Search

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) small-molecule inhibitors in breast cancer arises through mechanisms that are yet uncharacterized. In this study, we used a kinome-wide siRNA screen to identify kinases that, when downregulated, yield sensitivity to the CDK4/6 inhibitor ribociclib. In this manner, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a key modifier of ribociclib sensitivity in estrogen receptor–positive MCF-7 breast cancer cells. Pharmacologic inhibition of PDK1 with GSK2334470 in combination with ribociclib or palbociclib, another CDK4/6 inhibitor, synergistically inhibited proliferation and increased apoptosis in a panel of ER-positive breast cancer cell lines. Ribociclib-resistant breast cancer cells selected by chronic drug exposure displayed a relative increase in the levels of PDK1 and activation of the AKT pathway. Analysis of these cells revealed that CDK4/6 inhibition failed to induce cell-cycle arrest or senescence. Mechanistic investigations showed that resistant cells coordinately upregulated expression of cyclins A, E, and D1, activated phospho-CDK2, and phospho-S477/T479 AKT. Treatment with GSK2334470 or the CDK2 inhibitor dinaciclib was sufficient to reverse these events and to restore the sensitivity of ribociclib-resistant cells to CDK4/6 inhibitors. Ribociclib, in combination with GSK2334470 or the PI3Kα inhibitor alpelisib, decreased xenograft tumor growth more potently than each drug alone. Taken together, our results highlight a role for the PI3K–PDK1 signaling pathway in mediating acquired resistance to CDK4/6 inhibitors. Cancer Res; 77(9); 2488–99. ©2017 AACR.

E2F4 gene signature modulation and association with overall survival in ER+/HER2- breast cancer treated with endocrine therapy. a) E2F4 gene signature expression at baseline (pre) and after 2-weeks of aromatase inhibitor treatment (post), data from ACOSOGZ1031b study. Tumors were divided according to the E2F4 score at baseline in high, medium or low tertiles. After two-weeks of aromatase inhibitor treatment, there was a decrease in the E2F4 score in all groups. However, the proportion of tumors still expressing a high post-treatment E2F4 score was greater in patients with a high E2F4 score at baseline (60%) than the other groups. b) Overall survival in patients with ER+ breast cancer treated with adjuvant endocrine therapy in the METABRIC database (n=1498) according to E2F4 signature score tertiles. c) Overall survival in patients with ER+ tumors from METABRIC according to E2F4 score tertiles in Luminal A and d) Luminal B tumors.

Supplemental Table S1: The Preoperative Endocrine Prognostic Index (PEPI score) Supplemental Table S2:* Baseline Ki67: 7 patients had missing values Supplemental Table S3a: Driver Mutations Supplemental Table S3b: Driver Copy Number Alterations Supplemental Table S3c: Mutations clinically actionable. Drug prescription Supplemental Table S4: Actionable CNAs. Drug prescription Supplemental Table S5: Overlap between genes upregulated in resistant tumors and transcription factor targets genes Suplemental Table S6: Change from baseline to surgery in gene expressions of the 47 most upregulated genes in the tumors resistant to prolonged neoadjuvant letrozole from 60 HR-positive/HER2-negative breast cancers in POP trial. A decreased gene expression translates into a geometric mean change < 1 . An increased or stable gene expression translates into a geometric mean change {greater than or equal to} 1. Supplemental Table S7: Mutations detected in this study(post-filtering) Supplemental Table S8: Gene expression values (FPKM)

Supplementary Figure 4 - PDF file 1098K, KRT19 inhibition does not down-regulate PTEN protein level through modulation of PTEN stability

Supplementary data information

Purpose: We describe herein a novel P447_L455 deletion in the C2 domain of PIK3CA in a patient with an ER+ breast cancer with an excellent response to the PI3Kα inhibitor alpelisib. Although PIK3CA deletions are relatively rare, a significant portion of deletions cluster within amino acids 446–460 of the C2 domain, suggesting these residues are critical for p110α function.Experimental Design: A computational structural model of PIK3CAdelP447-L455 in complex with the p85 regulatory subunit and MCF10A cells expressing PIK3CAdelP447-L455 and PIK3CAH450_P458del were used to understand the phenotype of C2 domain deletions.Results: Computational modeling revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Coimmunoprecipitation experiments showed reduced binding of the C2 deletion mutants with p85 compared with wild-type p110α. The MCF10A cells expressing PIK3CA C2 deletions exhibited growth factor–independent growth, an invasive phenotype, and higher phosphorylation of AKT, ERK, and S6 compared with parental MCF10A cells. All these changes were ablated by alpelisib treatment.Conclusions: C2 domain deletions in PIK3CA generate PI3K dependence and should be considered biomarkers of sensitivity to PI3K inhibitors. Clin Cancer Res; 24(6); 1426–35. ©2017 AACR.

Supp Figure S1-S5 with legends, Supp Tables S1-S4

Supplementary Figure 6 - PDF file 1339K, Statistical analyses of western blots and RT-PCR data

Distribution of clinically actionable mutations according to PEPI score, PCNA proliferation signature and breast cancer subtypes

Supplementary Information - PDF file 163K, Legends to Supplementary data and Materials and Methods

Supplementary Fig. 7A - PDF file 188K, Correlation between KRT19 and HER2 expression in breast cancer patient samples

CONSORT diagram

Supplementary Figure S1. RNAi screen to identify kinases that sensitize cells to CDK4/6 inhibition. Supplementary Figure S2. Calculation of the sensitivity index (SI) value. Supplementary Figure S3. PDK1 and CDK4/6 inhibitors are synergistic against a panel of cancer cell lines. Supplementary Figure S4. Inhibition of the PI3K/PDK1 pathway results in durable responses and is well-tolerated in vivo. Supplementary Figure S5. Ribociclib-resistant cells are cross-resistant to other CDK4/6 inhibitors and upregulate P-PDK1. Supplementary Figure S6. Inhibition of PI3K/PDK1/AKT/mTOR signaling pathway resensitizes ribociclib-resistant cells to CDK4/6 inhibition.

Variant filtering algorithm and mutation allele frequency distribution a) Variant filter algorithm. b) Mutation Allele Frequency (MAF) histogram of 11861 non-synonymous variants. c) MAF of 330 non-synonymous variants after filtering out potential germline variants.

Purpose: Keratin19 (KRT19) is the smallest known type I intermediate filament and is used as a marker for reverse transcriptase PCR–mediated detection of disseminated tumors. In this study, we investigated the functional analysis of KRT19 in human breast cancer.Experimental Design: Using a short hairpin RNA system, we silenced KRT19 in breast cancer cells. KRT19 silencing was verified by Western blot analysis and immunocytochemistry. We further examined the effect of KRT19 silencing on breast cancer cells by cell proliferation, migration, invasion, colony formation assay, cell-cycle analysis, immunocytochemistry, immunohistochemistry, and mouse xenograft assay.Results: Silencing of KRT19 resulted in increased cell proliferation, migration, invasion, and survival. These effects were mediated by upregulation of Akt signaling as a result of reduced PTEN mRNA expression. Silencing of KRT19 decreased the nuclear import of early growth response-1 (Egr1), a transcriptional factor for PTEN transcription, through reduced association between Egr1 and importin-7. We also confirmed that silencing of KRT19 increased tumor formation in a xenograft model.Conclusions: KRT19 is a potential tumor suppressor that negatively regulates Akt signaling through modulation of Egr1 nuclear localization. Clin Cancer Res; 19(16); 4335–46. ©2013 AACR.

Supplementary Materials and Methods and Figure Legends for Supplementary Figures S1-6

Supplementary Figure 5 - PDF file 2097K, KRT19 regulated nuclear localization of Egr1 transcriptional factor

Supplementary Fig. 3 - PDF file 2033K, KRT19 inhibition induces Akt activity through down-regulation of PTEN expression.

Supplementary Fig. 2 - PDF file 2822K, KRT19 inhibition up-regulates colony formation, cell migration and invasion

Purpose: This study aimed to identify biomarkers of resistance to endocrine therapy in estrogen receptor–positive (ER+) breast cancers treated with prolonged neoadjuvant letrozole.Experimental Design: We performed targeted DNA and RNA sequencing in 68 ER+ breast cancers from patients treated with preoperative letrozole (median, 7 months).Results: Twenty-four tumors (35%) exhibited a PEPI score ≥4 and/or recurred after a median of 58 months and were considered endocrine resistant. Integration of the 47 most upregulated genes (log FC > 1, FDR < 0.03) in letrozole-resistant tumors with transcription-binding data showed significant overlap with 20 E2F4-regulated genes (P = 2.56E−15). In patients treated with the CDK4/6 inhibitor palbociclib before surgery, treatment significantly decreased expression of 24 of the 47 most upregulated genes in letrozole-resistant tumors, including 18 of the 20 E2F4 target genes. In long-term estrogen-deprived ER+ breast cancer cells, palbociclib also downregulated all 20 E2F4 target genes and P-RB levels, whereas the ER downregulator fulvestrant or paclitaxel only partially suppressed expression of this set of genes and had no effect on P-RB. Finally, an E2F4 activation signature was strongly associated with resistance to aromatase inhibitors in the ACOSOG Z1031B neoadjuvant trial and with an increased risk of relapse in adjuvant-treated ER+ tumors in METABRIC.Conclusions: In tumors resistant to prolonged neoadjuvant letrozole, we identified a gene expression signature of E2F4 target activation. CDK4/6 inhibition suppressed E2F4 target gene expression in estrogen-deprived ER+ breast cancer cells and in patients' ER+ tumors, suggesting a potential benefit of adjuvant CDK4/6 inhibitors in patients with ER+ breast cancer who fail to respond to preoperative estrogen deprivation. Clin Cancer Res; 24(11); 2517–29. ©2018 AACR.

Background: CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have prolonged survival of patients with ER+ metastatic breast cancer. However, this combination is not curative mainly due to acquired drug resistance. Knowledge about mechanisms of such resistance remains quite incomplete. We report herein a forward-genetics screen to discover a broad spectrum of novel somatic mutations causal to CDK4/6i resistance. Methods: We used CRISPR/Cas9 to delete the DNA mismatch repair (MMR) gene MSH2 in MCF7 and T47D ER+ breast cancer cells. Deficiency of DNA MMR proteins such as MSH2 results in a high nucleotide substitution rate which, in turn, predisposes cells to acquire drug resistance-associated mutations. MSH2-/- MCF7 and T47D cells were infected with a lentiviral barcode library containing ~1000 unique DNA barcodes. MSH2-/- barcoded cells were expanded for ~25 doublings to allow the accumulation of random mutations. Clones resistant to CDK4/6i were selected in the presence of IC90 of palbociclib (200 nM) or abemaciclib (500 nM) for 4-6 weeks. CDK4/6i resistant clones with unique barcode IDs were subjected to whole exome sequencing (WES). Results: Following drug selection, ~73 uniquely barcoded resistant colonies emerged from MCF7 and T47D MSH2-/- clonal lines. As expected, MCF7 and T47D MSH2-/- clones harbored a high mutation burden compared to parental cells. Candidate variants were distilled based on (a) functionality prediction and (b) mutation frequency in Project GENIE. We observed RB1 (5/73 clones; 6.8%) mutations in CDK4/6i resistant clones, providing proof-of-principle that this approach can identify clinically-relevant drug resistant alterations. Overall, we identified non-synonymous alterations in 2,206 genes in T47D palbociclib-resistant, 2,195 genes in T47D abemaciclib-resistant, and 1,312 genes in MCF7 palbociclib-resistant lines. A secondary screen of the 10 genes recurrently mutated in all three CDK4/6i resistant groups identified loss of ASXL1 as top hit. ASXL1 encodes a polycomb repressive complex protein that regulates chromatin accessibility. Loss of ASXL1 has been implicated in myeloid transformation through epigenetic reprogramming. WES of 76 CDK4/6i resistant tumor biopsies (DFCI/MBCproject cohort) identified ASXL1 alterations in two and four patients with acquired and primary resistance, respectively (6/76=7.9%). One of the tumors that progressed after an initial response to palbociclib had acquired the same ASXL1 R549C mutation that was identified in our screen. Among 1,769 tumors from patients treated with CDK4/6i (TEMPUS database), 37 exhibited ASXL1 alterations (4 frameshift, 6 truncating, 3 in-frame del, 24 missense mutations). DNAseq of patient-derived organoids established from post-CDK4/6i metastases identified ASXL1 mutations in 2/7 organoids (29%). ASXL1-/- MCF7 and T47D cells were cross-resistant to fulvestrant. GSEA analysis of RNA-seq data showed upregulation of E2F targets in palbociclib-treated cells stably transduced with ASXL1 shRNA but not control shRNA (Enrichment score=0.75, q=1.00E-09). This was associated with maintenance of RB phosphorylation in the presence of CDK4/6i, markedly higher levels of CDK2, CDK6, cyclins E and A, and downregulation of p21 and p27. Finally, siRNAs targeting CDK2 or cyclin A reduced the viability of ASXL1-deficient T47D cells by 50% and 90%, respectively. Conclusions: An accelerated mutagenesis approach using MMR-deficient ER+ breast cancer cells identified loss of ASXL1 as a novel mechanism of resistance to CDK4/6i. ASXL1 alterations were found in ~8% of tumors from patients with de novo or acquired resistance to CDK4/6i. Knockdown of CDK2 and cyclin A restored sensitivity to CDK4/6i and reduced viability of ASXL1 deficient cells, suggesting CDK2 inhibitors are a treatment approach against these drug-resistant tumors. Citation Format: Dhivya R. Sudhan, Sumanta Chatterjee, Jiwoong Kim, Yunguan Wang, Vishal Kandagatla, Dan Ye, Chang-Ching Lin, Jorge Gomez Tejeda Zanudo, Esha Jain, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Juan Manuel Povedano, David McFadden, Alex Barrett, Nikhil Wagle, Ariella B. Hanker, Carlos L. Arteaga. Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-09.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease group with variable clinico-pathologic features. Based on gene expression profiles, TNBCs are grouped into 6 major subtypes. The Luminal androgen receptor (LAR) subtype is enriched for potentially targetable biomarkers, including high androgen receptor (AR) expression, high rates of PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combinations of CDK4/6, AR, and PI3K-AKT inhibitors in pre-clinical models of LAR TNBC for future clinical trial design. Methods: MDA-MB-453 and MFM-223 (both Rb-intact/PTEN-intact/PIK3CA-mutant) and CAL-148 (Rb-null/PTEN-null/PIK3CA-mutant) LAR TNBC cell lines were treated with the CDK4/6 inhibitor palbociclib, the PI3Kα inhibitor alpelisib, the AKT inhibitor capivasertib, and the AR antagonist enzalutamide, each alone or in different combinations. Drug sensitivity was determined by coulter counter cell counts in 2D, colony formation, and the CellTiterGlo cell viability assay. The combination index (CI) which defines synergism (CI < 1), additive effect (CI = 1) and antagonism (CI > 1), calculated by the CompuSyn method, was used to evaluate the synergistic effects of drug combinations. Expression of cell cycle and PI3K-AKT downstream signaling molecules was measured by western blot analysis. An androgen response element (ARE) luciferase-based reporter assay was used to evaluate AR transcriptional activity. Results: Rb-intact LAR TNBC cell lines were sensitive to single-agent palbociclib, alpelisib or capivasertib (IC50, ~500 nM). Enzalutamide had minimal growth inhibitory activity (IC50, 15-25 μM). Palbociclib combined with either alpelisib or capivasertib synergistically inhibited proliferation of LAR TNBC cells (CI values, 0.07-0.86). Treatment of Rb-intact LAR TNBC cells with palbociclib monotherapy suppressed Rb phosphorylation and resulted in adaptive phosphorylation/activation of S473 AKT and AKT substrates GSKβ and PRAS40 at 24h. These responses were not observed in Rb-null CAL-148 cells. Palbociclib-induced phosphorylation of AKT substrates as well as induction P-S6 and P-4EBP1 were better suppressed by capivasertib than by alpelisib over a dose range. Addition of the PI3Kβ/δ inhibitor AZD8186 to alpelisib markedly enhanced the inhibition of P-AKT, P-PRAS40 and P-Sin, suggesting inhibition of PI3Kα is inadequate to block the adaptive response to palbociclib in these cells. Mean CI values showed that the combination of palbociclib/capivasertib was more synergistic against LAR TNBC cells compared to palbociclib/alpelisib (mean CI, 0.29 vs. 0.78). ARE reporter activity did not change upon inhibition of PI3K or AKT with alpelisib or capivasertib, respectively. Conclusions: Our results suggest that addition of an AKT inhibitor to palbociclib suppresses the rebound activation of AKT following treatment with the CDK4/6i and is effective in LAR TNBC with wild type Rb. In vivo studies are underway to investigate the antitumor activity of the combination of palbociclib and capivasertib in LAR TNBC xenografts. Citation Format: Gun Min Kim, Kyung-min Lee, Dhivya Sudhan, Albert Lin, Arnaldo Marin, Sumanta Chatterjee, Dan Ye, Vishal Kandagatla, Saurabh Mendiratta, Ariella Hanker, Carlos Arteaga. Combined inhibition of CDK4/6 and AKT is effective in Rb-intact triple-negative breast cancer of the luminal androgen receptor (LAR) subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-07.

Background: CDK4/6 inhibitors (CDK4/6i) have improved survival of patients with advanced estrogen receptor-positive (ER+) breast cancer. However, this benefit is transient as virtually all these tumors eventually develop drug resistance and recur. Clinical studies have reported an association of RB1 loss-of-function genomic alterations with acquired resistance to CDK4/6i. Given the enrichment of RB1 alterations post CDK4/6i treatment, ER+/RB1-deficient breast cancer will become a rising patient population in need of discovery of novel treatment strategies. In this study, we sought to identify actionable vulnerabilities for this refractory breast cancer subtype using a genome-wide CRISPR screen. Methods: RB1 was knocked out in ER+ MCF-7 and T47D breast cancer cells using CRISPR-Cas9; complete gene knockout was confirmed by PCR-based genotyping, Sanger sequencing, and immunoblot analysis. Isogenic RB1 knockout (RBKO) and wild-type (WT) T47D cells were used for the genome-wide CRISPR screen. MAGeCKFlute was used to identify differentially essential genes in T47D RBKO vs WT cells; Gene Ontology (GO) analysis was used to prioritize hits. MCF-7 and T47D RBKO cells were used for validating and studying the function of the identified genes. Results: Knockout of RB1 in MCF-7 and T47D cells increased IC50 of abemaciclib, palbociclib, and ribociclib 10-200 fold compared to WT cells. RNA-seq analysis showed upregulation of E2F target gene expression in RBKO vs WT cells. The CRISPR screen revealed that CCND1 and CDK4 lost their essentiality in T47D RBKO cells, suggesting that loss of RB1 uncouples the CDK4/Cyclin D1 complex from E2F-regulated transcription. GO analysis of the top 50 differentially essential hits of RBKO vs WT cells showed an enrichment of protein arginine methyltransferase activity, primarily PRMT5, which post-translationally mono-methylates and symmetrically di-methylates protein arginine. In agreement with this finding, PRMT5 knockout by three individual sgRNAs resulted in more potent growth inhibition of MCF-7 and T47D RBKO cells than WT cells. Further, transfection of PRMT5 siRNA or treatment with the PRMT5 small molecule inhibitor GSK3326595 - currently in clinical trials - resulted in G1 arrest of MCF-7 and T47D RBKO cells as assayed by propidium iodide staining but did not induce caspase 3/7 or PARP cleavage (apoptosis). RNA-seq of PRMT5 siRNA vs control siRNA in MCF-7 and T47D RBKO cells exhibited significant downregulation of E2F Hallmark gene signature, further suggesting PRMT5 inhibition as a strategy to suppress E2F-regulated gene expression when cells lose Rb. The CRISPR screen also revealed that transcription factors that drive ER signaling, such as FOXA1, GATA3, MYC, SPDEF, and ESR1 (the gene encoding ERα), were commonly essential in both T47D WT and RBKO cells. Estrogen deprivation or treatment with fulvestrant inhibited estrogen responsive element (ERE) luciferase reporter activity, expression of putative E2F target genes, and proliferation of both WT and RBKO cells, suggesting that ER+ cells still rely on ERα irrespective of RB1 status. Treatment of MCF-7 and T47D RBKO cells with fulvestrant and GSK3326595 resulted in more potent growth inhibition than each drug alone, suggesting a novel approach to treat ER+/RB1-deficient breast cancer. We are currently testing the antitumor activity of fulvestrant plus GSK3326595 against RBKO xenografts as well as the requirement of arginine methyltransferase activity associated with PRMT5 for growth of ER+/RB1-deficient breast cancer cells. Conclusion: PRMT5 is essential for proliferation of ER+/RB1-deficient breast cancer cells. Targeting PRMT5 in combination with anti-estrogens is a novel and testable strategy to suppress E2F-regulated cell cycle progression of this CDK4/6 inhibitor-resistant breast cancer subtype. Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Alberto Servetto, Kyung-min Lee, He Zhang, Yunguan Wang, Dan Ye, Sumanta Chatterjee, Dhivya R Sudhan, Hiroaki Akamatsu, Yang Xie, Joshua T Mendell, Ariella B Hanker, Carlos L Arteaga. A genome-wide CRISPR screen identifies PRMT5 as a novel therapeutic target in ER+/RB1-deficient breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-09.

We investigated the tumor microenvironment (TME) in estrogen receptor positive (ER+) primary breast cancers from stage I-III patients treated with estrogen deprivation (ED, induced with letrozole) for 10-21 days. We used primary breast cancer biopsies, collected from 198 patients, to assess tumor-infiltrating lymphocytes (TILs), transcriptomic profiles, and TME composition. Diagnostic biopsy and surgical on-treatment tumor specimens were collected; Ki67, ERα, PR, and HER2 expression were measured using automated quantitative analysis (AQUA). Tumors were categorized as estrogen deprivation-sensitive (ED-S) or -resistant (ED-R) based on the natural log (ln) of the Ki67 score (ln ≤1.0 or ≤2.7% Ki67+ cells vs. ln ≥2.0 or ≥7.4%, respectively) in the on-treatment (surgical) biopsy. Scoring of stromal (s)TILs, using hematoxylin and eosin staining of on-treatment specimens, revealed that sTILs were elevated in ED-R compared to ED-S tumors (p Citation Format: Fabiana Napolitano, Dhivya R. Sudhan, Yunguan Wang, Paula I. González-Ericsson, Luigi Formisano, Kyung-Min Lee, Chang-Ching Lin, María Rosario Chica-Parrado, Hongli Ma, Nathaniel Evans, Alberto Servetto, Saurabh Mendiratta, Spencer Barnes, Yisheng V. Fang, Justin M. Balko, Gordon B. Mills, Marilyne Labrie, Ariella B. Hanker, Carlos L. Arteaga. Immune cells infiltration and activation are higher in breast cancers resistant to antiestrogen therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3445.

RB1 loss-of-function genomic alterations confer resistance to CDK4/6 inhibitors (CDK4/6i) and are enriched post treatment of CDK4/6i in estrogen receptor-positive (ER+) metastatic breast cancer. ER+/Rb-deficient breast cancer is a rising patient population in need of novel therapeutic strategies. Herein, we used a genome-wide CRISPR screen and identified protein arginine methyltransferase 5 (PRMT5) as a molecular vulnerability in this refractory breast cancer subtype. sgRNA-induced depletion of PRMT5 arrested growth of MCF-7 and T47D RB1 knockout (RBKO) cells. PRMT5 catalyzes symmetric dimethylation of arginine (SDMA). In RBKO cells carrying doxycycline-inducible shRNA targeting the 3’UTR of PRMT5, rescue with wild-type but not an enzymatically dead mutant of PRMT5 restored cell growth, supporting that PRMT5 methyltrasferase activity is essential for growth of these cells. Gene set enrichment analysis (GSEA) of RNA-seq data revealed significant downregulation of cell cycle-related Hallmark gene signatures in RBKO cells treated with PRMT5 siRNA versus control siRNA. Both gene silencing and pharmacological blockade of PRMT5 with the small molecule inhibitor pemrametostat impeded G1-to-S cell cycle progression in MCF-7 and T47D RBKO cells and in lung, prostate, and triple-negative breast cancer cells with natural RB1 mutations or deletions, suggesting that PRMT5 inhibition can block the G1-to-S transition even in the absence of Rb. To identify the protein interactome of PRMT5 and the mechanism by which it promotes cell cycle progression in Rb-deficient cells, we performed proteomics analysis of Co-IP mass spectrometry and an SDMA post-translational modification scan and pinpointed FUS (fused in sarcoma) as a putative downstream effector of PRMT5. FUS is known to regulate RNA polymerase II (Pol II)-mediated transcription. Inhibition of PRMT5 with pemrametostat significantly reduced SDMA levels on FUS and dissociated FUS from Pol II as evidenced by FUS Co-IP and immunoblot analysis. ChIP-seq analysis revealed that treatment of RBKO cells with pemrametostat derepressed phosphorylation of Ser2 in the C-terminus of Pol II at transcription start sites (TSS) of genes involved in cell cycle progression. In accordance with the abnormal accumulation of pSer2 Pol II at TSS, pemrametostat treatment also resulted in an increased Pol II pausing index and an enrichment of intron retention splicing variants. Finally, therapeutic inhibition of PRMT5 with pemrametostat synergized with fulvestrant (a selective ER degrader) against growth of ER+/Rb-deficient breast cancer cell line- and patient-derived xenografts in mice, suggesting this combination as a novel therapeutic strategy for ER+/Rb-deficient metastatic breast cancers. Citation Format: Chang-Ching Lin, Tsung-Cheng Chang, Yunguan Wang, Yanfeng Zhang, Andrew Lemoff, Yisheng V. Fang, He Zhang, Dan Ye, Isabel Soria-Bretones, Alberto Servetto, Kyung-min Lee, Xuemei Luo, Joseph J. Otto, Hiroaki Akamatsu, David W. Cescon, Lin Xu, Yang Xie, Joshua T. Mendell, Ariella B. Hanker, Carlos L. Arteaga. PRMT5 is an actionable target in CDK4/6 inhibitor-resistant ER+/Rb-deficient breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3934.

Anticancer chemotherapy is an essential part of cancer treatment, but the emergence of resistance due to continued drug usage remains a major hurdle. Among the multiple mechanisms behind the acquisition of drug resistance, metabolic plasticity is receiving increasing attention. Recent proteomic and transcriptomic studies identified metabolic enzymes as potential targets for overcoming the resistance. Yet, these studies mainly focused on differentially expressed genes and overlooked the complexity of the metabolic network, especially its ability to reorganize the metabolic state in response to gene downregulation. Here, we employ the genome-scale metabolic model (GEM) to simulate the metabolic flow and identify candidates that can act as a metabolic switch for drug resistance. Using adriamycin- and paclitaxel-resistant MCF-7 breast cancer cells as model systems, we perform proteomics analysis followed by GEM construction to analyze the differential metabolic flow in the resistant cells. Moreover, we perform GEM simulations after individually knocking out every metabolic enzyme and identify a metabolic switch whose knockout restores the metabolic state of drug-sensitive MCF-7 cells. Notably, downregulating the candidate metabolic switch predicted by our model results in sensitization to the drug. We further show that the cotreatment of adriamycin or paclitaxel with a small chemical inhibitor of the metabolic switch synergistically induces cell death in drug-resistant cells. Collectively, our study demonstrates the application of GEM simulation and subsequent modulation of metabolic state as a strategy to restore drug sensitivity to overcome drug-resistant cancer. Citation Format: JinA Lim, Hae Deok Jung, Kyung-min Lee, Moonhyeon Jeon, Han Suk Ryu, Hyun Uk Kim, Yoosik Kim. Genome-scale metabolic modeling reveals a metabolic switch that restores sensitivity to anticancer chemotherapy in drug-resistant breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1737.

Background: FGFR1 amplification occurs in ~ 15% of estrogen receptor-positive (ER+) breast cancers. In these tumors, nuclear FGFR1 has been shown to interact with DNA, but its engagement in transcription regulation remains unclear. Thus, we investigated the mechanisms underpinning the genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Methods: FGFR1-ChIP-Seq was performed in CAMA1 ER+/FGFR1-amplified human breast cancer cells to identify genomic distribution of FGFR1. IP with FLAG antibody followed by Mass Spectrometry (MS) was carried out on nuclear plus chromatin fractions of CAMA1 cells overexpressing 3XFLAG-FGFR1 to uncover the nuclear FGFR1 interactome. Results: FGFR1-ChIP-Seq detected 4408 peaks in CAMA1 cells cultured in estrogen-free conditions, with marked enrichment of GC-rich consensus motifs. 67% of peaks were enriched at promoter regions. ChIP-PCR confirmed FGFR1 binding to several genomic loci in ER+/FGFR1-amplified cell lines (CAMA1 and MDA-MB-134) and PDX (HCI-011). Further, MS uncovered RNA Polymerase II subunits among the top nuclear FGFR1 interacting proteins. FGFR1 mainly bound Pol II phosphorylated on Ser5 (Pol II S5P), a marker of transcription initiation, in CAMA1, MDA-MB-134 and HCI-011 cell extracts. Pol II S5P-ChIP-Seq revealed that 65% (2867/4408) of FGFR1 peaks were shared with Pol II S5P in CAMA1 cells. Also, ChIP-Seq revealed that 95% of FGFR1 peaks overlapped with both H3K4me3 and H3K27ac, markers of active transcription. Consistent with these results, RNA-Seq of CAMA1 cells showed that expression of FGFR1-bound genes was markedly higher than non FGFR1-bound genes (p1), whose expression is likely regulated by nuclear FGFR1. A high signature score correlated with resistance to letrozole (p Conclusions: These findings support a prominent role for FGFR1 in the transcriptional machinery of breast cancer cells. Whether this transcriptional action is causal to antiestrogen resistance in ER+/FGFR1-amplified breast cancer is currently under investigation. Citation Format: Alberto Servetto, Rahul Kollipara, Luigi Formisano, Kyung-min Lee, Albert Lin, Dhivya R. Sudhan, Ariella B. Hanker, Sumanta Chatterjee, Hiroaki Akamatsu, Arnaldo Marin, Saurabh Mendiratta, Ralf Kittler, Carlos L. Arteaga. FGFR1 associates with gene promoters and regulates transcription in ER+/FGFR1-amplified breast cancer: Implications for endocrine resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1304.

Background/Purpose: Next-generation sequencing (NGS) is being incorporated rapidly into clinical practice. Fine-needle aspiration biopsy (FNAB) specimens have been used feasibly in molecular analysis including direct sequencing and microarrays. They are readily available and enriched in malignant cells, thus providing opportunities for genomic analysis for more clinical samples. In this study, we assessed the feasibility and sensitivity of FNAB for the detection of somatic mutations by NGS compared to bulk tissue. Methods: Bulk tissue and FNAB was sampled via skin superficial to the palpable tumor from surgically resected breast cancer specimen. DNA was extracted from the bulk tissues and FNAB samples obtained from twelve patients. Somatic mutations detected from whole exome sequencing (WES) by next-generation sequencing (NGS) (HiSeq 2500, Illumina) were analyzed for corresponding pairs of bulk tissue and FNAB. Verification of somatic mutations detected exclusively from FNAB and known to be clinically relevant to breast cancer was carried out by Sanger sequencing. Invasive tumor percentages of bulk tissues were evaluated using hematoxylin and eosin (H&E)-stained sections. Results: Average depth of coverage were 158.8x and 158.3x for bulk tissue and FNAB, respectively. Number of detected somatic mutations ranged from 2 to 153 (median 18.5) and 19 to 210 (median 39.5) for bulk tissue and FNAB, respectively. Ten specimens had more mutations detected exclusively from FNAB than from bulk tissue. Allele fractions plotting of corresponding pairs of bulk tissue and FNAB showed good, intermediate, and poor correlation in five, two, and five specimens, respectively. H&E-stained sections of bulk tissue from the five specimens with good correlation contained an invasive tumor percentage of 45 to 98%, whereas those from five specimens with poor correlation contained 0 to 25%. Three of the poorly correlated bulk tissues were judged to have 0% of invasive tumor. Among mutations detected exclusively from FNAB, eighteen different genes of interest in 22 foci were evaluated for both FNAB and corresponding bulk tissue by Sanger sequencing. In the results, three mutations (PIK3CA, TP53 x2) were verified in FNAB samples but not in the bulk tissue. Conclusion: WES was successfully carried out in all pairs of bulk tissue and FNAB from twelve breast cancer patients. In samples with high tumor content somatic mutation profiles showed high correlation between the two samples whereas samples with low tumor content failed to show correlation. The failure was mostly due to the scarcity of tumor portions in the bulk tissues, indicating that FNAB more reliably retained malignant tumor portion. This study suggests that FNAB is an easy and feasible method, and furthermore, provides a more reliable specimen for NGS analysis where somatic mutations could be identified for potential prognostic or therapeutic benefits. Citation Format: Han-Byoel Lee, Jisun Kim, Kyung-Min Lee, Je-Gun Joung, Hae-ock Lee, Min Kyoon Kim, Eunshin Lee, Jongjin Kim, Tae-Kyung Yoo, Yun-Gyoung Kim, Young Joon Kang, Han Suk Ryu, In-Ae Park, Hyeong-Gon Moon, Dong-Young Noh, Woong-Yang Park, Wonshik Han. Feasibility and sensitivity of fine-needle aspiration biopsies for the detection of somatic mutations using next-generation sequencing in breast cancer [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 P2-05-07.

Background: FGFR1 amplification occurs in about 15% of estrogen receptor-positive (ER+) breast cancers and is associated with resistance to endocrine therapy. In these tumors, nuclear FGFR1 has been shown to interact with ERα. In addition, FGFR1 has been demonstrated to alter gene expression through binding to chromatin. However, the mechanisms underpinning nuclear FGFR1-mediated gene transcription remain unclear. Thus, we sought to elucidate the genomic and non-genomic role of FGFR1 in ER+/FGFR1-amplified breast cancer. Methods: FGFR1 ChIP-Seq and ERα ChIP-Seq were performed on CAMA1 ER+/FGFR1-amplified breast cancer cells. ChIP-qPCR was employed to quantify DNA binding events. For ChIP-Seq, immunoblot, RT-qPCR, Estrogen Response Element (ERE) luciferase reporter and growth assays, CAMA1 cells were plated in estrogen-free media for 24 hours and then stimulated with 100 ng/ml FGF3 or 1 nM β-Estradiol. Results: FGFR1 ChIP-Seq detected 2211 DNA binding sites in CAMA1 cells cultured in estrogen-free conditions. Gene Set Enrichment Analysis (GSEA) revealed that the TNFα signaling via NF-KB, MYC targets, G2M checkpoints, ERE early and ERE late response genes (all FDR Conclusions: These findings suggest a FGFR1 kinase-dependent role on ER-mediated transcription in ER+/FGFR1-amplified breast cancer cells. We are currently performing mass spectrometry analysis to identify binding partners of nuclear FGFR1 that mediate its transcriptional function. Citation Format: Alberto Servetto, Luigi Formisano, Rahul Kollipara, Dhivya R. Sudhan, Kyung-min Lee, Sumanta Chatterjee, Ariella B. Hanker, Saurabh Mendiratta, Ralf Kittler, Carlos L. Arteaga. FGFR1 signaling modulates estrogen-independent ER transcriptional activity in ER+/FGFR1-amplified breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4402.

Purpose: Although recent studies have suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) is important in the progression of various malignancies, its role in gallbladder carcinoma (GBC) remains unknown. We investigated CXCL12 expression in GBC and its biologic and prognostic role in GBC tumorigenesis. Experimental Design: We examined CXCL12 expression in tumor specimens from 72 patients with GBC by immunohistochemistry and analyzed the correlation between CXCL12 expression and clinicopathologic factors or survival. The functional significance of CXCL12 expression was investigated by CXCL12 treatment and suppression of CXCR4, a major receptor of CXCL12, as well as by CXCL12 overexpression in in vitro and in vivo studies. Results: CXCL12 was differentially expressed in GBC tissues. CXCL12 expression was significantly associated with a high histologic grade (P = 0.042) and nodal metastasis (P = 0.015). Multivariate analyses showed that CXCL12 expression (HR, 8.675; P = 0.014) was an independent risk factor for patient survival. CXCL12 significantly increased anchorage-dependent and -independent growth, migration, invasion, adhesiveness, and survival of GBC cells in vitro, and these effects were dependent on CXCR4. Consistent with these results, overexpression of CXCL12 significantly promoted GBC tumorigenicity in a xenograft model. Conclusions: Our results indicate that GBC cells express both CXCL12 and its receptor CXCR4, and CXCL12 may have a role in GBC progression through an autocrine mechanism. In addition, CXCL12 is a novel independent poor prognostic factor in patients with GBCs. Thus, targeting CXCL12 and CXCR4 may provide a novel therapeutic strategy for GBC treatment. Clin Cancer Res; 18(12); 3270–80. ©2012 AACR.

Many somatic mutations, structural alterations, and gene expression changes are causally implicated in oncogenesis and tumor progression, and as a result, affect clinical outcome. Although majority of breast cancer patients have benefits from therapeutics targeting tumor biology still many patients suffer from disease recurrence and metastasis. To find novel therapeutic target in breast cancer, here we examine the both whole exome and whole transcriptome of fresh-frozen primary breast cancer tissues from 120 patients whose clinical, pathological, and survival data are available. We identified 11,684 putative somatic mutations in 7,373 genes. We hypothesized that therapeutic target has to have recurrent mutation and gene expression at least more than average expression. With a cut-off of 3 or more mutations in each gene, 1,116 genes were selected. After the filtering of Volume>0.3, 696 genes were selected. Finally, 55 genes were selected which are druggable or potentially druggable using drug database. Interestingly, 18 of the 55 were genes involved in metabolism. Among them we further focused PSMD2 and interrogated their oncogenic roles, a subunit of the 19S regulatory complex of proteasomes, as a constituent of a signature is a candidate driven factor in breast cancer. PSMD2 is required for proliferation both in vitro and in vivo, as shRNA-mediated PSMD2 silencing attenuated tumor growth. Further, PMSD2 is sufficient to induce stem cell characteristics, as forced PSMD2 expression facilitated mammosphere formation and increased the CD44+ breast CSC-like population. Mechanistically, PSMD2 inhibition were associated with changes in epithelial-mesenchymal transition (EMT) phenotype. Knockdown of PSMD2 induced dramatic morphological changes, the typical spindle-like appearance cells were replaced with the round shape, like epithelial morphology. Moreover, treatment of TGF-β1, the main inducer of EMT, lead to increased expression of PSMD2. PSMD2 silencing up-regulated E-cadherin and down-regulated expression of vimentin, N-cadherin and snail, mesenchymal markers. Clinically, PSMD2 expression was elevated in basal-like TNBC (triple negative breast cancer), correlated with poor prognosis, insilico analysis on a dataset comprising over 2413 microarrays of the web application bc-GenExMiner. Altogether, our findings suggest that PSMD2 may be a good molecular target candidate and PSMD2 inhibitor may kills breast cancer stem cells and ameliorates poor prognosis of breast cancer. Citation Format: Kyung-Min Lee, Dong-Young Noh, Wonshik Han. Integrated eoxme and transcriptome sequencing identifies a novel candidate for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4786. doi:10.1158/1538-7445.AM2017-4786

Introduction: Copy number alteration of genome is common in breast cancer and tend to have more driver role than single point mutations. Traditionally, genome-wide analysis of DNA copy number changes were done by array CGH or SNP array method. Here, we did DNA whole exome sequencing (WES) and RNA-seq using Next Generation Sequencing (NGS) technology to find common genes or chromosomal regions of which DNA copy was highly amplified and at the same time RNA expression was also upregulated. Materials and Method: RNA and DNA were extracted fromfresh frozen tissues of 93 breast cancer patients. WES and RNA-seq were done using NGS technology (Illumina HiSeq 2000). As a control, normal DNA from all matched patients were also sequenced. GATK was used to gain mean depth and coverage data for targeted regions.CNVs were calculated with ExomeCNV, a statistical method to detect somatic CNVs using depth-of-coverage information from mapped short sequence reads.To estimate expression levels, the relative transcript abundances were measured in FPKM using Cufflinks. Results and Discussion: DNA of 1,737 genes were highly amplified (log R>1.0) in two or more samples. The two most commonly amplified chromosomes were chromosome 8 and 17. We applied a cut-off for higher gene expression as relative FPKM >1.5. ERBB2 amplifications and high expression were most common (21.5%) of all genes and it was in agreement withHER-2 IHC and FISH result. Among previously reported amplified genes, FGFR1 (5.4%) and PVT1 (8.6%) in chromosome 8, CCND1, PAK1 (3.2%) and EMSY (4.3%) in chromosome 11, CCNE1 (4.3%) in chromosome 19 were also identified in this study. IGF1R high amplification and expression was found in two samples, and ESR1, MDM2, KIT was found in only one sample each. We found uncommon but novel and recurrent highly amplified and expressed genes: CLK4 in 5q (3.2%), AHI/MYB in 6q (3.2%), MMP7(2.2%) andMALAT1 in 11q (1.1%), and NEK8 in 17q (4.3%) We designed FISH probe for this 5 new genes and confirmed the high amplifications in each sample with FISH. Functional study of these genes will be followed for the driver role of these genes in carcinogenesis and progression of breast cancer cells. Citation Format: Eunshin Lee, Woosung Lim, Kyung-Min Lee, Tae-kyung Yoo, Jongjin Kim, Han-Byoel Lee, Yun-Gyoung Kim, YoungJoon Kang, Min Kyoon Kim, Hyeong-Gon Moon, Dong-Young Noh, Wonshik Han Han. Discovery of novel amplified genes in primary breast cancer with copy number and gene expression analysis of whole exome and transcriptome sequencing data [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 P2-03-18.

Intratumoral heterogeneity has emerged as a problem to be resolved for precise diagnosis and effective treatment of cancer. Breast cancer is one of the most heterogeneous cancer in its molecular subtypes and its intratumoral genomic diversity has been demonstrated as well. Single-cell sequencing approach would reveal heterogeneity in gene expression signatures and the common gene expression characteristics across tumor cells within a tumor. In this study, we isolated live individual cells and amplified cDNAs using C1™Single-cell Auto Prep System (Fluidigm) then performed RNA sequencing using HiSeq2500 System (Illumina). Single-cell transcriptomes were acquired from four primary tumor (ER+/HER2-, ER-/HER2+, ER+/HER2+ and triple negative breast cancer) and two matched lymph node metastases (ER+/HER2+ and triple negative breast cancer). Because the tumor tissue isolates may include stromal and immune cells in the tumor microenvironment, we defined ‘epithelial cell adhesion molecule signature’ to filter out non-tumor populations (Poster# presented by Woosung Chung et. al) After the removal of non-tumor cells, collected tumor single-cell transcriptomes largely recapitulated bulk tumor-expression profiles. The ER+/HER2- tumor cells showed highly activated estrogen receptor-associated pathways. Although both ER-/HER2+ and ER+/HER2+ tumors had the HER2 gene amplification and overexpression, HER2/HER3 pathway is activated only in ER-/HER2+ tumor cells. The association of gene expression for cancer stem cells and chemoresistance, such as IL8 and CXCL1, with HER2/HER3 signaling pathway activation was prominent. By contrast, the ER+/HER2+ tumor cells had highly activated estrogen receptor signaling, presenting them as the luminal type. The triple negative breast cancer cells highly expressed adhesion molecules and proteases suggesting their invasive nature, yet demonstrated extensive gene expression heterogeneity. Collectively these data revealed the power of single cell gene expression profiling in the precise characterization of tumor cell properties. The explicit gene expression signatures for patient specific tumors may be defined as subtype-specific signatures and helpful to unmask targets for molecular directed therapies. Citation Format: Hye Hyeon Eum, Hae-Ock Lee, Woosung Chung, Kyu-Tae Kim, Kyung-Min Lee, Arum Jo, WonShik Han, Woong-Yang Park. Single-cell RNA sequencing presents explicit expression signatures in primary breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 161.

In an attempt to identify biomarkers and/or therapeutic targets for malignant breast cancer, the present study showed a comparative proteome profiling of invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein DS-20, a crucial regulator of H-Ras activation, as a potential marker for invasive breast cancer. DS-20 (amino acid residues 1-38) interacted with H-Ras (residues 166-189) in lipid rafts and their interaction was important for H-Ras activation. DS-20 knockdown inhibited H-Ras activation and invasion in Hs578T triple-negative breast cancer cells (TNBC) and T24 bladder carcinoma cells in which H-Ras is endogenously activated, suggesting a crucial role of DS-20 in the invasive program which relies on the activation of H-Ras. We further showed that DS-20 was required for epidermal growth factor-induced H-Ras activation, but not that of N-Ras, in MDA-MB-231 TNBC cells. Intravasation of MDA-MB-231 cells treated with shRNA DS-20 in the chick chorioallantoic membrane model was markedly reduced, indicating that DS-20 is required for an invasive capacity in vivo. In a xenograft mice tumor model, DS-20 was essential for in vivo tumor aggressiveness of Hs578T cells, suggesting DS-20 as a potential target for the treatment of breast cancer. Using human breast cancer samples, we provide clinical evidence for the tumorigenic potential of DS-20 and its association with H-Ras. Taken together, our findings provide a new insight into the molecular basis of Ras isoform-specific interplay with plasma membrane leading to cell invasion. Citation Format: Hae-Young Yong, Eun-Sook Kim, Minsoo Koh, Hwajin Son, You Rim Jeon, Jin-Sun Hwang, Myeong-Ok Kim, Yujin Cha, Wahn Soo Choi, Dong-Young Noh, Kyung-Min Lee, Ki-Bum Kim, Jae-Seon Lee, Hyung Joon Kim, Hong-Hee Kim, Eun Joo Kim, So Yeon Park, Hyeong-Reh Choi Kim, Aree Moon. Identification of a lipid raft protein that is required for H-Ras activation and breast cancer aggressiveness. [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 5263. doi:10.1158/1538-7445.AM2014-5263

Breast cancer cells with mutations in breast cancer-associated 1 (BRCA1) or BRCA2 have defects in the homologous recombination (HR) DNA repair pathway and this confers sensitivity to inhibitors of poly (ADP) ribose polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. The transcription factor Forkhead box M1 (FoxM1) is a key regulator of cell proliferation and is overexpressed in many forms of primary cancers, leading to uncontrolled cell division and genomic instability. FoxM1 is implicated in genotoxic drug resistance but its role and mechanism of action remain unclear. Our results revealed that triple-negative breast cancer (TNBC) cell lines depleted in FoxM1 levels by small interfering RNA transfection display increase DNA damage, as evidence by activation of checkpoint kinase 2 (Chk2) and phosphorylation of histone H2AX. These results shows a role for FoxM1 in the transcriptional response during DNA damage/checkpoint signaling and we hypothesized that FoxM1 inhibition would result in HR impairment and subsequent sensitization to PARP inhibitor. We found that knockdown of FoxM1 led to sensitization to AZD2281, PARP inhibitor, in MDA-MB-231 (BRCA-wild-type) and MDA-MB-436 (BRCA-mutated) TNBC cells. Combined PARP and FoxM1 inhibition had a synergistic anti-proliferative effect on BRCA1-defective and BRCA-proficient TNBC cell lines in vitro. Furthermore, the combination treatment of AZD2281 and thiostrepton (FoxM1 inhibitor) increased DNA repair-related gene expression such as H2AX, chk1, chk2 and p53 and downregulated BRCA2 expression in TNBC cell lines. Here, we show that depletion or inhibition of FoxM1 compromises the ability of cells to repair DNA by HR and sensitization to PARP inhibition in TNBCs without BRCA mutations, providing a rationale to combine FoxM1 and PARP inhibitors. Therefore, this combination may be effective for treating both BRCA1-defective and BRCA-proficient TNBC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C76. Citation Format: Jun Woo Lee, Kyung-Min Lee, Wonshik Han. FoxM1 inhibition sensitized BRCA-proficient triple-negative breast cancer to PARP inhibition. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C76.

Tumor metastasis is multiple step pathological process involved in the final phase of tumor development. During this process, tumor cells obtain invasiveness and metastatic potential. Periostin, a mesenchyme-specific gene, has emerged as critical contributor of tumor metastasis and angiogenesis. Clinical evidence has supported that periostin are highly expressed in several cancers including breast, where it is associated with a poor outcome. Thus, periostin is a potential therapeutic target for the regulation of breast cancer growth and metastasis. In this study, we generated and characterized a DNA aptamer directed against human periostin (PNDAs) through utilization of Advanced-SELEX process. Aptamers have been selected that bind to periostin with nanomolar binding affinity (Kd: 1nM) and specificity. Furthermore, PNDA specifically binds to periostin which is expressed in breast cancer tissues and serum. In addition, PNDA treatment led to a reduction of integrin-mediated breast cancer cell migration and adhesion by 50%. Also, PNDA can significantly decrease activation of various components of integrin signal transduction pathways and their downstream effector molecules. Together, our data suggest that DNA aptamer targeting periostin may have potential as an agent to inhibit breast cancer growth and metastasis. 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 5190. doi:1538-7445.AM2012-5190