Mothaffar F. Rimawi

Tamoxifen Resistance in Breast Tumors Is Driven by Growth Factor Receptor Signaling with Repression of Classic Estrogen Receptor Genomic Function

Not all breast cancers respond to tamoxifen, and many develop resistance despite initial benefit. We used an in vivo model of estrogen receptor (ER)-positive breast cancer (MCF-7 xenografts) to investigate mechanisms of this resistance and develop strategies to circumvent it. Epidermal growth factor receptor (EGFR) and HER2, which were barely detected in control estrogen-treated tumors, increased slightly with tamoxifen and were markedly increased when tumors became resistant. Gefitinib, which inhibits EGFR/HER2, improved the antitumor effect of tamoxifen and delayed acquired resistance, but had no effect on estrogen-stimulated growth. Phosphorylated levels of p42/44 and p38 mitogen-activated protein kinases (both downstream of EGFR/HER2) were increased in the tamoxifen-resistant tumors and were suppressed by gefitinib. There was no apparent increase in phosphorylated AKT (also downstream of EGFR/HER2) in resistant tumors, but it was nonetheless suppressed by gefitinib. Phosphorylated insulin-like growth factor-IR (IGF-IR), which can interact with both EGFR and membrane ER, was elevated in the tamoxifen-resistant tumors compared with the sensitive group. However, ER-regulated gene products, including total IGF-IR itself and progesterone receptor, remained suppressed even at the time of acquired resistance. Tamoxifens antagonism of classic ER genomic function was retained in these resistant tumors and even in tumors that overexpress HER2 (MCF-7 HER2/18) and are de novo tamoxifen-resistant. In conclusion, EGFR/HER2 may mediate tamoxifen resistance in ER-positive breast cancer despite continued suppression of ER genomic function by tamoxifen. IGF-IR expression remains dependent on ER but is activated in the tamoxifen-resistant tumors. This study provides a rationale to combine HER inhibitors with tamoxifen in clinical studies, even in tumors that do not initially overexpress EGFR/HER2.

TBCRC 001: Randomized Phase II Study of Cetuximab in Combination With Carboplatin in Stage IV Triple-Negative Breast Cancer

PURPOSE Epidermal growth factor receptor (EGFR) is a targetable receptor frequently overexpressed in basal-like breast cancer, which comprises most triple-negative breast cancers (TNBCs), the only subtype without established targeted therapy. PATIENTS AND METHODS In this randomized phase II trial, patients with metastatic TNBC received anti-EGFR antibody cetuximab (400 mg/m(2) load then 250 mg/m(2) per week intravenously [IV]) alone, with carboplatin (area under the curve of 2, once per week IV) added after progression or as concomitant therapy from the beginning. Response rate (RR) was the primary end point; others included time to progression (TTP), overall survival (OS), and toxicity. Embedded correlative studies included molecular subtyping on archival tissue. Fresh tumor tissue before and after 7 to 14 days of therapy was used for microarray analyses exploring EGFR pathway activity and inhibition. RESULTS In 102 patients with TNBC, RRs were 6% (two of 31) to cetuximab and 16% (four of 25) to cetuximab plus carboplatin after progression. RR to those treated from the beginning with cetuximab plus carboplatin was 17% (12 of 71); 31% of patients responded or had prolonged disease stabilization. The cetuximab plus carboplatin regimen was well tolerated, but both TTP and OS were short at 2.1 months (95% CI, 1.8 to 5.5 months) and 10.4 months (95% CI, 7.7 to 13.1 months), respectively. Of 73 patients with archival tissue for analysis, 74% had basal-like molecular subtype. Sixteen patients had tumor biopsies before and 1 week after therapy; genomic patterns of the EGFR pathway showed activation in 13 and inhibition by therapy in five. CONCLUSION Despite strong preclinical data, combination cetuximab plus carboplatin in metastatic TNBC produced responses in fewer than 20% of patients. EGFR pathway analysis showed that most TNBCs involved activation. However, cetuximab blocked expression of the EGFR pathway in only a minority, suggesting that most had alternate mechanisms for pathway activation.

Loss of Phosphatase and Tensin Homolog or Phosphoinositol-3 Kinase Activation and Response to Trastuzumab or Lapatinib in Human Epidermal Growth Factor Receptor 2–Overexpressing Locally Advanced Breast Cancers

PURPOSE Phosphatase and tensin homolog (PTEN) loss or activating mutations of phosphoinositol-3 (PI3) kinase (PIK3CA) may be associated with trastuzumab resistance. Trastuzumab, the humanized human epidermal growth factor receptor 2 (HER2) monoclonal antibody, and lapatinib, an epidermal growth factor receptor/HER2 tyrosine kinase inhibitor, are both established treatments for HER2-overexpressing breast cancers. Understanding of the cellular response to HER2-targeted therapies is needed to tailor treatments and to identify patients less likely to benefit. METHODS We evaluated the effect of trastuzumab or lapatinib in three HER2-overexpressing cell lines. We confirmed the in vitro observations in two neoadjuvant clinical trials in patients with HER2 overexpression; 35 patients received trastuzumab as a single agent for the first 3 weeks, then docetaxel every 3 weeks for 12 weeks (trastuzumab regimen), whereas 49 patients received lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before primary surgery (lapatinib regimen). Apoptosis, Ki67, p-MAPK, p-AKT, and PTEN were assessed by immunohistochemistry. Genomic DNA was sequenced for PIK3CA mutations. RESULTS Under low PTEN conditions, in vitro data indicate that lapatinib alone and in combination with trastuzumab was effective in decreasing p-MAPK and p-AKT levels, whereas trastuzumab was ineffective. In the clinical trials, we confirmed that low PTEN or activating mutation in PIK3CA conferred resistance to the trastuzumab regimen (P = .015), whereas low PTEN tumors were associated with a high pathologic complete response rate (P = .007). CONCLUSION Activation of PI3 kinase pathway is associated with trastuzumab resistance, whereas low PTEN predicted for response to lapatinib. These observations support clinical trials with the combination of both agents.

Multicenter Phase II Study of Neoadjuvant Lapatinib and Trastuzumab With Hormonal Therapy and Without Chemotherapy in Patients With Human Epidermal Growth Factor Receptor 2–Overexpressing Breast Cancer: TBCRC 006

PURPOSE We previously reported the eradication of human epidermal growth factor receptor 2 (HER2)- amplified human xenografts in mice by inhibition of the HER2 pathway with lapatinib and trastuzumab to block all homo- and heterodimer signaling as well as by blockade of estrogen receptor (ER) when expressed. In this clinical trial, we sought to translate these findings to patients using targeted therapy without chemotherapy. PATIENTS AND METHOD Women with stages II to III HER2-positive breast cancers were eligible. They received trastuzumab once per week (4 mg/kg loading, then 2 mg/kg) and lapatinib 1000 mg once per day for 12 weeks. Women with ER-positive tumors also received letrozole (plus a luteinizing hormone-releasing hormone [LHRH] agonist if premenopausal). Pathologic response was assessed by ER status. Biopsies were obtained at baseline, weeks 2 and 8, and time of surgery. RESULTS Sixty-six patients were enrolled, and 64 were eligible and evaluable for response. Median tumor size was 6 cm (range, 1.5 to 30 cm). Adverse events were mainly grades 1 to 2 (GI, 63%; skin, 46%). Grade 3 metabolic, GI, and liver (18%; 12 patients) and grade 4 liver toxicities (one patient) were also observed. Overall, in-breast pathologic complete response (pCR; ypT0-is) was 27% (ER positive, 21%; ER negative, 36%). The rate of low-volume residual disease (ypT1a-b) was 22% (ER positive, 33%; ER negative, 4%). CONCLUSION In patients with locally advanced HER2-positive breast cancer, our approach of targeted therapy only resulted in a high pCR rate without chemotherapy. Our data support the hypothesis that selected patients with HER2-positive tumors may not need chemotherapy, and more-complete blockade of HER receptors and ER is an effective strategy worthy of further study.

Advanced concepts in estrogen receptor biology and breast cancer endocrine resistance: implicated role of growth factor signaling and estrogen receptor coregulators

Estrogen receptor (ER), mediating estrogen-signaling stimuli, is a dominant regulator and a key therapeutic target in breast cancer etiology and progression. Endocrine therapy, blocking the ER pathway, is one of the most important systemic therapies in breast cancer management, but de novo and acquired resistance is still a major clinical problem. New research highlights the role of both genomic and nongenomic ER activities and their intimate molecular crosstalk with growth factor receptor and other signaling kinase pathways in endocrine resistance. These signaling pathways, when overexpressed and/or hyperactivated, can modulate both activities of ER, resulting in endocrine resistance. Thus, these signal transduction receptors and signaling molecules may serve as both predictive markers and novel therapeutic targets to circumvent endocrine resistance. Compelling experimental and clinical evidence suggest that the epidermal growth factor/HER2/neu receptor (EGFR/HER2) pathway might play a distinct role in endocrine resistance, and especially in resistance to selective estrogen receptor modulators (SERMs) such as tamoxifen. Results from preclinical studies of treatment combinations with various endocrine therapy drugs together with several potent anti-EGFR/HER2 inhibitors are very promising, and clinical trials to see whether this new strategy is effective in patients are now ongoing.

Mechanisms of Tumor Regression and Resistance to Estrogen Deprivation and Fulvestrant in a Model of Estrogen Receptor–Positive, HER-2/neu-Positive Breast Cancer

HER-2/neu in breast cancer is associated with tamoxifen resistance, but little data exist on its interaction with estrogen deprivation or fulvestrant. Here, we used an in vivo xenograft model of estrogen receptor (ER)-positive breast cancer with HER-2/neu overexpression (MCF7/HER-2/neu-18) to investigate mechanisms of growth inhibition and treatment resistance. MCF7/HER-2/neu-18 tumors were growth inhibited by estrogen deprivation and with fulvestrant, but resistance developed in 2 to 3 months. Inhibited tumors had reductions in ER, insulin-like growth factor-I receptor (IGF-IR), phosphorylated HER-2/neu (p-HER-2/neu), and phosphorylated p42/44 mitogen-activated protein kinase (p-MAPK). p27 was increased especially in tumors sensitive to estrogen deprivation. Tumors with acquired resistance to these therapies had complete loss of ER, increased p-HER-2/neu, increased p-MAPK, and reduced p27. In contrast, IGF-IR and phosphorylated AKT (p-AKT) levels were markedly reduced in these resistant tumors. The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib, which can block EGFR/HER-2/neu signaling, significantly delayed the emergence of resistance to both estrogen deprivation and fulvestrant. Levels of p-MAPK and p-AKT decreased with gefitinib, whereas high ER levels were restored. Eventually, however, tumors progressed in mice treated with gefitinib combined with estrogen deprivation or fulvestrant accompanied again by loss of ER and IGF-IR, increased p-HER-2/neu, high p-MAPK, and now increased p-AKT. Thus, estrogen deprivation and fulvestrant can effectively inhibit HER-2/neu-overexpressing tumors but resistance develops quickly. EGFR/HER-2/neu inhibitors can delay resistance, but reactivation of HER-2/neu and signaling through AKT leads to tumor regrowth. Combining endocrine therapy with EGFR/HER-2/neu inhibitors should be tested in clinical breast cancer, but a more complete blockade of EGFR/HER-2/neu may be optimal.

Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

IntroductionThe human epidermal growth factor receptor 2 (HER2)-targeted therapies trastuzumab (T) and lapatinib (L) show high efficacy in patients with HER2-positive breast cancer, but resistance is prevalent. Here we investigate resistance mechanisms to each drug alone, or to their combination using a large panel of HER2-positive cell lines made resistant to these drugs.MethodsResponse to L + T treatment was characterized in a panel of 13 HER2-positive cell lines to identify lines that were de novo resistant. Acquired resistant lines were then established by long-term exposure to increasing drug concentrations. Levels and activity of HER2 and estrogen receptor (ER) pathways were determined by qRT-PCR, immunohistochemistry, and immunoblotting assays. Cell growth, proliferation, and apoptosis in parental cells and resistant derivatives were assessed in response to inhibition of HER or ER pathways, either pharmacologically (L, T, L + T, or fulvestrant) or by using siRNAs. Efficacy of combined endocrine and anti-HER2 therapies was studied in vivo using UACC-812 xenografts.ResultsER or its downstream products increased in four out of the five ER+/HER2+ lines, and was evident in one of the two intrinsically resistant lines. In UACC-812 and BT474 parental and resistant derivatives, HER2 inhibition by T reactivated HER network activity to promote resistance. T-resistant lines remained sensitive to HER2 inhibition by either L or HER2 siRNA. With more complete HER2 blockade, resistance to L-containing regimens required the activation of a redundant survival pathway, ER, which was up-regulated and promoted survival via various Bcl2 family members. These L- and L + T-resistant lines were responsive to fulvestrant and to ER siRNA. However, after prolonged treatment with L, but not L + T, BT474 cells switched from depending on ER as a survival pathway, to relying again on the HER network (increased HER2, HER3, and receptor ligands) to overcome Ls effects. The combination of endocrine and L + T HER2-targeted therapies achieved complete tumor regression and prevented development of resistance in UACC-812 xenografts.ConclusionsCombined L + T treatment provides a more complete and stable inhibition of the HER network. With sustained HER2 inhibition, ER functions as a key escape/survival pathway in ER-positive/HER2-positive cells. Complete blockade of the HER network, together with ER inhibition, may provide optimal therapy in selected patients.

Epidermal growth factor receptor expression in breast cancer association with biologic phenotype and clinical outcomes

Epidermal growth factor receptor (EGFR) expression is associated with aggressive phenotypes in preclinical breast cancer models, but in clinical studies, EGFR has been inconsistently linked to poor outcome. We hypothesized that EGFR expression in human breast tumors, when centrally and uniformly assessed, is associated with an aggressive phenotype and resistance to systemic therapy.

β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

IntroductionThe overexpression of human epidermal growth factor receptor (HER)-2 in 20% of human breast cancers and its association with aggressive growth has led to widespread use of HER2-targeted therapies, such as trastuzumab (T) and lapatinib (L). Despite the success of these drugs, their efficacy is limited in patients whose tumors demonstrate de novo or acquired resistance to treatment. The β1 integrin resides on the membrane of the breast cancer cell, activating several elements of breast tumor progression including proliferation and survival.MethodsWe developed a panel of HER2-overexpressing cell lines resistant to L, T, and the potent LT combination through long-term exposure and validated these models in 3D culture. Parental and L/T/LT-resistant cells were subject to HER2 and β1 integrin inhibitors in 3D and monitored for 12 days, followed by quantification of colony number. Parallel experiments were conducted where cells were either stained for Ki-67 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or harvested for protein and analyzed by immunoblot. Results were subjected to statistical testing using analysis of variance and linear contrasts, followed by adjustment with the Sidak method.ResultsUsing multiple cell lines including BT474 and HCC1954, we reveal that in L and LT resistance, where phosphorylation of EGFR/HER1, HER2, and HER3 are strongly inhibited, kinases downstream of β1 integrin--including focal adhesion kinase (FAK) and Src--are up-regulated. Blockade of β1 by the antibody AIIB2 abrogates this up-regulation and functionally achieves significant growth inhibition of L and LT resistant cells in 3D, without dramatically affecting the parental cells. SiRNA against β1 as well as pharmacologic inhibition of FAK achieve the same growth inhibitory effect. In contrast, trastuzumab-resistant cells, which retain high levels of phosphorylated EGFR/HER1, HER2, and HER3, are only modestly growth-inhibited by AIIB2.ConclusionsOur data suggest that HER2 activity, which is suppressed in resistance involving L but not T alone, dictates whether β1 mediates an alternative pathway driving resistance. Our findings justify clinical studies investigating the inhibition of β1 or its downstream signaling moieties as strategies to overcome acquired L and LT resistance.

High IGF-IR Activity in Triple-Negative Breast Cancer Cell Lines and Tumorgrafts Correlates with Sensitivity to Anti–IGF-IR Therapy

Purpose: We previously reported an insulin-like growth factor (IGF) gene expression signature, based on genes induced or repressed by IGF-I, which correlated with poor prognosis in breast cancer. We tested whether the IGF signature was affected by anti–IGF-I receptor (IGF-IR) inhibitors and whether the IGF signature correlated with response to a dual anti–IGF-IR/insulin receptor (InsR) inhibitor, BMS-754807. Experimental Design: An IGF gene expression signature was examined in human breast tumors and cell lines and changes were noted following treatment of cell lines or xenografts with anti–IGF-IR antibodies or tyrosine kinase inhibitors. Sensitivity of cells to BMS-754807 was correlated with levels of the IGF signature. Human primary tumorgrafts were analyzed for the IGF signature and IGF-IR levels and activity, and MC1 tumorgrafts were treated with BMS-754807 and chemotherapy. Results: The IGF gene expression signature was reversed in three different models (cancer cell lines or xenografts) treated with three different anti–IGF-IR therapies. The IGF signature was present in triple-negative breast cancers (TNBC) and TNBC cell lines, which were especially sensitive to BMS-754807, and sensitivity was significantly correlated to the expression of the IGF gene signature. The TNBC primary human tumorgraft MC1 showed high levels of both expression and activity of IGF-IR and IGF gene signature score. Treatment of MC1 with BMS-754807 showed growth inhibition and, in combination with docetaxel, tumor regression occurred until no tumor was palpable. Regression was associated with reduced proliferation, increased apoptosis, and mitotic catastrophe. Conclusions: These studies provide a clear biological rationale to test anti–IGF-IR/InsR therapy in combination with chemotherapy in patients with TNBC. Clin Cancer Res; 17(8); 2314–27. ©2011 AACR.