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These figures and tables contain supporting (and essential) data to confirm the clinical utility of the miR-200 family and signature in early gastric cancer, as well as its regulation by STAT3. Supplementary Figure S1. The association of WHO intestinal-type GC sub-classes and individual TNM stage with the expression of miR-200 family members and miR-21 in TCGA GC patients. Supplementary Figure S2. The association between patient survival and expression of miR- 200s and a complete network map of GSEA results generated from TCGA GC patients. Supplementary Figure S3. miR-429 promotes AGS cells proliferation and clonigenicity. Supplementary Figure S4. The association between patient survival and expression of individual genes from the miR-200 family-associated 15-gene signature. Supplementary Figure S5. STAT3 activation correlates with miR-429 and other miR-200 members in mouse and human gastric epithelial cells. Supplementary Figure S6. Generation of STAT3 knockout human GC lines, and STAT3- independent transcriptional regulation of miR-200 family members. Supplementary Table S1. Clinicopathological features and demographics of GC patients from TCGA cohort used for expression profiling of hsa-miR-429. Supplementary Table S2. Chi-square analysis between the miR-200 family 15-gene signature and conventional key prognostic factors using two independent cohorts. 2 Supplementary Table S3. Multivariate analysis of overall survival comparing the miR-200 family 15-gene signature and other conventional clinical parameters including histology and tumor grade (differentiation) using updated TCGA data.

Purpose: The majority of gastric cancer patients are diagnosed with late-stage disease, for which distinct molecular subtypes have been identified that are potentially amenable to targeted therapies. However, there exists no molecular classification system with prognostic power for early-stage gastric cancer (EGC) because the molecular events promoting gastric cancer initiation remain ill-defined.Experimental Design: miRNA microarrays were performed on gastric tissue from the gp130F/F preclinical EGC mouse model, prior to tumor initiation. Computation prediction algorithms were performed on multiple data sets and independent gastric cancer patient cohorts. Quantitative real-time PCR expression profiling was undertaken in gp130F/F-based mouse strains and human gastric cancer cells genetically engineered for suppressed activation of the oncogenic latent transcription factor STAT3. Human gastric cancer cells with modulated expression of the miR-200 family member miR-429 were also assessed for their proliferative response.Results: Increased expression of miR-200 family members is associated with both tumor initiation in a STAT3-dependent manner in gp130F/F mice and EGC (i.e., stage IA) in patient cohorts. Overexpression of miR-429 also elicited contrasting pro- and antiproliferative responses in human gastric cancer cells depending on their cellular histologic subtype. We also identified a miR-200 family–regulated 15-gene signature that integrates multiple key current indicators of EGC, namely tumor invasion depth, differentiation, histology, and stage, and provides superior predictive power for overall survival compared with each EGC indicator alone.Conclusions: Collectively, our discovery of a STAT3-regulated, miR-200 family–associated gene signature specific for EGC, with predictive power, provides a molecular rationale to classify and stratify EGC patients for endoscopic treatment. Clin Cancer Res; 24(6); 1459–72. ©2018 AACR.

Purpose: The majority of gastric cancer patients are diagnosed with late-stage disease, for which distinct molecular subtypes have been identified that are potentially amenable to targeted therapies. However, there exists no molecular classification system with prognostic power for early-stage gastric cancer (EGC) because the molecular events promoting gastric cancer initiation remain ill-defined. Experimental Design: miRNA microarrays were performed on gastric tissue from the gp130F/F preclinical EGC mouse model, prior to tumor initiation. Computation prediction algorithms were performed on multiple data sets and independent gastric cancer patient cohorts. Quantitative real-time PCR expression profiling was undertaken in gp130F/F-based mouse strains and human gastric cancer cells genetically engineered for suppressed activation of the oncogenic latent transcription factor STAT3. Human gastric cancer cells with modulated expression of the miR-200 family member miR-429 were also assessed for their proliferative response. Results: Increased expression of miR-200 family members is associated with both tumor initiation in a STAT3-dependent manner in gp130F/F mice and EGC (i.e., stage IA) in patient cohorts. Overexpression of miR-429 also elicited contrasting pro- and antiproliferative responses in human gastric cancer cells depending on their cellular histologic subtype. We also identified a miR-200 family–regulated 15-gene signature that integrates multiple key current indicators of EGC, namely tumor invasion depth, differentiation, histology, and stage, and provides superior predictive power for overall survival compared with each EGC indicator alone. Conclusions: Collectively, our discovery of a STAT3-regulated, miR-200 family–associated gene signature specific for EGC, with predictive power, provides a molecular rationale to classify and stratify EGC patients for endoscopic treatment. Clin Cancer Res; 24(6); 1459–72. ©2018 AACR.

In a recent report (Paroni et al, Oncogene, 31:3431-43, 2012), we demonstrated that 25% of all ERBB2+ breast cancers present with a co-amplification of the RARA locus, encoding the nuclear retinoic acid receptor, RARα. ERBB2+/RARA+ breast cancer cell lines, like SKBR3 and AU565, are sensitive to the anti-proliferative and cyto-differentiating effects of all-trans retinoic acid (ATRA), whereas the ERBB2+/RARA− counterparts are refractory. Simultaneous targeting of RARα with ATRA and ERBB2 with lapatinib in SKBR3 and AU565 cells leads to synergistic interactions as to growth inhibition and cyto-differentiation. In addition, combinations of ATRA and lapatinib exert an apoptotic effect, which is not observed with the single components. This suggests that ERBB2+/RARA+ breast cancer patients may benefit from lapatinib+ATRA regimens and represent the rationale of an ongoing clinical trial. The use of lapatinib and ATRA for the stratified therapy of ERBB2+/RARA+ patients requires knowledge of the molecular mechanisms underlying the cross-talk between the two compounds. We evaluated the effects of lapatinib and/or ATRA on the expression profiles of more than 1,000 microRNAs in SKBR3 cells using an appropriate microarray platform. Both lapatinib and ATRA caused up- and down-regulation of numerous microRNAs. The combination of the two compounds enhanced the action of the single components of the mixture and resulted in the up- and down-regulation of novel target microRNAs as well. We integrated these results with microRNA-target-gene prediction algorithms and whole-genome gene-expression data looking for significant correlations between microRNA and potential/established target mRNA levels. Network analysis led to the identification of two highly interconnected microRNA/target-mRNA modules controlled in opposite directions by lapatinib+ATRA. The first one consists of six microRNAs (miR-1207-5-p, miR-874, miR-134, miR-762, miR-1181 and miR-320c) which are up-regulated by lapatinib+ATRA and control a common set of down-regulated target mRNAs in a coordinate fashion. Most of the mRNAs of the module control cell proliferation in a positive fashion. The second network consists of seven microRNAs (miR-203, miR-125a-5p, miR-193a-5p, miR-210, miR-362-5p, miR-532-5p and miR-342-3p) which are down-regulated by lapatinib+ATRA and control a series of up-regulated target-mRNAs. In this case, the majority of regulated genes is involved in negative regulation of cell proliferation. Coordinated regulation of microRNA sets controlling a common group of genes and functions represents a new concept that deserves further studies. Currently, we are in the process of establishing the functional significance of the identified microRNA modules for the anti-tumor effects of lapatinib and ATRA. Citation Format: Enrico Garattini, James N. Fisher, Gabriela Paroni, Floriana Centritto, Gregory J. Goodall, Anna Tsykin, Maddalena Fratelli, Mineko Terao. Combinations of lapatinib and all-trans retinoic acid control the coordinate expression of microRNA modules and target-mRNAs in ERBB2+/RARA+ breast carcinoma cells: Significance for the antitumor activity. [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 3088. doi:10.1158/1538-7445.AM2013-3088