Sarmistha Nanda

Au Nanomatryoshkas as Efficient Near-Infrared Photothermal Transducers for Cancer Treatment: Benchmarking against Nanoshells

Au nanoparticles with plasmon resonances in the near-infrared (NIR) region of the spectrum efficiently convert light into heat, a property useful for the photothermal ablation of cancerous tumors subsequent to nanoparticle uptake at the tumor site. A critical aspect of this process is nanoparticle size, which influences both tumor uptake and photothermal efficiency. Here, we report a direct comparative study of ∼90 nm diameter Au nanomatryoshkas (Au/SiO2/Au) and ∼150 nm diameter Au nanoshells for photothermal therapeutic efficacy in highly aggressive triple negative breast cancer (TNBC) tumors in mice. Au nanomatryoshkas are strong light absorbers with 77% absorption efficiency, while the nanoshells are weaker absorbers with only 15% absorption efficiency. After an intravenous injection of Au nanomatryoshkas followed by a single NIR laser dose of 2 W/cm2 for 5 min, 83% of the TNBC tumor-bearing mice appeared healthy and tumor free >60 days later, while only 33% of mice treated with nanoshells survived the same period. The smaller size and larger absorption cross section of Au nanomatryoshkas combine to make this nanoparticle more effective than Au nanoshells for photothermal cancer therapy.

Sub-100 nm gold nanomatryoshkas improve photo-thermal therapy efficacy in large and highly aggressive triple negative breast tumors

There is an unmet need for efficient near-infrared photothermal transducers for the treatment of highly aggressive cancers and large tumors where the penetration of light can be substantially reduced, and the intra-tumoral nanoparticle transport is restricted due to the presence of hypoxic or necrotic regions. We report the performance advantages obtained by sub 100nm gold nanomatryushkas, comprising concentric gold-silica-gold layers compared to conventional ~150nm silica core gold nanoshells for photothermal therapy of triple negative breast cancer. We demonstrate that a 33% reduction in silica-core-gold-shell nanoparticle size, while retaining near-infrared plasmon resonance, and keeping the nanoparticle surface charge constant, results in a four to five fold tumor accumulation of nanoparticles following equal dose of injected gold for both sizes. The survival time of mice bearing large (>1000mm(3)) and highly aggressive triple negative breast tumors is doubled for the nanomatryushka treatment group under identical photo-thermal therapy conditions. The higher absorption cross-section of a nanomatryoshka results in a higher efficiency of photonic to thermal energy conversion and coupled with 4-5× accumulation within large tumors results in superior therapy efficacy.

Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase

IntroductionActivation of the phosphatidylinositol 3-kinase (PI3K) pathway in estrogen receptor α (ER)-positive breast cancer is associated with reduced ER expression and activity, luminal B subtype, and poor outcome. Phosphatase and tensin homolog (PTEN), a negative regulator of this pathway, is typically lost in ER-negative breast cancer. We set out to clarify the role of reduced PTEN levels in endocrine resistance, and to explore the combination of newly developed PI3K downstream kinase inhibitors to overcome this resistance.MethodsAltered cellular signaling, gene expression, and endocrine sensitivity were determined in inducible PTEN-knockdown ER-positive/human epidermal growth factor receptor 2 (HER2)-negative breast cancer cell and/or xenograft models. Single or two-agent combinations of kinase inhibitors were examined to improve endocrine therapy.ResultsModerate PTEN reduction was sufficient to enhance PI3K signaling, generate a gene signature associated with the luminal B subtype of breast cancer, and cause endocrine resistance in vitro and in vivo. The mammalian target of rapamycin (mTOR), protein kinase B (AKT), or mitogen-activated protein kinase kinase (MEK) inhibitors, alone or in combination, improved endocrine therapy, but the efficacy varied by PTEN levels, type of endocrine therapy, and the specific inhibitor(s). A single-agent AKT inhibitor combined with fulvestrant conferred superior efficacy in overcoming resistance, inducing apoptosis and tumor regression.ConclusionsModerate reduction in PTEN, without complete loss, can activate the PI3K pathway to cause endocrine resistance in ER-positive breast cancer, which can be overcome by combining endocrine therapy with inhibitors of the PI3K pathway. Our data suggests that the ER degrader fulvestrant, to block both ligand-dependent and -independent ER signaling, combined with an AKT inhibitor is an effective strategy to test in patients.

HER2 Reactivation through Acquisition of the HER2 L755S Mutation as a Mechanism of Acquired Resistance to HER2-targeted Therapy in HER2+ Breast Cancer

Purpose: Resistance to anti-HER2 therapies in HER2+ breast cancer can occur through activation of alternative survival pathways or reactivation of the HER signaling network. Here we employed BT474 parental and treatment-resistant cell line models to investigate a mechanism by which HER2+ breast cancer can reactivate the HER network under potent HER2-targeted therapies. Experimental Design: Resistant derivatives to lapatinib (L), trastuzumab (T), or the combination (LR/TR/LTR) were developed independently from two independent estrogen receptor ER+/HER2+ BT474 cell lines (AZ/ATCC). Two derivatives resistant to the lapatinib-containing regimens (BT474/AZ-LR and BT474/ATCC-LTR lines) that showed HER2 reactivation at the time of resistance were subjected to massive parallel sequencing and compared with parental lines. Ectopic expression and mutant-specific siRNA interference were applied to analyze the mutation functionally. In vitro and in vivo experiments were performed to test alternative therapies for mutant HER2 inhibition. Results: Genomic analyses revealed that the HER2L755S mutation was the only common somatic mutation gained in the BT474/AZ-LR and BT474/ATCC-LTR lines. Ectopic expression of HER2L755S induced acquired lapatinib resistance in the BT474/AZ, SK-BR-3, and AU565 parental cell lines. HER2L755S-specific siRNA knockdown reversed the resistance in BT474/AZ-LR and BT474/ATCC-LTR lines. The HER1/2–irreversible inhibitors afatinib and neratinib substantially inhibited both resistant cell growth and the HER2 and downstream AKT/MAPK signaling driven by HER2L755S in vitro and in vivo. Conclusions: HER2 reactivation through acquisition of the HER2L755S mutation was identified as a mechanism of acquired resistance to lapatinib-containing HER2-targeted therapy in preclinical HER2-amplified breast cancer models, which can be overcome by irreversible HER1/2 inhibitors. Clin Cancer Res; 23(17); 5123–34. ©2017 AACR.

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.

GPCRs profiling and identification of GPR110 as a potential new target in HER2+ breast cancer

PurposeG protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC.MethodsGene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur− population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and anchorage-independent cell growth, mammosphere formation, and migration/invasion were evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+ trastuzumab-resistant (LTR) cells.ResultsAdhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells.ConclusionOur data suggest a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Abstract P3-05-13: Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells

Background: The HER2 pathway can be inhibited by potent targeting agents such as lapatinib (L), trastuzumab (T), or their combination (LT), but acquired and de novo resistance still occur. Resistance to these drugs remains a major hurdle in the management of HER2+ breast cancer. Consequently, elucidation of mechanisms of acquired therapeutic resistance to HER2-directed therapies is of critical importance. Methods: To obtain clues to the mechanisms for resistance we developed a panel of HER2+ breast cancer cell lines resistant to L, T, or LT. Parental cells and resistant derivatives of the HER2+ BT474 cell line were characterized by RNA-seq. Genes that were overexpressed in resistant compared to parental cells were confirmed by RT-PCR, Western blotting, and immunohistochemistry (IHC). Cell growth and cell signaling were assessed in parental and resistant cell lines after down-regulation (by siRNA) or overexpression (via an inducible cDNA) of IRS4 in the presence or absence of treatment. The effect of IRS4 overexpression on L resistance was assessed in a BT474 xenograft model. The proteins that interact with IRS4 were identified by co-immunoprecipitation with IRS4 followed by separation of the associated proteins by SDS-PAGE and microsequencing by mass spectrometry. Results: RNA-seq analysis revealed that IRS4 was the most up-regulated gene in BT474 L or LT resistant derivatives in which HER2 signaling is effectively inhibited, but not T alone, where HER2 signaling is reactivated. Western blotting and IHC validated this result and identified membrane localization of IRS4. Knockdown of IRS4 in L- or LT-resistant cells reversed resistance and restored growth inhibition. IRS4 knockdown also inhibited downstream signaling, with a reduction in pAKT but not in pMAPK. Induction of the cell cycle regulator p27 and down-regulation of survivin were observed after IRS4 knockdown. Overexpression of IRS4 cDNA in parental BT474 and SKBR3 cells led to resistance to L/LT, increased pAkt, and decreased the apoptotic marker cleaved PARP in the presence of L or the LT combination. The BT474 xenograft model showed that IRS4 overexpression in the absence of treatment had no effect on tumor growth but it significantly reduced the inhibitory effect of lapatinib (p=0.002). A group of proteins that interact with IRS4 in BT474 L-resistant cells were identified by mass spectrometry. The roles of these proteins in IRS4-mediated resistance to lapatinb-containing regimens are under investigation. Conclusion: IRS4 overexpression is a critical factor in causing resistance to lapatinib-containing regimens in BT474 cells. Investigation of IRS4 and its signaling partners in HER2+ human tumors resistant to lapatinib will be important to determine if this mechanism is also operative in patients. Citation Format: Lanfang Qin, Maria B Hahn, Xiaoyong Fu, Martin J Shea, Mario Giuliano, Sarmistha Nanda, Xiaowei Xu, Huizhong Hu, Sung Yun Jung, Laura M Heiser, Nicholas Wang, Joe W Gray, Susan G Hilsenbeck, Chad Creighton, Chad A Shaw, Gary C Chamness, Dean P Edwards, Sabrina Herrera, Carolina Gutierrez, C Kent Osborne, Rachel Schiff. Overexpression of insulin receptor substrate 4 can mediate acquired resistance to lapatinib-containing regimens in HER2+ breast cancer cells [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 P3-05-13.

Abstract P5-05-03: Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib

Background: Targeting HER2 with lapatinib (L), trastuzumab (T), or the LT combination, is effective in HER2+ breast cancer (BC), but acquired resistance commonly occurs. In our 12-week neoadjuvant trial (TBCRC006) of LT without chemotherapy in HER2+ BC, the overall pathologic complete response rate (pCR) was 27%. To investigate resistance mechanisms our lab developed 10 HER2+ BC cell lines resistant (R) to these drugs (LR/TR/LTR). To discover potential predictive markers/therapeutic targets to circumvent resistance, we completed genomic profiling of the cell line panel and a subset of pre-treatment baseline specimens from TBCRC006. Methods: Parental (P) lines and LR/TR/LTR derivatives of 9 HER2+ BC cell line models were profiled with whole exome and RNA sequencing. Mutations detected in R lines but not in same-model P lines were identified. cDNAs were assessed by targeted Sanger sequencing. Single cells of the BT474AZ-LR line were cloned and their cDNAs were sequenced. Mutant-specific Q-PCR was designed to sensitively quantify mutations. Whole exome sequencing (minimum depth 100X) of 17 baseline tumor/normal pairs from TBCRC006 were performed on Illumina HiSeq. Results: We found and validated the HER2 L755S mutation in the BT474ATCC-LTR line and the BT474AZ-LR line (∼30% of DNA/RNA/cDNA in BT474AZ-LR), in which the HER pathway was reactivated to cause resistance. Overexpression of this mutation was previously shown to induce L resistance in HER2-negative BC cell lines, suggesting a role as an acquired L/LT resistance driver in HER2+ BC. Sanger sequencing of BT474AZ-LR single cell clones found the HER2 L755S mutation in every clone but only in ∼30% of the HER2 copies. Using sensitive mutant-specific Q-PCR, we found statistically higher levels of HER2 L755S expression in BT474ATCC-P and BT474AZ-P compared to parentals of other HER2+ BC cell lines (UACC812/AU565/SKBR3/SUM190). These data suggest that this mutation exists subclonally within BT474 parental lines and was selected to become the more dominant population in the two resistant lines. The HER1/2 irreversible tyrosine kinase inhibitor (TKI) afatinib (Afa) robustly inhibited growth of both BT474ATCC-LTR/AZ-LR cells (IC50: Afa 0.02µM vs. L 3 µM). Western blots confirmed inhibition of the HER and downstream Akt and MAPK signaling in the LR cells by Afa. Sequencing of TBCRC006 baseline samples found the HER2 L755S mutation in 1/17 subjects. This patient did not achieve pCR after neoadjuvant LT treatment. The variant was present in 2% of the reads, indicating it as a subclonal event in this patient’s baseline tumor. Conclusion: Acquired resistance in two of our BT474 LR/LTR lines is due to selection of HER2 L755S subclones present in the parental cell population. The higher HER2 L755S levels detected in BT474 parentals compared with other HER2+ BC parental lines, and detection of its subclonal presence in a pre-treatment HER2+ BC patient, suggest that sensitive mutation detection methods will be needed to identify patients with potentially actionable HER family mutations in primary tumor. Treating this patient group with an irreversible TKI like Afa may prevent resistance and improve clinical outcome of this subset of HER2+ BC. Citation Format: Xiaowei Xu, Agostina Nardone, Huizhong Hu, Lanfang Qin, Sarmistha Nanda, Laura M Heiser, Nicholas Wang, Kyle R Covington, Edward S Chen, Alexander Renwick, Tao Wang, Carmine De Angelis, Alejandro Contreras, Carolina Gutierrez, Suzanne AW Fuqua, Gary C Chamness, Chad Shaw, David A Wheeler, Joe W Gray, Susan G Hilsenbeck, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Clonal evolution of the HER2 L755S mutation leads to acquired HER-targeted therapy resistance that can be reversed by the irreversible HER1/2 inhibitor afatinib [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 P5-05-03.

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.