorage independent growth assays. These data suggest S961 is an effective inhibitor of insulin receptor activation but only when little IGF1R is expressed. Thus, insulin receptor targeting might be useful in the management of endocrine resistant breast cancer. Citation Format: Kelly LaPara, Douglas Yee. Targeting the insulin receptor with a small peptide (S961) in cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-06-03.

Abstract IA17: Targeting downstream effectors of growth factor signaling

Transmembrane growth factor receptors mediate signaling through multiple intracellular pathways. In breast cancer cells, the type I insulin-like growth factor receptor (IGF1R) has been implicated in the malignant phenotype. However, clinical trials with anti-IGF1R antibodies have been disappointing, in part, due to adaptive feedback pathways stimulated when IGF1R is blocked. To determine whether IGF1R inhibition could be enhanced by disrupting other pathways, we evaluated gene expression induced by receptor activation. In previous work, we found that xCT (SLC7A11) mRNA expression was increased by IGF-I in estrogen receptor (ER) positive breast cancer cell lines (MCF-7, T47D, and ZR-75-1) in an insulin receptor substrate-1 (IRS-1) dependent manner. xCT encodes the functional subunit of the heterodimeric plasma membrane transport system xC- critical for the cellular uptake of cystine to generate glutathione to modulate cellular redox control. IGF-I increased xC- transporter expression and function to control cellular redox levels. In MCF-7 cells, IGF-I-stimulated monolayer and anchorage-independent growth was suppressed by infecting cells with xCT shRNA or by treating cells with the xC- chemical inhibitor sulfasalazine (SASP). Anchorage-independent growth assays showed that disruption of xC- function by SASP sensitized cellular response of MCF-7 cells to anti-IGF-IR inhibitors (monoclonal antibody huEM164 and tyrosine kinase inhibitor NVP-AEW-541). IGF1R also activates PI3K/Akt/mTORC1 signaling to affect ER phosphorylation and mRNA cap dependent translation. In tamoxifen resistant cells, IGF1R is lost yet PI3K signaling is maintained. Since mRNA cap dependent translation is increased by PI3K signaling, we determined if inhibition of the eIF4F translation pathway would affect endocrine responsive and tamoxifen resistant cells. The eIF4F translation pathway is activated by IGF1R in wild-type cells and hyperactive in tamoxifen resistant MCF-7L (TamR) breast cancer cells. Targeting the eIF4E subunit of the eIF4F complex through its degradation using an antisense oligonucleotide (ASO) or via sequestration using a mutant 4E-BP1 inhibited the proliferation and colony formation of parental and TamR cells. Use of these agents also resulted in cell cycle arrest and induction of apoptosis in TamR cells. Finally, pharmacologic inhibition of the eIF4E-eIF4G interaction also decreased the proliferation and anchorage dependent colony formation in TamR cells. Taken together, these data show that IGF1R activation stimulates multiple downstream effectors important for breast cancer cell biology. Inhibition of selected downstream signaling molecules is likely to have synergy with anti-IGF-IR drugs. Citation Format: Yuzhe Yang, Dedra Fagan, Lynsey Fettig Anderson, Kelly LaPara, Xihong Zhang, Aleksandra Ochnik, Jie Ying Chan, Heather Beckwith, Douglas Yee. Targeting downstream effectors of growth factor signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA17.

Abstract CN07-02: Disrupting insulin‐like growth factor signaling with monoclonal antibodies

The insulin‐like growth factor (IGF) system is composed of a network of ligands, receptors, and binding proteins. The type I IGF receptor (IGF1R) is a transmembrane tyrosine kinase receptor and shares a high degree of homology with the insulin receptor (InsR). Data from many sources implicate the IGF system in cancer biology. Because of the role for InsR in glucose homeostasis, specific targeting of IGF1R with monoclonal antibodies has been deemed desirable. Numerous monoclonal antibodies have been developed against this receptor and share similar properties in preclinical model systems. To date, all of the antibodies have been shown to bind IGF1R and cause its internalization and downregulation. Early clinical studies have shown that IGF1R antibodies have activity as single agents in some diseases (sarcoma) and have activity in combination with cytotoxic chemotherapy. These early promising results suggest that inhibition of this receptor will emerge as a new cancer therapy. In addition, these early clinical results raise additional questions regarding optimizing their use in cancer treatment. In a xenograft model, monoclonal antibodies to IGF1R do not suppress tumor growth of the MDA‐435/LCC6 cell line yet inhibit tumor metastasis. These data suggest that a commonly measured clinical phenotype, tumor growth, may not be detected in early phase II clinical trials. Clinical trials have also shown that IGF1R monoclonal antibody therapy results in elevation of growth hormone, IGF‐I, and insulin levels. Since InsR may also mediate signaling in the IGF system, it is possible that InsR is also a target for cancer therapy. To address this issue, we have used shRNA to downregulate InsR expression while leaving IGF1R levels intact. In the MDA‐435/LCC6 cells, disruption of InsR decreases tumor growth and inhibits pulmonary metastases. We have also selected cells in increasing concentrations of IGF1R antibodies. Finally, long term incubation of cells with IGF1R monoclonal antibodies results in cells with low levels of receptor expression, yet the receptor remains biochemically functional and can be inhibited by IGF1R tyrosine kinase inhibitors. Thus, disruption of IGF1R signaling has promise as a new cancer therapy. Future consideration of the role of the ligands, the InsR, and combination therapies with other signaling disruptors will allow us to optimize this therapeutic strategy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):CN07-02.