Ck Osborne

Development of Resistance to Targeted Therapies Transforms the Clinically Associated Molecular Profile Subtype of Breast Tumor Xenografts

The effectiveness of therapies targeting specific pathways in breast cancer, such as the estrogen receptor or HER2, is limited because many tumors manifest resistance, either de novo or acquired, during the course of treatment. To investigate molecular mechanisms of resistance, we used two xenograft models of estrogen receptor-positive (ER+) breast cancer, one with and one without HER2 overexpression (MCF7/HER2-18 and MCF7 wt, respectively). Mice with established tumors were assigned to the following treatment groups: estrogen supplementation (E2), estrogen deprivation (ED), ED plus tamoxifen (Tam), all with or without the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (G). Another group received ED plus the antiestrogen fulvestrant (MCF7 wt only). Tumors with acquired or de novo resistance to these endocrine therapies were profiled for gene expression and compared with tumors in the E2 control group. One class of genes underexpressed in endocrine-resistant tumors (relative to E2-treated tumors) were estrogen inducible in vitro and associated with ER+ human breast cancers (luminal subtype). Another class of genes overexpressed in tumors with acquired resistance in both models represented transcriptional targets of HER2 signaling and was associated with ER-/HER2+ human cancers (ERBB2+ subtype). A third class of genes overexpressed in MCF7/HER2-18 tumors exhibiting de novo resistance to tamoxifen was associated with ER+ human cancers but not with estrogen-regulated genes. Thus, in response to various endocrine therapy regimens, these xenograft breast tumors shut down classic estrogen signaling and activate alternative pathways such as HER2 that contribute to treatment resistance. Over time, the molecular phenotype of breast cancer can change.

PI3 kinase activation and response to trastuzumab or lapatinib in HER-2 overexpressing locally advanced breast cancer (LABC).

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #34 Background: Activating mutations of PI3 kinase ( PIK3CA ) and PTEN loss may be associated with trastuzumab resistance. Trastuzumab, a HER2 humanized monoclonal antibody, and lapatinib, an EGFR/HER2 tyrosine kinase inhibitor are both established treatments. Greater understanding of the cellular response to trastuzumab or lapatinib is needed to tailor targeted therapy for individual patients and identify those less likely to benefit. Material and Methods: We performed two sequential neoadjuvant clinical trials in HER-2 overexpressing LABC: 40 patients received weekly trastuzumab at standard doses given initially as a single agent for the first 3 weeks, then in combination with 3-weekly docetaxel for 12 weeks (T), while 49 patients received lapatinib as a single agent (1,500 mg daily, orally) for 6weeks then the combination of 3-weekly trastuzumab/docetaxel for 12 weeks, before primary surgery (L). Sequential core biopsies of the primary breast tumors were taken at initial, weeks 1 and 3 after the first dose of trastuzumab, and at initial, weeks 2, 4, and 6 after lapatinib. Apoptosis, Ki67 proliferation rate, and PTEN were assessed by immunohistochemistry. Low PTEN was defined as Allred score of <3. Genomic DNA (10-100ng) was sequenced using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) and an ABI 3730 automated capillary sequencer. Two sample and paired sample comparisons were performed using nonparametric tests. Results: There was a significant decrease in clinical tumor size after three weeks of trastuzumab (n=35, median=-20%), and six weeks of lapatinib (n=49, median=-74%) compared to pre-therapy (p<0.001). At surgery, pathologic complete response was observed in 38% in patients on upfront T and 70% patient on L. There was a significant increase in apoptosis (median=3.5% to 4.7%, p=0.006) within one week after trastuzumab, with no significant change in Ki67 at any of the time point. Lapatinib was associated with a no significant increase in apoptosis but a significant decrease in Ki67 at week 2, 4, and 6 of therapy (p<0.001). Cases with low PTEN or PIK3CA mutations were significantly less likely to have a pathologic complete response to T (p<0.005). Howver, low PTEN or PIK3CA mutations was not significantly associated with pathologic resistance to L. Conclusions: Activation of PI3 kinase pathway is associated with trastuzumab but not lapatinib resistance. Lapatinib may affect signalling through the Ras/Raf/MAPK/ERK pathway, inhibiting cell division. Low PTEN expression was not associated with lapatinib resistance, and may explain the clinical efficacy of lapatinib in trastuzumab-resistant patients, supporting clinical trials for the combination of both agents. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 34.

Abstract S4-01: A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models

Background: The selective estrogen receptor (ER) down-regulator (SERD) fulvestrant (Ful) antagonizes ER activity and degrades ER protein in a dose-dependent manner in the preclinical and clinical settings, though its efficacy is limited by an incomplete abolition of ER protein levels. Therefore additional strategies are needed to achieve a more complete suppression of ER level and activity. The bromodomain-containing protein 4 (BRD4), a member of the BET family that is required both for ESR1 gene expression and for ER-mediated gene transcription, represents an attractive therapeutic target for ER+ breast cancer (BC). Here, we investigated the efficacy of the novel BRD4 inhibitor GS-626510 (GS-6510) in a panel of ER+ BC parental and endocrine-resistant (EndoR) cell lines and in a patient-derived xenograft (PDX) model. Materials and Methods: The effects of GS-6510 (25nM – 290nM) alone or in combination with endocrine therapies were tested in ER+ MCF7, T47D, and ZR75-1 cell lines, as well as in their derivatives made resistant to estrogen (E2) deprivation (EDR), tamoxifen (TamR), or Ful (FulR). Cell growth (by methylene blue) after 6 days of GS-6510 and protein levels (by Western blot) after 2 days of GS-6510 were assessed. The in vivo efficacy of GS-6510, Ful, and the combination was tested in the ER+ HCBx34 PDX model. The mRNA levels of genes associated with cell cycle, ER signaling, and endocrine-resistance were assessed using the NanoString platform. Results: A dose-dependent inhibitory effect of GS-6510 was observed in all of the experimental settings. At a clinically relevant dose, GS-6510 reduced E2-stimulated cell growth and enhanced the efficacy of endocrine therapies in all parental cell lines. In the endocrine-sensitive HCBx34 PDX model, GS-6510 reduced tumor growth and, in combination with Ful, induced tumor regression and inhibited the expression of ER-dependent and cell cycle related genes including CCND1, MYC, and BCL2. Notably, the addition of GS-6510 to EndoR cell models that continue to rely on and express ER (MCF7 EDR and TamR) led to a substantial inhibition in cell growth (85%; 98%, respectively). Though the combination of GS-6510 and Ful was not associated with a significantly greater cell growth inhibition compared to GS-6510 alone in these models, a better suppression of ER levels was observed. Interestingly, GS-6510 also remained effective in EndoR models that exhibited an ER-independent growth, including all FulR lines, though its efficacy varied among the different cell lines and resistant derivatives. Conclusion: Our findings suggest that the epigenetic regulator BRD4 is a suitable target for therapeutic intervention in ER+ BC. The anti-tumor efficacy of GS-6510 in endocrine sensitive and especially in ER-dependent EndoR models is worthy of further clinical investigation. The growth inhibitory effects observed in some of the ER-independent EndoR models suggests that additional genes/pathways involved in endocrine resistance could be affected by GS-6510. Identifying these pathways and determining their predictive role are needed to guide patient selection for future clinical trials. Citation Format: De Angelis C, Nardone A, Cataldo ML, Fu X, Trivedi M, Yi S, Breckenridge D, Chamnsess GC, Vitorino P, Osborne CK, Schiff R. A novel BRD4 inhibitor enhances endocrine therapy efficacy and circumvents endocrine-resistance in estrogen receptor-positive breast cancer models [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 S4-01.

Abstract PD01-01: Overcoming endocrine therapy resistance related to PTEN loss by strategic combinations with mTOR, AKT, or MEK inhibitors

Background: Hyperactive PI3K signaling is associated with a more aggressive subtype of estrogen receptor (ER) positive breast cancer (BC) and with endocrine resistance. Loss or downregulation of PI3K9s inhibitor PTEN is more common in basal and luminal B vs. luminal A BC. However, the role of PTEN in modulating response to various endocrine therapies is unclear. Here we investigated the effects of PTEN knockdown (KD) on endocrine sensitivity and the potential of multiple kinase inhibitors to restore and improve responses. Methods: Nude mice bearing ER+ BC xenograft tumors of MCF7 cells stably expressing a doxycycline (Dox)-inducible PTEN-shRNA were randomized to four endocrine treatment groups [continued estrogen (E2) supplementation, or E2-deprivation (ED) alone or in combination with tamoxifen (Tam) or fulvestrant (Ful)]; all -/+ Dox. The effects of single or combined kinase inhibitors on these endocrine treatments -/+ Dox were studied in vitro using inhibitors (i) to mTOR (AZD2014, 0.2 μM), AKT (AZD5363, 1 μM), or MEK (Selumetinib/ARRY-142886, 1 μM). Cell growth, apoptosis, and ER and progesterone receptor (PR) signaling were analyzed using cell cytometry, qRT/PCR, and Western blotting. Synergism tests were used to examine the growth effects of the most promising combinatorial therapy with multiple kinase inhibitors in different endocrine settings. Results: In wild-type (WT) PTEN xenograft tumors, endocrine therapies were very effective, inducing frequent tumor regression. In PTEN KD tumors endocrine therapies were less effective — PTEN KD delayed tumor regression in all endocrine regimens and accelerated tumor progression in the Tam treated group. Furthermore, at day 250, only 1/8 and 0/7 tumors had developed resistance in the ED and the Ful (−Dox) groups, respectively, while with PTEN KD (+Dox), 7/15 and 5/15 tumors developed resistance to ED and to Ful. In vitro PTEN KD also induced resistance to all endocrine therapies. mRNA and/or protein levels of ER and PR were suppressed by PTEN KD and restored by mTORi and AKTi. In cells with WT PTEN, mTORi was highly effective with or without endocrine therapy. However, AKTi and MEKi were more effective in combination with endocrine therapy. All three inhibitors were less effective upon PTEN KD. The mTORi plus AKTi combination resulted in a potent synergistic inhibition in PTEN KD cells in the presence of E2 or with ED. In contrast, in the presence of Tam, AKTi plus MEKi, independent of PTEN status, was the most effective combination at the doses chosen. Finally, these inhibitors and combinations were more effective in the presence of Ful than ED or Tam in WT PTEN cells. AKTi combined with Ful was still highly effective even in PTEN KD cells, but mTORi and MEKi were less effective. Conclusions: Our results suggest that PTEN loss renders endocrine therapy less effective in in vitro and in vivo experimental models. Single AKT/MEK kinase inhibitors are more potent in the presence of endocrine therapy. In PTEN KD cells, the activity of all three kinase inhibitors is largely diminished, except for AKTi in the presence of fulvestrant. Kinase inhibitor combinations are generally more effective, but the optimal combinations vary by PTEN status and type of endocrine therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-01.

Abstract P4-06-02: Identification of novel G-protein coupled receptor targets in HER2-positive breast cancer.

Background: HER2 is a member of human epidermal growth factor receptor (HER) superfamily and HER2-overexpressing (HER2+) breast cancer (BC) is an aggressive tumor. Despite the clinical success of anti-HER2 therapies, de novo and acquired drug resistance occur in many patients. Identification of novel drug targets to overcome anti-HER2 therapy resistance is an unmet need. Since G-protein coupled receptors (GPCRs) are known to cross-talk with the HER superfamily, it is possible that some GPCRs may signal to modulate the HER2 pathway. GPCRs are considered excellent drug targets due to their plasma membrane localization, unique ligand-binding pocket, and availability of high throughput assays for drug screening. The expression and function of the majority of GPCRs are largely unknown in HER2+ BC. The goal of this study was to identify novel GPCR targets in HER2+ BC, in the context of anti-HER2 therapy resistance. Methods: We examined the differential GPCRs expression in BC stem cells, suggested to be involved in resistance, as well as in anti-HER2 treatment-resistant BT474 cell line model of HER2+ BC. BC stem cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. Brightly fluorescent ALDH+ cells were separated from ALDH- cells using FACS Aria II cell sorter. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure to increasing drug concentration of trastuzumab (T), lapatinib (L), or their combination (T+L). RNA was isolated and subjected to profiling using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs from 50 different subfamilies. Only overexpressed GPCRs were considered of interest as drug targets, and the overexpression of GPCRs was verified by RT-PCR and western blotting for the selected targets. Publically available TCGA dataset for mRNA expression was also interrogated to determine differential expression of selected GPCRs in HER2+ vs. other subtypes of BC. Results: Nine GPCRs [BAI3, EDNRA, GPR110, GPR116, GPR124, MTNR1A, EDG2, EMR2, GCGR] were upregulated in ALDH+ compared to ALDH- BT474 cells. In addition, 11 GPCRs [CCBP2, CCR9, F2RL1, GALR2, GPR1, GPR24, GPR87, GPR110, GPR183, LGR4, OXER1] were over-expressed in the resistant derivatives (T, L, and T+L) compared to the parental BT474 cells. Out of these, 13 belong to Class A and 6 to Class B, designated by The International Union of Basic and Clinical Pharmacology (IUPHAR). GPR110 was the only GPCR common to BC stem cells as well as resistant derivatives of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of BC compared to ER+ luminal A and B subtypes. GPR110 as well as other differentially expressed GPCRs are currently being investigated as potential targets by determining the effects of their downregulation or exogenous over-expression on the growth and drug-sensitivity of these BC cells. Conclusions: We report for the first time the differential expression of a large panel of GPCRs in the BC stem cell population and in anti-HER2 resistant derivatives of the BT474 cell line model of HER2+ BC. Results of the functional studies will guide novel strategies to improve the treatment for HER2+ BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-02.

P1-08-04: Obesity, Adjuvant Therapy, and Survival Outcomes in Early-Stage Breast Cancer.

BACKGROUND: Obesity has risen to epidemic proportions and is associated with worse breast cancer (BC) prognosis in most studies. However, the effects of obesity according to adjuvant therapy choice are largely unknown. To address this issue, we examined the relationship between body mass index (BMI), adjuvant therapy, and survival outcomes in a large cohort of early-stage BC patients. METHODS: We retrospectively studied patients from the Baylor Breast Center Tumor Bank treated from 1970–1995. Patients were divided into 3 BMI classes: normal/underweight (N, BMI RESULTS: There were 4,368 patients. Median age was 58. 74% were postmenopausal. 72% had stage I-II disease, 28% stage III. 76% were estrogen receptor (ER)-positive, 24% ER-negative. Patients distributed into BMI classes as follows: N 48%, Ov 30%, Ob 22%. Higher BMI was associated with postmenopausal status and increasing age, tumor size, positive lymph nodes, and stage, as well as a higher likelihood of receiving treatment. Median follow-up was 5 years. Kaplan-Meier analysis showed that TTR was significantly shorter in the Ov and Ob groups as compared to the N group (p=0.019), due to distant (p=0.001) rather than local (p=0.970) recurrences. DFS was also significantly worse in the Ov and Ob groups (p=0.002), as was OS (p=0.001). The Table shows the hazard ratios for the various survival outcomes after adjustment for age, tumor size, nodal status, and treatment groups. For all patients, TTR, DFS, and OS were significantly worse in the Ob vs. N groups. TTR and DFS were significantly worse in the chemo treated Ob vs. N groups. DFS and OS were significantly better in the endo treated Ov vs. N groups. DISCUSSION: In this large cohort of BC patients, survival outcomes (TTR, DFS, OS) were significantly worse in the obese group. This remained true after adjustment for multiple factors. Obesity was associated with worse survival outcomes in the chemo treated (CMF) group. Overweight was associated with better survival outcomes in the endo treated (tamoxifen) group. These results confirm and extend the results of previous studies. Further studies to discover the reasons for these differences in outcomes are underway. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-08-04.

Abstract P3-04-07: The new oral SERD AZD9496 is efficacious in antagonizing ER and circumventing resistance to endocrine therapy

Background: The selective estrogen receptor (ER) degrader (SERD) fulvestrant (Ful) is a potent ER antagonist that upon binding to ER induces its degradation. Ful has shown clinical efficacy in metastatic disease upon progression on previous endocrine therapies and superior activitycompared to an aromatase inhibitor as first line therapy when given at a high dose, 500mg. However, major clinical limitations of Ful are its low bioavailability and its route of administration. Here, we assess the efficacy and the mechanism of action of the new oral SERD AZD9496 compared to Ful in our panel of endocrine-sensitive and -resistant (EndoR) in vitro and in vivo models. Methods: The effects of AZD9496 and Ful were studied in vitro in various ER+ MCF7, ZR75-1, T47D, 600MPE, and MDAMB415 parental lines and in MCF7 and T47D derivatives made resistant (R) to estrogen deprivation (ED), tamoxifen (Tam), or Ful. Cell growth, Western blot, Q-RT-PCR, and ERE-reporter assays were conducted to assess treatment efficacy as well as ER levels and activity. Xenografts of parental MCF7 cells were established in ovariectomized nude mice with exogenous estrogen (E2). Mice were then randomized to continued E2 or ED, with and without AZD9496 or Ful. Mice bearing transplantable MCF7 EDR and TamR xenografts were randomized to continue original treatment or to switch to Ful or AZD9496, and tumor size was followed. Expression of classic and nonclassic/indirect ER-regulated genes was evaluated in RNA extracts of short-term-treated xenografts using the BioMark FLUIDIGM platform. Results: AZD9496 inhibited cell growth (50-100%) of all ER+ parental cells and greatly, though not fully, degraded ER protein levels. AZD9496 also potently reduced ER-dependent exogenous and endogenous gene/protein expression in presence and absence of E2. In parental MCF7 xenograft-bearing mice, 10 days of AZD9496 resulted in a greater inhibition of tumor growth and in a greater reduction of levels of ER-dependent targets in comparison to Ful in the presence of E2. The effects of the 2 SERDs were similar in the absence of E2. In EndoR models that retain ER, AZD9496 inhibited cell growth in vitro by degrading ER, similar to Ful. Both SERDs also delayed tumor growth of EDR and TamR xenografts and effectively reduced levels of ER and ER-induced proteins, though no tumor regression was observed in the TamR model. Notably, AZD9496 failed to inhibit growth of FulR cells and xenografts. Expression analysis showed that the 2 SERDs potently inhibited classic ER activity, while simultaneously increasing expression of some genes known to be regulated by the nonclassic/indirect ER activity, including genes involved in escape pathways of endocrine resistance. Conclusions: The oral SERD AZD9496 is a potent antiestrogen that antagonizes and degrades ER. AZD9496, like Ful, inhibits ER-dependent transcription and tumor growth in both naive and resistant EDR and TamR models, but shows cross-resistance in FulR models. Both AZD9496 and Ful failed to completely reduce ER protein expression and to induce TamR tumor regression, suggesting that additional strategies to reduce ER levels and to enhance the inhibition of ER signaling and/or of co-operating survival mechanisms may be needed to improve treatment outcome. Citation Format: Nardone A, Weir H, De Angelis C, Cataldo ML, Fu X, Shea MJ, Mitchell T, Trivedi M, Chamness GC, Osborne CK, Schiff R. The new oral SERD AZD9496 is efficacious in antagonizing ER and circumventing resistance to endocrine therapy [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 P3-04-07.

Abstract PD6-2: FoxA1 gene amplification in ER+ breast cancer mediates endocrine resistance by increasing IL-8

Background: ER transcriptional programming is associated with fundamental changes when endocrine resistance develops. The Forkhead transcription factor, FoxA1, is a pioneer factor for ER-DNA binding. We hypothesize that FoxA1 plays a critical role in ER transcriptional reprogramming in endocrine resistance by augmenting itself and the specific downstream effectors. Methods: Next generation sequencing was applied to characterize a panel of endocrine-resistant (Endo-R) cell models. Genomic PCR amplification and FISH assays were developed to measure FoxA1 copy number gain (CNG). Q-RT-PCR, Western blots, IHC, ELISA, and cytokine arrays were used to determine the levels of FoxA1 and IL-8 in cell culture and in vivo xenograft tumors. Effects of gene knockdown (ER, FoxA1, or IL-8) or inducible FoxA1 overexpression on ER and growth factor receptor (GFR) downstream signaling were determined by cell growth and Western blots. ER and FoxA1 binding at the IL-8 gene locus was measured by ChIP-qPCR. ChIP-seq analysis was integrated with RNA-seq data. Kaplan-Meier analysis evaluated the predictive role of FoxA1in ER+ breast tumors. Results: Exome-seq revealed that FoxA1 is the most highly amplified gene in TamR vs. P cells from two independent MCF7 models. Genomic PCR and FISH also indicate FoxA1 CNG in Endo-R models of ZR75-1 and BT474. Increased FoxA1 expression was found in multiple Endo-R cells and in MCF7L Endo-R xenograft tumors. Cytokines, especially IL-8, are more highly expressed in multiple Endo-R cell models, similar to our previous microarray data from MCF7 Endo-R xenograft tumors. FoxA1 forced overexpression significantly induced IL-8 expression in MCF7L-P cells. It also activated multiple GFR downstream signaling pathways, and conferred endocrine resistance. Conversely, knockdown of either FoxA1 or ER significantly decreased IL-8 levels in TamR cells, and inhibited cell growth in both P and TamR cells. Knockdown of IL-8 in TamR cells substantially inhibited GFR downstream signaling, and was more cytotoxic than in P cells. A novel FoxA1-binding site (10 kb at 5’UTR of IL-8) recruited more FoxA1 and p300 in MCF7L-TamR than -P cells. ChIP-seq shows a general enhancement of FoxA1 binding around the genes (within 20 kb) that are differentially expressed in TamR vs. P cells. We identified a FoxA1 CNG-associated gene signature from TCGA breast tumors that predicts worse relapse-free survival (RFS) in Tam-treated ER+ tumors (from Loi et al). Meta-analysis showed that FoxA1 mRNA levels in the top 25th percentile predict worse RFS in ER+ patients treated with Tam (N=615), but not in systemically untreated patients (N=500). FoxA1 CNG and overexpression in clinical specimens by using our newly developed FISH and IHC assays are currently being investigated. Conclusions: FoxA1 gene amplification was enriched in two independent MCF7 Tam-R cell models. Clonal selection of FoxA1 gene amplification may occur and lead to endocrine resistance. High levels of FoxA1 may mediate endocrine resistance by directly inducing IL-8. The data suggest that IL-8 signaling is a component of a cytokine loop controlled by the FoxA1/ER transcriptional reprogramming, which might be exploited in therapeutics to overcome endocrine resistance. Citation Format: Xiaoyong Fu, Rinath Jeselsohn, Emporia F Hollingsworth, Dolores Lopez-Terrada, Chad J Creighton, Agostina Nardone, Martin Shea, Laura M Heiser, Pavana Anur, Nicholas Wang, Catie Grasso, Paul Spellman, Carolina Gutierrez, Mothaffar F Rimawi, Susan G Hilsenbeck, Joe W Gray, Myles Brown, C K Osborne, Rachel Schiff. FoxA1 gene amplification in ER+ breast cancer mediates endocrine resistance by increasing IL-8 [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 PD6-2.

Abstract 5469: Trastuzumab resistant HER2+ breast cancer cells retain sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Overexpression of HER2 in breast cancer is associated with aggressive tumor features and poor patient survival. Current therapeutic agents targeting this receptor have increased the survival rate of patients with HER2+ breast cancer; however some patients eventually develop resistance to these therapies. We have previously reported that HER2+ breast cancers are susceptible to poly (ADP-Ribose) polymerase inhibitors (PARPi) alone independent of a basal or induced DNA repair deficiency but instead via suppression of NF-κB signaling. In this study, we investigated PARPi sensitivity in HER2+ breast cancer cells that are resistant to the HER2 targeted agent trastuzumab. Materials and Methods: HER2+ breast cancer cell lines BT-474, UACC812, SKBRR3 and their trastuzumab resistant counterparts were used in this study. Cells were treated with vehicle or 10µM ABT-888 or transfected with scrambled or PARP1 siRNA. NF-κB transcriptional activity was measured using a NF-κB-driven luciferase reporter assay. Protein expression was measured by Western blot analysis. Cellular cytotoxicity and viability following PARPi was assessed with colony formation and ATPlite assays, respectively. Results: HER2+ parent and trastuzumab resistant breast cancer cells showed similar susceptibility to the PARPi ABT-888. There was a dose response reduction in survival fraction (greater than 70% at 10μM) and a greater than 40% decrease in cell viability with 10μM ABT-888. This cytotoxicity was associated with more than 50% attenuation of NF-κB transcriptional activity and reduced expression of the NF-κB activator IKKα. Suppression of NF-κB signaling was also observed utilizing PARP1 siRNA. Conclusions: HER2+ breast cancer cells resistant to trastuzumab continue to be sensitive to PARP inhibition through attenuation of the NF-κB signaling pathway. These results support the use of PARPi as part of a therapeutic strategy for patients with HER2+ breast cancer. Citation Format: Monica E. Wielgos, Tiffiny Cooper, Andres Forero, James A. Bonner, Francisco J. Esteva, C K. Osborne, Rachel Schiff, Albert F. LoBuglio, Eddy S. Yang. Trastuzumab resistant HER2+ breast cancer cells retain sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5469. doi:10.1158/1538-7445.AM2014-5469

Abstract P6-04-05: GPR110 overexpression increases tumorigenic potential of HER2+ breast cancer cells

Human epidermal growth factor receptor-2-overexpressing (HER2+) breast cancer is an aggressive tumor. Despite the clinical success of anti-HER2 drugs such as lapatinib (L) and trastuzumab (T), intrinsic and acquired drug resistance occurs in many patients. Identification of novel drug targets in HER2+ breast cancer is an unmet clinical need. In this context, G-protein coupled receptors (GPCRs) may be excellent drug targets because they cross-talk with the HER family members. However, the expression and function of the majority of GPCRs are unknown in HER2+ breast cancer. In a preliminary study, we examined the differential gene expression of GPCRs in anti-HER2 treatment-resistant derivatives as well as in the tumorigenic cell population, suggested to be involved in resistance, of a BT474 cell line model of HER2+ breast cancer. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure of parental cells to increasing concentrations of L, T, or their combination (L+T). Tumorigenic cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. RNA was profiled using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs. The publically available TCGA dataset was interrogated to determine differential mRNA expression of selected GPCRs in HER2+ and other subtypes of breast cancer. To determine the functional role of GPR110, BT474 cells were infected with lentiviral GPR110 construct (GPR110-OE) or empty vector (EV), and stable pools were obtained. Anchorage-dependent cell growth was evaluated using MTT cell proliferation assay over 8 days. Tumorigenic potential was determined by calculating the% of ALDH+ cells using Aldefluor assay and by evaluating the anchorage-independent cell growth using soft agar assay over 14 days. The influence of GPR110 overexpression on HER signaling pathway was investigated by measuring the levels of phosphorylated (active) and total protein levels of HER1 and HER2 using immunoblotting. GPR110 was the only GPCR overexpressed in resistant derivatives versus parental cells as well as in ALDH+ versus ALDH- cells of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of breast cancer compared to ER+ luminal A and B subtypes. Overexpression of GPR110 in BT474 cells (9-fold in GPR110-OE vs. EV cells) resulted in a marked 5-fold increase in the number of colonies when grown in soft agar compared to EV cells even though anchorage-dependent cell growth was not significantly different between EV and GPR110-OE cells. In addition, GPR110-OE cells had a significantly higher% of ALDH+ population compared to EV cells. Phosphorylated (but not total) HER1 and HER2 protein levels were significantly higher in GPR110-OE cells compared to EV cells, suggesting hyperactive HER signaling with GPR110 overexpression. Experiments with L and T treatment will reveal the role of GPR110 in drug efficacy and acquired resistance. In summary, we show for the first time a pro-tumorigenic role of GPR110 in HER2+ breast cancer. Therefore, GPR110 may be a novel pharmacological target in HER2+ breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-05.