Adam Schayowitz

Trastuzumab reverses letrozole resistance and amplifies the sensitivity of breast cancer cells to estrogen

In this study, we investigated adaptive mechanisms associated with aromatase inhibitor (AI) resistance in breast cancer cells and show that sensitivity to AIs can be extended through dual inhibition of estrogen receptor (ER) and human epidermal receptor-2 (Her-2) signaling. We used human ER-positive breast cancer cells stably transfected with the aromatase gene (MCF-7Ca). These cells grow as tumors in nude mice and are inhibited by AIs. Despite continued treatment, tumors eventually become insensitive to AI letrozole. The cells isolated from these long-term letrozole-treated tumors (LTLT-Ca) were found to have decreased ERalpha levels. Our results suggest that LTLT-Ca cells survive estrogen deprivation by activation of Her-2/mitogen-activated protein kinase (MAPK) pathway. Here, we show that trastuzumab (antibody against Her-2; IC(50) = 0.4 mg/mL) was very effective in restoring the ERalpha levels and sensitivity of LTLT-Ca cells to endocrine therapy by down-regulation of Her-2/MAPK pathway and up-regulation of ERalpha. In contrast, trastuzumab was ineffective in the parental hormone-responsive MCF-7Ca cells (IC(50) = 4.28 mg/mL) and xenografts. By blocking Her-2, trastuzumab also up-regulates ERalpha and aromatase expression and hypersensitized MCF-7Ca cells to E(2). We show that trastuzumab is beneficial in hormone-refractory cells and xenografts by restoring ER, implicating Her-2 as a negative regulator of ERalpha. In xenograft studies, the combination of trastuzumab plus letrozole is equally effective in inhibiting growth of MCF-7Ca tumors as letrozole alone. However, on the acquisition of resistance and increased Her-2 expression, the combination of letrozole plus trastuzumab provided superior benefit over letrozole or trastuzumab alone.

Synergistic effect of a novel antiandrogen, VN/124-1, and signal transduction inhibitors in prostate cancer progression to hormone independence in vitro

This study was carried out to determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer. We investigated the role of the androgen receptor and its relationship to other signal transduction proteins. A hormone-refractory prostate cancer cell line [high-passage LNCaP (HP-LNCaP)] was established in vitro. Cells were treated with inhibitors of mammalian target of rapamycin and tyrosine kinase receptors. Expression of these proteins and the androgen receptor were measured by Western immunoblotting. Analysis of the model and various treatments was also assessed through proliferation assays, luciferase activation assays, binding assays, and ELISA. Our novel antiandrogen, VN/124-1, effectively inhibited proliferation of hormone-resistant prostate cancer cell lines (HP-LNCaP), which were no longer sensitive to bicalutamide and had increased expression of the androgen receptor. Treatment with everolimus or gefitinib resulted in an increase in protein expression and activation of the androgen receptor. Conversely, inhibition of the androgen receptor resulted in increased expression of IGFR1β, pHER2, pmTOR, and pAkt. The addition of bicalutamide to everolimus or gefitinib inhibited cell proliferation in HP-LNCaP cells. However, the addition of VN/124-1 has proven to be superior to bicalutamide, and the combination was synergistic (P < 0.05) compared with either agent alone. This study suggests that compensatory cross-talk between the androgen receptor and various signaling pathways may account for decreased sensitivity to androgen receptor antagonists and the progression to hormone-resistant prostate cancer. Furthermore, these findings suggest that inhibition of both pathways may provide effective control in hormone-resistant prostate cancer and restore sensitivity to androgen antagonists in hormone-refractory patients. [Mol Cancer Ther 2008;7(1):121–32]

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway improves response of long-term estrogen-deprived breast cancer xenografts to antiestrogens.

Purpose: Aromatase inhibitors that block the synthesis of estrogen are proving to be superior to antiestrogens and may replace tamoxifen as first-line treatment for postmenopausal estrogen receptor (ER)–positive breast cancer patients. However, acquisition of resistance to all forms of treatments is inevitable and a major clinical concern. In this study, we have investigated the effects of long-term estrogen deprivation in the breast cancer xenograft model and whether sensitivity to antiestrogens can be restored in vivo. We also compared whether combining wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. Experimental Design: Long-term estrogen-deprived aromatase-transfected human ER-positive breast cancer cells (UMB-1Ca) were grown as tumors in ovariectomized athymic nude mice. Twelve weeks after inoculation, when tumors reached 300 mm3, animals were grouped and injected with vehicle, Δ4A, letrozole, tamoxifen, fulvestrant, wortmannin, tamoxifen plus wortmannin, and wortmannin plus fulvestrant. Tumor volumes were measured weekly. Results: Tumors of UMB-1Ca cells grew equally well with and without androstenedione, indicating the ability of the cells to proliferate in the absence of estrogen. The combination of wortmannin with tamoxifen or fulvestrant inhibited tumor growth better than either drug alone. The combination of wortmannin plus fulvestrant was the most effective treatment that maintained tumor regression for a prolonged time. Conclusion: These results suggest that blocking both ER and growth factor receptor pathways could provide effective control over tumor growth of long-term estrogen-deprived human breast cancers.

Stopping Treatment Can Reverse Acquired Resistance to Letrozole

Using the intratumoral aromatase xenograft model, we have observed that despite long-lasting growth inhibition, tumors eventually begin to grow during continued letrozole treatment. In cells isolated from these long-term letrozole-treated tumors (LTLT-Ca), estrogen receptor-alpha (ERalpha) levels were decreased, whereas signaling proteins in the mitogen-activated protein kinase cascade were up-regulated along with human epidermal growth factor receptor 2 (Her-2). In the current study, we evaluated the effect of discontinuing letrozole treatment on the growth of letrozole-resistant cells and tumors. The cells formed tumors equally well in the absence or presence of letrozole and had similar growth rates. After treatment was discontinued for 6 weeks, letrozole was administered again. Marked tumor regression was observed with this second course of letrozole treatment. Similarly, in MCF-7Ca xenografts, a 6-week break in letrozole treatment prolonged the responsiveness of the tumors to letrozole. To understand the mechanisms of this effect, LTLT-Ca cells were cultured in the absence of letrozole for 16 weeks. The resulting cell line (RLT-Ca) exhibited properties similar to MCF-7Ca cells. The cell growth was inhibited by letrozole and stimulated by estradiol. The expression of phosphorylated mitogen-activated protein kinase (MAPK) was reduced and ERalpha and aromatase levels increased compared with LTLT-Ca cells and were similar to levels in MCF-7Ca cells. These results indicate that discontinuing treatment can reverse letrozole resistance. This could be a beneficial strategy to prolong responsiveness to aromatase inhibitors for patients with breast cancer.

Toremifene–atamestane; alone or in combination: Predictions from the preclinical intratumoral aromatase model

Since most breast cancers occur in postmenopausal women and are hormone dependent, we developed a model system that mimics this situation. In this model, tumors of human estrogen receptor (ER) positive breast cancer cells stably transfected with aromatase (Ac-1) are grown in immune-compromised mice. Using this model we have explored a number of therapeutic strategies to maximize the antitumor efficacy of antiestrogens (AEs) and aromatase inhibitors (AIs). This intratumoral aromatase xenograft model has proved accurate in predicting the outcome of several clinical trials. In this current study we compared the effect of an AE toremifene and steroidal AI atamestane, alone or in combination, on growth of hormone-dependent human breast cancer. We have also compared toremifene plus atamestane combination with tamoxifen in this study. The growth of Ac-1 cells was inhibited by tamoxifen, toremifene and atamestane in vitro with IC(50) values of 1.8+/-1.3 microM, 1+/-0.3 microM and 60.4+/-17.2 microM, respectively. The combination of toremifene plus atamestane was found to be better than toremifene or atamestane alone in vitro. The effect of this combination was then studied in vivo using Ac-1 xenografts grown in ovariectomized female SCID mice. The mice were injected with toremifene (1000 microg/day), atamestane (1000 microg/day), tamoxifen (100 microg/day), or the combination of toremifene plus atamestane. In this study, our results indicate that the combination of toremifene plus atamestane was as effective as toremifene or tamoxifen alone but may not provide any additional benefit over toremifene alone or tamoxifen alone.

Abstract 3498: Functional profiling of Champions TumorGraft™ models from metastatic melanoma patients.

Introduction: Molecularly targeted agents, such as the BRAF inhibitor vemurafenib, may produce short-term responses in some patients; however, most patients are intrinsically resistant, or develop resistance through restructuring of signal transduction pathways. SnapPath™ is a live-cell-processing platform that utilizes ex vivo signal transduction modulation of live tumor samples to produce Functional Signaling Profiles (FSPs). Application of this technology to Champions TumorGraft models may provide novel insights to guide oncology drug development as these models preserve the biological properties of the original human tumor. Methods: Fresh melanoma tumor specimens were collected from patients and implanted into immunodeficient mice. Fine needle aspiration biopsies were performed on each melanoma TumorGraft model and processed on the SnapPath™ platform (BioMarker Strategies) to modulate tumor cell signal transduction networks through brief ex vivo exposure to the vemurafenib tool compound PLX-4720. Cell lysates were then analyzed using a multiplexed immunoassay to assess the inhibition of the downstream MAPK markers pMEK1 and pERK-1/2. FSPs were then created for each TumorGraft model based on baseline and modulated levels of each phosphoprotein. In parallel, the in vivo sensitivity to vemurafenib and BRAF mutation status was evaluated in each Champions TumorGraft model. FSPs were then compared with in vivo efficacy, gene expression and genotype data. Results: Functional profiling stratified the TumorGraft models into two distinct groups upon ex vivo exposure to a BRAF inhibitor: 1) MAPK markers suppressed and 2) MAPK markers not suppressed. As anticipated, TumorGraft models that showed resistance to ex vivo BRAF inhibition demonstrated vemurafenib resistance in vivo and were BRAF wild type. There were other models that displayed MAPK suppression with ex vivo BRAF inhibition and vemurafenib sensitivity in vivo or MAPK suppression with ex vivo BRAF inhibition but demonstrated vemurafenib resistance in vivo. One of these TumorGrafts contained a BRAF V600E mutation, suggesting the activation of an alternate pathway that conferred resistance. The other TumorGraft contained a novel BRAF insertion. The functional profiling suggests that this insertion may activate BRAF and is susceptible to vemurafenib inhibition, but the tumor may contain an alternate pathway that confers resistance. Analysis of gene expression data demonstrated hierarchical clustering of BRAF mutated TumorGraft models. Conclusions: These results demonstrate the capability of the SnapPath™ platform to generate FSPs from FNAs of Champions melanoma TumorGraft models. Overall, the combination of Champions TumorGraft models with functional profiling represents a powerful tool for pharmacodynamic assessment of targeted therapeutics in clinically relevant models and has the potential to guide oncology therapy. Citation Format: Elizabeth M. Bruckheimer, Adam Schayowitz, Kala Barnes, Greg Bertenshaw, Tin Khor, James Cotton, Jay Friedman, Dhanrajan Tiruchinapalli, Douglas P. Clark. Functional profiling of Champions TumorGraft™ models from metastatic melanoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3498. doi:10.1158/1538-7445.AM2013-3498

Abstract 3192: Translational research platform (SnapPath™) for enabling drug development and personalized medicine in metastatic melanoma

Introduction: The approved BRAF inhibitor, vemurafenib, produces short-term responses in many metastatic melanoma patients; however, most patients develop resistance through reactivation of signal transduction pathways. More sophisticated tools for the analysis of signal transduction in preclinical models and clinical samples would vastly improve drug development and personalized cancer therapy. SnapPath™ is a live-cell-processing platform that utilizes ex vivo signal transduction modulation of tumor samples to produce Functional Signaling Profiles (FSPs), which can be used in drug development and clinical studies. Methods: Melanoma cell line xenografts (SK-Mel-28, SK-Mel-3, Colo829, MALME3M and A2058) were generated in athymic nude mice. Fine needle aspiration biopsies (FNA) were performed on each tumor type when tumor volumes reached 500mm 3 . One FNA sample consisted of 4 passes of a 23G, 1′’ needle on a 10cc syringe. The live cell biopsies were processed on the SnapPath™ live-tumor-cell platform (BioMarker Strategies, LLC) to modulate tumor cell signal transduction networks through brief exposure to the BRAF inhibitor PLX-4720 (3uM) or IGF1 (100ng/ml). The resulting cell lysates were then analyzed using the Bio-Plex multiplexed immunoassay system for the following phosphoproteins: pIGF-1R, pMEK1, pErk-1/2, pAkt, pGSK3B, pp70S6 kinase and pStat3. Functional signaling profiles (FSP) were then created for each tumor based on baseline and modulated levels of each phosphoprotein. FSPs were then compared with known targeted drug sensitivity profiles. Results: Reproducible FSPs were obtained from FNA samples of each xenograft model. The FSPs obtained from FNA samples were similar to those derived from 2-dimensional in vitro cultures of the corresponding cell lines. Exposure of samples to the BRAF inhibitor PLX-4720 resulted in modulation of the MAPK pathway that corresponded to the anticipated activity of the drug. Responses of the xenograft samples to IGF1 growth factor stimulation differed between cell lines and may correlate with resistance mechanisms to BRAF inhibitors via PI3K, mTOR, or AKT, and may also correlate with responses to combination drug therapy. Conclusions: These results demonstrate the capability of the SnapPath™ platform to generate functional signaling profiles from fine needle aspiration biopsy samples of xenografted melanoma tumors. The derived FSPs correspond to anticipated drug effects on signal transduction and consequently represent a valuable tool for pharmacodynamic studies in preclinical xenograft and primary human tumorgraft models as well extension into the clinical setting. The elucidation of bypass pathways by SnapPath™ represents an important new tool for the analysis of resistance mechanisms in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3192. doi:1538-7445.AM2012-3192

Abstract 3144: Functional stratification of breast carcinoma cells enables predictive therapeutic strategies

Introduction: Most targeted therapies still lack effective predictive biomarkers. A major limitation of the existing classes of biomarkers is the lack of functional information about the signal transduction networks targeted by molecularly targeted drugs. We have developed a functional assay based on ex vivo biomarkers produced by live tumor cells. The profile is elicited by short-term epidermal growth factor (EGF) stimulation in the presence or absence of a MEK inhibitor. The resultant changes in signal transduction phosphoprotein levels are used to create functional signaling profiles that stratify tumor cell lines into functional groups. This functional signaling profile is feasible by an automated platform that is amenable to tumor biopsy processing. Methods: Breast Cancer Cell lines (BT-474, MDA-MD-231, SKBR3, HCC-1937, BT-20, T47D, MCF-7, BT-549) were propagated and removed from the plate by gentle scraping to simulate a FNA biopsy sample. Following removal, the cells were placed on the SnapPath™ live-tumor-cell processing platform (BioMarker Strategies, LLC) to evoke ex vivo biomarkers. SnapPath™ disperses the sample, enriches for tumor cells, aliquots into test wells, and applies ex vivo stimulation by EGF (200ng/ml) in the presence or absence of the MEK inhibitor, U0126 (1uM). Cell lysates were then analyzed using the BioPlex platform for the following phosphoproteins: p-Erk 1/2, p-Akt, p-EGFR, p-Stat3 (BioRad). Functional profiles were generated for each cell line based on the levels of phosphoproteins. Results: Functional signaling profiles of breast cancer cell lines stimulated with EGF in the presence of U0126 revealed distinct functional groups that enabled the stratification. Two functional groups were identified based on AKT phosphorylation levels: one group displayed variable, but low levels of p-AKT inhibition, whereas another group showed unanticipated up-regulation of p-AKT. This second group may be resistant to MEK inhibition but sensitive to the combination of MEK/AKT inhibition. Two other functional groups were identified based on EGFR phosphorylation levels: one group displayed variable, but low p-EGFR inhibition, whereas the other group showed unanticipated up-regulation of p-EGFR. This second group may be resistant to MEK inhibition, but sensitive to combined MEK/EGFR inhibition. Conclusion: Functional signaling profiles of human cancers reveal unique details of signal transduction networks that permit stratification of tumors unavailable through traditional biomarkers. These profiles may correlate with targeted drug sensitivity or resistance and may yield successful companion diagnostics, including combination therapies of targeted agents. Such functional profiles can be reproducibly elicited from small numbers of tumor cells on an automated platform, suggesting that this approach to predictive tests is possible for human tumor biopsy samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3144. doi:10.1158/1538-7445.AM2011-3144

Abstract 3990: Methods for Fine Needle Aspiration (FNA) biopsy sampling and "ex vivo" signal transduction analysis of murine xenografted human tumors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Fine Needle Aspiration (FNA) biopsy is a common procedure for sampling human tumors to enable diagnosis and, increasingly, molecular diagnostics on fixed samples. However, FNA is rarely used to sample murine xenografted human tumors. FNA sampling of xenografted tumors offers several advantages over surgical excision: 1) efficiency; 2) enables serial sampling of individual tumors over time; 3) provides live cells to perform “ex vivo” analysis of signal transduction pathways. Objectives: To define a protocol for FNA sampling of murine xenografted tumors using an HCT-116 human colon carcinoma model; to characterize the cellular and molecular composition of such samples and to present proof-of-concept studies for “ex vivo” analysis from FNA samples. Methods: FNA biopsies were performed on HCT-116 xenografts using a standardized protocol that will be defined. Samples were assessed by microscopic analysis of direct smears stained with the Diff-Quik or Papanicolaou stains. Tumor cell number was determined by hemocytometer count and cell viability by trypan blue exclusion. Samples were lysed and total protein and RNA were extracted and quantitated by spectrophotometry. RNA quality was determined by RIN calculation. “Ex vivo” stimulation was performed by treating tumor cells obtained from FNA with TPA and EGF and subsequently analyzing phosphoprotein levels relative to baseline using Western blots. Results: FNA sample: Cellular composition: 25 % tumor cells, 50% macrophages, 20% lymphocytes, and 5% degenerate cells (n=5). Tumor cell viability median: after 1 hour post-FNA: 42% (range 22-88%) n=15. Cell viability decreases by 50% after 2 h post-FNA, (n=5). Total Protein median/FNA: 555 ug/ml (range 36-4222 ug/FNA) n=33; Total RNA median 14.80 ug/FNA (range 3.24-26.37 ug) n=18; RIN number median/FNA: 8.40 (range 3.10-9.50) n=15. “Ex vivo” analysis of signal transduction: a 60%, transient increase in phospho-ERK was identified within the first 5 min. post-FNA procedure, which returned to baseline. No increase in p-AKT or p-JNK was noted during this time period. Following “ex vivo” EGF or PMA stimulation, FNA samples showed a 1-3 fold increase -respectively- in p-ERK relative to baseline. Conclusions: Murine xenografts can be efficiently sampled by FNA biopsy. Such samples yield sufficient high-quality biomolecules for molecular analysis. FNA samples also enable live cell analysis, such as “ex vivo” stimulation of signal transduction pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3990.