Phage Lentiviral Mediated GPR110 Overexpression in BT474 and SKBR3 Parental Cells: E 150 150 E

Presented at San Antonio Breast Cancer Symposium® - DecemberCollege of Pharmacy 4-8, 20182013 Role of GPR110 in Breast Cancer -Dox +Dox Raksha Bhat1, Lanfang Qin2, Carmine De Angelis2, Debashish Sahay1, Dharmendra Bhargava1, Chad J. Creighton2, Puja Yadav1, Sahar Yazdanfard1,A .Ahmed Al-rawi1, B. -Dox +Dox Clone 1 Clone 1 Vikas Yadav3, Suhas Vasaikar2, Sarmistha Nanda2, Vidyalakshmi Sethunath2, Xiaoyong Fu2, Bing Zhang2, Vihang Narkar3, Rachel Schiff2, Meghana V. Trivedi1,2 Clone 5 Clone 5

1University of Houston College of Pharmacy, Houston, TX; 2Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX; 3University of Texas McGovern Medical School, Houston, TX.

ABSTRACT METHODS RESULTS -Dox +Dox Our long-term goal is to discover adhesion GPCR targets in breast cancer. Our GPR110 gene expression analysis: GPR110 expression was measured using TaqMan RT-PCR (Life A. B. -Dox +Dox C. -Dox +Dox D. -Dox +Dox Technologies, Carlsbad, CA). A comparative C analysis was utilized to determine fold change in previous studies have found GPR110 to be overexpressed in tumorigenic cell T Clone 1 Clone 1 Clone 1 Clone 1 population as well as in anti-HER2 drug-resistant derivatives of HER2+ breast GPR110 mRNA expression based on the ΔΔCT approach using PPIA as a housekeeping gene. Clone 5 Clone 5 Clone 2 Clone 2

cancer cells. In subsequent studies, we found that GPR110 knockdown inhibited -Dox -Dox

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pHAGE lentiviral mediated GPR110 overexpression in BT474 and SKBR3 parental cells: e 150 150 e

-Dox e * -Dox

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* +Dox * t

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anchorage-independent cell growth, mammosphere formation, and r *

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a * +Dox +Dox g

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Overexpression of GPR110 was done using lentiviral pHAGE system under the control of * v i

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invasion/migration of HER2+ breast cancer cells. Conversely, overexpression of

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40 s

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inducible Tet-on promoter. The overexpressed GPR110 had an HA tag on the c-terminus to allow l

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20 e C

C 50 50

GPR110 by lentiviral delivery of cDNA enhanced anchorage-independent cell C

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detection using western blotting. o

# # growth, mammosphere formation, and invasion/migration in HER2+ breast cancer # 0 0 0 # 0 cells. In addition, GPR110 overexpression led to increase in the % of Aldefluor- MTT assay: Cell proliferation assay was performed using the MTT kit from ATCC (catalog # 30- Clone 1 Clone 5 Clone 1 Clone 5 Clone 1 Clone 2 Clone 1 Clone 2 BT474 GPR110 FL OE clone BT474 GPR110 FL OE clone positive tumorigenic cell population, further emphasizing the role of GPR110 as a 1010K). The cells were plated in 96 well plates and the absorbance was read at 72 hours. SKBR3 GPR110 FL OE clone SKBR3 GPR110 FL OE clone -Dox +Dox mediator of tumorigenesis in addition to the metastatic processes in HER2+ C. D. -Dox +Dox Soft agar assay: The effect of GPR110 overexpression using Dox on anchorage-independent cell Figure 5. Effects of GPR110 overexpression on invasion and migration of HER2+ breast cancer cells. BT474 clones 1 and 5 and breast cancer. Among various subtypes of breast cancer, GPR110 expression was Clone 1 Clone 1 growth was evaluated in BT474 and SKBR3 OE clones (5,000 cells/well, 24-well plates) using the SKBR3 clones 1 and 2 were grown in absence (-) or presence (+) of doxycycline (dox) (N=3). Invasion and migration of cells was higher in HER2+ and basal subtypes, most of which are triple-negative (negative soft agar assay as described before [Bhat et al., BCRT, 2018]. Number of colonies (at least 50 µm measuredClone 2 using the transwell insertClowithne 2(A and C) or without (B and D) matrigel, respectively. * indicates statistically significant for ER, PR, and HER2), compared to luminal A and B subtypes. GPR110 was either in size) were counted on Day 7 using GelcountTM (Oxford Optromix, Germany). difference by Two-way ANOVA, p<0.05. N=3-4. gene amplified or upregulated in 4% of all breast cancers based on the publicly A. BT474 NES B. D. available TCGA dataset. GPR110 overexpression predicted poorer recurrence-free Mammosphere assay: Mammosphere assay was conducted in GPR110-overexpressing clones -Dox +Dox -Dox +Dox survival in triple-negative breast cancer. Furthermore, GPR110 was overexpressed (5, 000 cells/well, 24 well plates) as described before [Bhat et al., BCRT, 2018]. Cl. 1 Cl. 5 HALLMARK_E2F_TARGETS -2 0 Ki67 Ki67

in brain metastatic lesions compared to mammary tumors in patient-derived 1

HALLMARK_G2M_CHECKPOINT

Migration and Invasion assay: The migration and invasion of cells was assessed using Corning e

xenograft models of triple-negative breast cancer (WHIM2 and WHIM30). HALLMARK_MYC_TARGETS_V1 n o BioCoat™ Tumor Cell Migration and Invasion Systems as described before [Bhat et al., BCRT, HALLMARK_ESTROGEN_RESPONSE_EARLY l

Knocking down GPR110 reduced anchorage-dependent and -independent cell C 2018]. The number of migrated and invaded cells were imaged and counted using ImageJ. HALLMARK_HYPOXIA growth, mammosphere formation, and invasion/migration of triple-negative HALLMARK_TNFA_SIGNALING_VIA_NFKB breast cancer cells. Overall, our results suggest that GPR110 may be a potential HALLMARK_MYC_TARGETS_V2

HALLMARK_UNFOLDED_PROTEIN_RESPONSE 5 drug target in HER2+ and triple-negative breast cancer. Drug discovery efforts to RESULTS NFkB NFkB HALLMARK_MTORC1_SIGNALING e

HALLMARK_ESTROGEN_RESPONSE_LATE n identify GPR110 antagonists will provide useful pharmacological tools for o HALLMARK_UV_RESPONSE_DN l A. C validating GPR110 as a drug target in breast cancer. Since GPR110 is also HALLMARK_KRAS_SIGNALING_DN overexpressed in various other types of cancer, understanding the mechanism of HALLMARK_IL2_STAT5_SIGNALING HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION GPR110 upregulation and signaling in cancer is an important future direction. 100 6 G0/1 100 4 G0/1 HALLMARK_P53_PATHWAY 5 14 7 S 12 S

HALLMARK_XENOBIOTIC_METABOLISM 80 18 G2/M 80 18 G2/M

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HALLMARK_ALLOGRAFT_REJECTION l e

C. e 60 87 60 91

BACKGROUND HALLMARK_GLYCOLYSIS f

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HALLMARK_DNA_REPAIR 40 40

% % Figure 1. Candidate GPCR selection HALLMARK_SPERMATOGENESIS Phospho STAT3 Cancer Anti-HER2 HALLMARK_PI3K_AKT_MTOR_SIGNALING 20 20 in Aldefluor+ vs. Aldefluor- cells B. C. D. stem cells resistant cells HALLMARK_MITOTIC_SPINDLE 0 0 and in anti-HER2 resistant cells. 11 Cells BT474 SKBR3 +Dox HALLMARK_CHOLESTEROL_HOMEOSTASIS -Dox +Dox -Dox +Dox (LTR and LR/TR) Total STAT3 BT474 GPR110 FL Clone 1 BT474 GPR110 FL Clone 5 GPCRs were upregulated in Clones Cl 1 Cl 5 Cl 1 Cl 2 HALLMARK_ANDROGEN_RESPONSE 350 * 350

ADORA1 Dox - + - + - + - + s

-Dox s -Dox

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tumorigenic Aldefluor+ cells 300 l 300 e

EDG2 CCBP2 +Dox e +Dox * c

250 GPR110-HA c

Figure 6. Dox-induced overexpression of GPR110 inhibits in vivo tumor growth, downregulates cell cycle pathways, and induces +

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EFNRA CCR9 compared to Aldefluor- population r o

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200 u 200 cell cycles arrest. (A) RNAseq analysis and gene set enrichment analysis shows GPR110 overexpression leads to downregulation of ﬂ

GPR87 GPR110 EBI2 of BT474 cells. Additionally, 10 75 ﬂ

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MTNR1A F2RL1 50 l E2F targets, G2M checkpoint pathway and MYC targets (with FDR q <5%). RPPA analysis also revealed key proteins/pathways in

A A

GPCRs were overexpressed in the f

100 f 100 o GALR2 o

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* cell cycle regulation which were validated to show (B) a reduction in Ki67+ cells and lower NF-B staining as well as (C) % LTR and LR or TR derivatives % BAI3 GPR1 50 50 * compared to parental BT474 cells. 25 0 0 downregulation of phospho-STAT3 pathway. (D) GPR110 overexpression also causes cell cycle arrest at G0/G1 phase. EMR2 GPR24 Clone 1 Clone 5 Clone 1 Clone 2 GPR116 LGR4 GPR110 was the only GPCR Actin BT474 GPR110 FL OE clone SKBR3 GPR110 FL OE clone GPR124 OXER1 overexpressed in both tumorigenic as well as resistant derivatives of Figure 3. GPR110-overexpression using pHAGE lentiviral mediated infection of BT474 and Figure 7: GPR110 knockdown decreases

SKBR1.43 parental cells. A-Dox. Map1.4of pHAGE lentiviral-Dox plasmid. B. Western blot analysis to detect the anchorage-dependent cell growth, colony and m

BT474 cells. m n

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1.2 1.2

0 7

7 mammosphere formation, and invasion/migration 5

expression5 1.0 of full-length GPR1.0110 using anti-HA antibodies in clones 1 and 5 of BT474 cells and

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e in TNBC cells. MDA-MB-231 cells were selected c

clonesc 1 and 2 of SKBR3 cells in absence (-) or presence (+) of doxycycline (Dox). C, D. qRT-PCR n

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r because they have gene amplification and o

analysiso 0.4 to detect the expression0.4 of full-length GPA110 using Taqman probes specific for GPR110

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b 0.2 0.2 A in clonesA 1 and 5 of BT474 cells and clones 1 an 2 of SKBR3 cells in absence (-) or presence (+) of overexpression of GPR110. The cells were reverse 0.0 0.0 Clone 1 Clone 5 Clone 1 Clone 5 transfected with non-targeting [si-control] or with Dox. *BT474indicates GPR110 FL OEstatistically clone significantSKBR3 GPR110 differenceFL OE clone by Two-way ANOVA, p<0.05. two independent GPR110-targeting [si-GPR110 (1) A. B. 400 -Dox 1000 -Dox

* +Dox * +Dox and (2)] siRNAs. (A) GPR110 knockdown efficiency. t t 800 * n 300 n Figure 4. Effects of GPR110

u (B) Anchorage-dependent cell growth by MTT assay.

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200 y overexpression on anchorage– n

n (C) Anchorage-independent cell growth by soft agar

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o C 100 C independent cell growth, mammosphere assay. (D) Mammosphere formation assay. (E) 200 formation, and Aldefluor positivity. 0 0 Migration and Invasion assay. * indicates Clone 1 Clone 5 Clone 1 Clone 5 BT474 GPR110 FL OE clone SKBR3 GPR110 FL OE clone BT474 clones 1 and 5 and SKBR3 clones 1 statistically significant difference by Two-way -Dox +Dox C. -Dox +Dox D. 18.6% 42.1% and 2 were grown in absence (-) or ANOVA, p<0.05. N=3-4.

Clone 1 Figure 2. GPR110 expression in publicly available datasets with different BC Clone 1 presence (+) of doxycycline (dox) for 72

subtypes. Publicly available TCGA dataset [Ciriello, et al. Cell, 2015] was used to 26.8% 49.7% hours before conducting subsequent plot the overexpression/gene amplification of GPR110 in different BC subtypes. (A) Clone 5 Clone 5 assays. GPR110 overexpression with DISCUSSION & FUTURE DIRECTIONS FUNDING Box and Whisker plot showing differential expression of GPR110 in basal, HER2+, addition of Dox increased (A, B) 40 * -Dox 80 * -Dox

t Department of Defense Grants W81XWH-14-1-0340 and

s n +Dox l +Dox luminal A, and luminal B subtypes of BC in human patients. Gene expression of l anchorage-independent cell growth by

u • GPR110 has a pro-tumorigenic and pro-metastatic role in

c 30 60

W81XWH-14-1-0341 to Drs. Trivedi and Schiff, respectively.

+ e GPR110 was significantly higher in HER2+ and basal-like BC compared to luminal A r r * soft agar assay, (C) mammosphere

o HER2+ BC and TNBC.

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* ﬂ

p 20 40 e and B subtypes. * indicates P<0.05, t test on log-transformed data. Box plots s formation, and (D) % of Aldefluor-positive

d • This presentation is the intellectual property of the author/ l

o GPR110 overexpression leads to cell cycle arrest.

f m 10 o 20 represent 5, 25, 50, 75, and 95%. Log2 FPKM expression values are normalized to cells. * indicates statistically significant

a • Future in vivo studies using GPR110 OE clones will assess its presenter. Please contact Dr. Trivedi at the following email

% M standard deviation from the median. (B) Percentage of GPR110 gene amplification 0 0 difference by Two-way ANOVA, p<0.05. role as a novel drug target to overcome anti-HER2 drug address for permission to reprint and/or distribute: Clone 1 Clone 5 Clone 1 Clone 5 in HR+/HER2-, HER2+, and TN subtypes of BC in human patients. BT474 GPR110 FL OE clone BT474 GPR110 FL OE clone N=3-4. resistance and combat metastasis in HER2+ breast cancer. [email protected]