Suk-Young Yoo

Epithelial–Mesenchymal Transition Predicts Polo-Like Kinase 1 Inhibitor–Mediated Apoptosis in Non–Small Cell Lung Cancer

Purpose: To identify new therapeutic targets for non–small cell lung cancer (NSCLC), we systematically searched two cancer cell line databases for sensitivity data on a broad range of drugs. We identified polo-like kinase 1 (PLK1) as the most promising target for further investigation based on a subset of sensitive NSCLC cell lines and inhibitors that were in advanced clinical development. Experimental Design: To identify potential biomarkers of response of NSCLC to PLK1 inhibition and mechanisms of PLK1 inhibitor–induced apoptosis, integrated analysis of gene and protein expression, gene mutations, and drug sensitivity was performed using three PLK1 inhibitors (volasertib, BI2536, and GSK461364) with a large panel of NSCLC cell lines. Results: The NSCLC cell lines had different sensitivities to PLK1 inhibition, with a minority demonstrating sensitivity to all three inhibitors. PLK1 inhibition led to G2–M arrest, but only treatment-sensitive cell lines underwent substantial apoptosis following PLK1 inhibition. NSCLC lines with high epithelial–mesenchymal transition (EMT) gene signature scores (mesenchymal cell lines) were more sensitive to PLK1 inhibition than epithelial lines (P < 0.02). Likewise, proteomic profiling demonstrated that E-cadherin expression was higher in the resistant cell lines than in the sensitive ones (P < 0.01). Induction of an epithelial phenotype by expression of the miRNA miR-200 increased cellular resistance to PLK1 inhibition. Also, KRAS mutation and alterations in the tight-junction, ErbB, and Rho signaling pathways correlated with drug response of NSCLC. Conclusions: In this first reported large-scale integrated analysis of PLK1 inhibitor sensitivity, we demonstrated that EMT leads to PLK1 inhibition sensitivity of NSCLC cells. Our findings have important clinical implications for mesenchymal NSCLC, a significant subtype of the disease that is associated with resistance to currently approved targeted therapies. Clin Cancer Res; 22(7); 1674–86. ©2015 AACR.

Biphasic components of sarcomatoid clear cell renal cell carcinomas are molecularly similar to each other, but distinct from, non‐sarcomatoid renal carcinomas

Sarcomatoid transformation, wherein an epithelioid carcinomatous tumour component coexists with a sarcomatoid histology, is a predictor of poor prognosis in clear cell renal cell carcinoma. Our understanding of sarcomatoid change has been hindered by the lack of molecular examination. Thus, we sought to characterize molecularly the biphasic epithelioid and sarcomatoid components of sarcomatoid clear cell renal cell carcinoma and compare them to non‐sarcomatoid clear cell renal cell carcinoma. We examined the transcriptome of the epithelioid and sarcomatoid components of advanced stage sarcomatoid clear cell renal cell carcinoma (n=43) and non‐sarcomatoid clear cell renal cell carcinoma (n=37) from independent discovery and validation cohorts using the cDNA microarray and RNA‐seq platforms. We analyzed DNA copy number profiles, generated using SNP arrays, from patients with sarcomatoid clear cell renal cell carcinoma (n=10) and advanced non‐sarcomatoid clear cell renal cell carcinoma (n=155). The epithelioid and sarcomatoid components of sarcomatoid clear cell renal cell carcinoma had similar gene expression and DNA copy number signatures that were, however, distinct from those of high‐grade, high‐stage non‐sarcomatoid clear cell renal cell carcinoma. Prognostic clear cell renal cell carcinoma gene expression profiles were shared by the biphasic components of sarcomatoid clear cell renal cell carcinoma and the sarcomatoid component showed a partial epithelial‐to‐mesenchymal transition signature. Our genome‐scale microarray‐based transcript data were validated in an independent set of sarcomatoid and non‐sarcomatoid clear cell renal cell carcinomas using RNA‐seq. Sarcomatoid clear cell renal cell carcinoma is molecularly distinct from non‐sarcomatoid clear cell renal cell carcinoma, with its genetic programming largely shared by its biphasic morphological components. These data explain why a low percentage of sarcomatoid histology augurs a poor prognosis; suggest the need to modify the pathological grading system and introduce the potential for candidate biomarkers to detect sarcomatoid change preoperatively without specifically sampling the histological sarcomatoid component.

Abstract PR-09: Low-fat diet reduces NF-κB regulated inflammatory cytokines and angiogenic factors in plasma of men with prostate cancer

Purpose: Diet and certain dietary supplements are postulated to influence the development and progression of prostate cancer. Angiogenesis and inflammation are central to tumor growth and progression, but the effect of diet on these processes remains uncertain. Procedure: We examined changes in 50 plasma cytokines and angiogenic factors (CAFs) in 145 men with prostate cancer enrolled in a pre-operative, randomized controlled phase-II trial with four arms: control (usual diet); low-fat (LF) diet; flaxseed-supplemented (FS) diet; and flaxseed-supplemented, low-fat diet. The mean duration of dietary intervention was 30 days. While two CAFS changed significantly in the FS arm (eotaxin and IL-16), 11 CAFs, including proangiogenic factors (vascular endothelial growth factor [VEGF], stromal-cell derived-1α) and myeloid factors (granulocyte-colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage-colony-stimulating factor) changed significantly in the LF arm compared to controls with virtually all of these factors decreasing. Results: Significant decreases in body mass index occurred in the LF arms and correlated with VEGF decreases (P Conclusions: Low-fat diets may reduce levels of specific inflammatory cytokines and angiogenic factors via the NF-κB mediated pathway. Citation Information: Cancer Prev Res 2010;3(12 Suppl):PR-09.

Recognition of Recurrent Protein Expression Patterns in Pediatric Acute Myeloid Leukemia Identified New Therapeutic Targets

Heterogeneity in the genetic landscape of pediatric acute myeloid leukemia (AML) makes personalized medicine challenging. As genetic events are mediated by the expression and function of proteins, recognition of recurrent protein patterns could enable classification of pediatric AML patients and could reveal crucial protein dependencies. This could help to rationally select combinations of therapeutic targets. To determine whether protein expression levels could be clustered into functionally relevant groups, custom reverse-phase protein arrays were performed on pediatric AML (n = 95) and CD34+ normal bone marrow (n = 10) clinical specimens using 194 validated antibodies. To analyze proteins in the context of other proteins, all proteins were assembled into 31 protein functional groups (PFG). For each PFG, an optimal number of protein clusters was defined that represented distinct transition states. Block clustering analysis revealed strong correlations between various protein clusters and identified the existence of 12 protein constellations stratifying patients into 8 protein signatures. Signatures were correlated with therapeutic outcome, as well as certain laboratory and demographic characteristics. Comparison of acute lymphoblastic leukemia specimens from the same array and AML pediatric patient specimens demonstrated disease-specific signatures, but also identified the existence of shared constellations, suggesting joint protein deregulation between the diseases. Implication: Recognition of altered proteins in particular signatures suggests rational combinations of targets that could facilitate stratified targeted therapy. Mol Cancer Res; 16(8); 1275–86. ©2018 AACR. See related article by Hoff et al., p. 1263

Abstract 1597: Transcriptomic architecture of the field of cancerization in the adjacent normal-appearing airway: Early mechanisms in lung carcinogenesis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Increasing our understanding of early events in the pathogenesis of lung cancer is crucial for identification of new targets for prevention and treatment of this malignancy. Earlier work has shown that seemingly normal cells adjacent to the tumor carry specific molecular alterations that are characteristic of the tumor itself suggestive of a field of cancerization. By sampling and studying normal-appearing tissue, the molecular field of cancerization provides biological insights into early phases in cancer development. In this study, we sought to characterize molecular field effects in the normal-appearing airway that are most representative of the nearby lung tumor, and thus, are most likely to denote early events in lung carcinogenesis. To achieve this, we performed genome-wide expression profiling of resected field cancerization specimens (n=20 patients) comprised of matched early-stage non-small cell lung cancers (NSCLCs), cytologically normal airways with varying spatial distance from the tumors and distant (relative to location of tumors) normal lung tissues (n=194 samples). Using ordinal logistic regression, we identified 422 genes that were progressively modulated in expression in normal-appearing airways by spatial distance from tumors. Notably, when examined in paired NSCLC and normal lung tissues, these genes were found to recapitulate tumor expression profiles. We then sought to examine the role of lysosomal protein transmembrane 4 beta (LAPTM4B), a putative oncogene that was found to be up-regulated in airways by shorter spatial distance from tumors, in lung oncogenesis. LAPTM4B was significantly elevated in NSCLC tissues compared to paired distant normal lung and was predictive of poor survival in lung adenocarcinoma. Moreover, LAPTM4B promoted anchorage-dependent and -independent lung cancer cell growth and was crucial for cellular survival and the autophagy response under nutrient- and serum-deprived conditions. In addition, pathways analysis of a LAPTM4B-dependent gene expression profile revealed decreased activation of the canonical nuclear factor erythroid 2-like 2 (NRF2)-mediated pathway following LAPTM4B knockdown. Further, we found that LAPTM4B augmented the expression and nuclear translocation of the NRF2 transcription factor following serum deprivation pointing to the probable role of the novel LAPTM4B/NRF2 signaling axis in promoting lung cancer cell survival. All in all, our study points to molecular field of cancerization profiles in the normal-appearing airway that highly signify the nearby lung tumor and comprise early mechanisms (e.g. LAPTM4B) in lung carcinogenesis. Citation Format: Yuho Maki, Junya Fujimoto, Suk-Young Yoo, Melinda Garcia, Adam Gower, Li Shen, Chi-Wan Chow, Carmen Behrens, Neda Kalhor, Cesar Moran, Jing Wang, Avrum Spira, Kevin R. Coombes, Ignacio I. Wistuba, Humam Kadara. Transcriptomic architecture of the field of cancerization in the adjacent normal-appearing airway: Early mechanisms in lung carcinogenesis. [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 1597. doi:10.1158/1538-7445.AM2014-1597

Abstract 2367: Transcriptomic architecture of the airway field cancerization in early-stage non-small cell lung cancer .

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Earlier work has identified in lung cancer a field cancerization (FC) phenomenon in which tumors and adjacent normal appearing tissues share specific molecular abnormalities (e.g., loss of heterozygosity) that may be highly pertinent to cancer pathogenesis. We sought to characterize the global molecular airway FC adjacent to early-stage non-small cell lung cancer (NSCLC) in an attempt to unravel profiles that may help to explain the development of the disease. We performed whole-transcript expression profiling of a set of resected early-stage NSCLC specimens (n=20 patients) with matched histologically normal airways of varying distance from the tumor and paired uninvolved normal lung tissue (n=194 samples). Using linear mixed-effects models, we derived FC profiles signifying genes concordantly differentially expressed between tumors and airways compared to normal lung tissues. Gene set enrichment analysis demonstrated that a subset of the genes (n=299) was significantly and congruently modulated between large airways of smokers with and without lung cancer. We then questioned whether the airway FC exhibits site from tumor-dependent expression patterns. Ordinal regression analysis identified airway profiles (n=422 genes) that were significantly progressively expressed by distance from tumors and topologically organized into canonical cancer-associated pathways, such as eukaryotic initiation factor, p70S6K kinase, polo-like kinase and mammalian target of rapamycin signaling (all p<0.001). In addition, the site-dependent airway profiles recapitulated NSCLC expression patterns and were concordantly modulated between tumors and uninvolved normal lung tissues pinpointing their probable roles in lung cancer pathogenesis. Quantitative real-time PCR (QRTPCR) analysis confirmed the differential expression of FC markers selected by both pathways analysis and statistical criteria. Notably, lysosome associated protein transmembrane 4 beta (LAPTM4B), a putative oncogene with no known role in lung carcinogenesis, was among the top 5 site-dependent FC markers and was significantly elevated in NSCLC and immortalized bronchial epithelial cell lines compared to normal cells. Furthermore, transient or stable knockdown of LAPTM4B by RNA interference decreased NSCLC cell growth as well as anchorage-dependent and -independent colony formation. In conclusion, our efforts in understanding the adjacent molecular FC in NSCLC unraveled airway profiles that 1) are, in part, relevant to lung cancer detection; 2) are modulated by distance from corresponding tumors; 3) recapitulate NSCLC expression patterns and 4) harbor markers engaged in mediating the lung malignant phenotype. Profiling the adjacent airway FC in conjunction with tumors, may provide additional insights into the molecular pathology of NSCLC. Funded in part by Department of Defense award W81XWH-10-1-1007. Citation Format: Yuho Maki, Junya Fujimoto, Suk-Young Yoo, Adam Gower, Li Shen, Melinda M. Garcia, Mohamed Kabbout, Chi-Wan Chow, Waun Ki Hong, Neda Kalhor, Jing Wang, Cesar Moran, Avrum Spira, Kevin R. Coombes, Ignacio I. Wistuba, Humam Kadara. Transcriptomic architecture of the airway field cancerization in early-stage non-small cell lung cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2367. doi:10.1158/1538-7445.AM2013-2367

Abstract 1205: Circulating micro-RNAs can detect adaptive response to therapy and aggressive subset of prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Circulating markers that inform of the adaptive biology and the clinical virulence of prostate cancer do not exist. Small cell carcinoma of the prostate (SCPC) is an aggressive, androgen receptor (AR)-negative, morphological variant that arises often during the castration-resistant progression of typical adenocarcinoma. Its presence is associated with a poor response to hormonal therapies and predicts for a short survival. Micro-RNAs (miRNAs) regulate a range of processes, including establishment and maintenance of tissue differentiation, and are well-preserved in serum. Based on the observation that SCPC are characterized by a loss of prostate epithelial marker expression (such as AR and HOXB13) and an increase in the expression of proneural transcription factors (such as MYCN and ASCL1) we formulated the hypothesis that the SCPC display a miRNA profile distinct from that of typical AR-driven prostate carcinomas. We profiled the miR extracted from the serum samples of 13 men with biopsy-proven SCPC, and of 9 men with bone-predominant metastatic CRPC. Because SCPC is associated with large tumor burdens, we selected patients with bone-predominant CRPC that had abnormally high alkaline phosphatase levels and PSA > 20ng/mL. 322 miR were detected in at least one of the 22 samples. We identified two miR that were present in 10 and 11 of 13 (77% and 85%) patients with SCPC and 3 and 1 of 9 (33% and 11%) of men with unselected typical bone-predominant CRPC. Both of these miRs have been shown to control multiple genes regulating neuronal differentiation and to induce the conversion of human fibroblasts into neurons, a mechanism which may be shared by prostate adenocarcinoma cells in their transdifferentiation to small cell prostate cancer cells. Moreover, one of them was recently shown to silence AR expression. Therefore we conclude that two circulating miRs may serve to monitor adaptive responses and identify virulent subsets of prostate cancer, thus serving to guide therapy and selection of patients for clinical trials. Validation of these findings is ongoing in a larger cohort of patients. Citation Format: Ana M. Aparicio, Emily Gallichotte, Heather Cheng, Suk-Young Yoo, Sankar Maity, Christopher Logothetis, Muneesh Tewari. Circulating micro-RNAs can detect adaptive response to therapy and aggressive subset of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1205. doi:10.1158/1538-7445.AM2013-1205

Abstract 2607: Genetic variants in the vitamin D pathway and breast cancer disease free survival

Objective. Vitamin D deficiency has been associated with poor outcomes in patients with breast cancer. The objective of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in 7 Vitamin D-related genes and disease free survival (DFS). We hypothesized that vitamin D-related genes may modify breast cancer DFS. Methods. A total of 102 SNPs in Vitamin D pathway genes were explored as predictors of DFS among 1030 stage I/II breast cancer patients treated at MD Anderson Cancer Center between 1985 and 2000. Genotyping was performed using the Illumina GoldenGate array. We applied recursive partitioning tree analysis (RPART) to explore interactions of variables predictive of DFS including vitamin D-related SNPs, age at diagnosis, race, stage, nuclear grade, estrogen (ER) and progesterone (PR) receptor status, chemotherapy, hormone therapy and BMI. Results. Results from partitioning identified chemotherapy as the optimum first split for DFS where patients receiving chemotherapy experienced longer DFS. In patients receiving chemotherapy (n=540), SNP rs1118569 (RXRA) followed by rs10881583 (RXRA) were associated with greater DFS. Patients with two major alleles of rs1118569 had the best outcome, whereas those with two minor alleles of rs10881583 and one or two minor rs1118569 alleles had poorer DFS. In patients who did not receive chemotherapy (n=482), stage I patients had longer DFS than stage II. Further, stage I patients who were homozygous for any allele of rs2248098 (VDR) had longer DFS. Among the stage II patients, those with ER/PR negative tumors had poorer DFS with heterozygous carriers of rs344781 in PLAUR having the worst DFS. Conclusion. While exploratory, we show that chemotherapy, stage, ER and PR status and vitamin D-related pathway polymorphisms but not race/ethnicity modified disease free survival in early stage breast cancer patients. Validation of vitamin D-related pathway genes as genetic determinants of patient outcomes would offer insights on alternative approaches for the secondary prevention of breast cancer recurrence considering clinical pathological and treatment characteristics of the patient population. 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 2607. doi:1538-7445.AM2012-2607

Abstract 4000: Trastuzumab targeting of HER2 upregulates miRNA-194 and downregulates profilin 2 and DNMT3A in HER2 positive breast cancer

Trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of oncoprotein HER2, is widely used in standard therapy for women with HER2-positive breast cancer. Although the effects of trastuzumab on cancer cell proliferation, angiogenesis, and apoptosis have been intensively investigated, the effect of trastuzumab on microRNA (miRNA) is unknown. Since miRNA provides a novel layer of gene regulation and may play role in trastuzumab action, we have consequently performed miRNA microarray profiling before and after trastuzumab treatment in both SKBr3 and BT474 breast cancer cells that overexpress HER2. Trastuzumab significantly decreased five human miRNAs and increased three others in SKBr3 cells, whereas in BT474 cells it significantly decreased two miRNAs and increased ten. The only miRNA that shared the same change in both cell lines was miRNA-194 (miR194), which was upregulated following trastuzumab treatment. Upregulation of miR194 by trastuzumab was further validated in vitro and in vivo by quantitative reverse transcription PCR (QRT-PCR) or Northern blotting. miR194 expression in 92 cases of breast cancer and 21 normal breast tissues (all were FFPE samples) was examined by QRT-PCR. miR194 levels were significantly higher in breast cancer samples versus normal tissues (p=0.0003), in estrogen-positive breast cancer samples (p=0.002), and in HER2-negative breast cancer samples (p=0.032). Importantly, expression of precursor miR194 specifically downregulated the protein levels of DNA methytransferase DNMT3A and actin cytoskeleton organizer profilin 2. miR-194 specifically inhibited the luciferase activity of wild type DNMT3A 3’-UTR construct, but not that of mutant vector, indicating that DNMT3A is a direct downstream target of miR-194. Both trastuzumab treatment and overexpression of miR-194 inhibited cell motility of breast cancer cells, which may result from the inhibition of DNMT3A and profilin 2. In summary, trastuzumab treatment upregulates miR194 expression and may exert its therapeutic effects through downregulation of miR194-downstream targets DNMT3A and profilin 2 in HER2-positive breast cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4000. doi:10.1158/1538-7445.AM2011-4000