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Supplementary Materials and Methods, Supplementary Table S1. List of 9 PRLR gene isoforms in the UCSC Genome Browser based on RNASeqV2 data. Supplementary Table S2. Protein expression profile by RPPA analysis in Ishikawa tumors treated with mannitol or G129R. Supplementary Table S3. Pathological staining results for PRLR using 2 scoring metrics in normal uterine tissue (n=3 patients) and UCEC-EEA (n=3 patients). Supplementary Figure S1 (supporting Figure 1). Expression of PRLR_SF (uc003jjl.3) in UCEC-EEA patients. Supplementary Figure S2 (supporting Figure 1). Expression of PRLR in human uterine cancer cell lines with both PTENmut and PTENWT backgrounds. Supplementary Figure S3 (supporting Figure 2). Representative images from the Ishikawa model showed the different sizes of primary tumors at the uterine horn in different groups. Supplementary Figure S4 (supporting Figure 3). In vitro efficacy of G129R in Ishikawa and Hec1-A cells under hormone-depleted condition. Supplementary Figure S5 (supporting Figure 3). G129R reduced the viabilities of Ishikawa and Hec1A cells in comparison with PRL stimulation at hormone-depleted condition. Supplementary Figure S6 (supporting Figure 5). siRNAs knock down the expression of FOXO3a or EIF-4EBP1 in Ishikawa and Hec1A cells. Supplementary Figure S7 (supporting Figure 4C & 4D, Supplementary Figure S2, and Supplementary Figure S6). Uncropped western blots produced in this study.

Abnormal activity of human prolactin (PRL) and its membrane-associated receptor (PRLR) contributes to the progression of uterine carcinoma. However, the underlying mechanisms are not well understood, and current means of targeting the PRL/PRLR axis in uterine cancer are limited. Our integrated analyses using The Cancer Genome Atlas and Genotype-Tissue Expression (GTEx) databases demonstrated that a short form of PRLR (PRLR_SF) is the isoform predominantly expressed in human uterine cancers; expression of this PRLR_SF was elevated in uterine cancers in comparison with cancer-free uterine tissues. We hypothesized that the overexpression of PRLR_SF in uterine cancer cells contributes, in part, to the oncogenic activity of the PRL/PRLR axis. Next, we employed G129R, an antagonist of human PRL, to block the PRL/PRLR axis in both PTENwt and PTENmut orthotopic mouse models of uterine cancer. In comparison with control groups, treatment with G129R as monotherapy or in combination with paclitaxel resulted in a significant reduction of growth and progression of orthotopic uterine tumors. Results from protein profiling of uterine cancer cells and in vivo tumors revealed a set of new downstream targets for G129R. Our results showed that G129R induced sub-G0 population arrest, decreased nascent protein synthesis, and initiated FOXO3a/EIF-4EBP1–mediated cell death in both PTENwt and PTENmut uterine cancer cells. Collectively, our results show a unique pattern of PRLR_SF expression predominantly in uterine cancer. Moreover, FOXO3a and EIF-4EBP1 are important mediators of cell death following G129R treatment in uterine cancer models.

Supplementary Table from Gene Body Methylation of the Lymphocyte-Specific Gene CARD11 Results in Its Overexpression and Regulates Cancer mTOR Signaling

EP-100 is a synthetic lytic peptide that specifically targets the gonadotropin-releasing hormone receptor on cancer cells. To extend the utility of EP-100, we aimed to identify effective combination therapies with EP-100 for ovarian cancer and explore potential mechanisms of this combination. A series of in vitro (MTT assay, immunoblot analysis, reverse-phase protein array, comet assay, and immunofluorescence staining) and in vivo experiments were carried out to determine the biological effects of EP-100 alone and in combination with standard-of-care drugs. EP-100 decreased the viability of ovarian cancer cells and reduced tumor growth in orthotopic mouse models. Of five standard drugs tested (cisplatin, paclitaxel, doxorubicin, topotecan, and olaparib), we found that the combination of EP-100 and olaparib was synergistic in ovarian cancer cell lines. Further experiments revealed that combined treatment of EP-100 and olaparib significantly increased the number of nuclear foci of phosphorylated histone H2AX. In addition, the extent of DNA damage was significantly increased after treatment with EP-100 and olaparib in comet assay. We performed reverse-phase protein array analyses and identified that the PI3K/AKT pathway was inhibited by EP-100, which we validated with in vitro experiments. In vivo experiment using the HeyA8 mouse model demonstrated that mice treated with EP-100 and olaparib had lower tumor weights (0.06 ± 0.13 g) than those treated with a vehicle (1.19 ± 1.09 g), EP-100 alone (0.62 ± 0.78 g), or olaparib alone (0.50 ± 0.63 g). Our findings indicate that combining EP-100 with olaparib is a promising therapeutic strategy for ovarian cancer.

Supplementary Table 1. Dataset for the reverse-phase protein array (RPPA) and processed data for Ingenuity Pathway Analysis.

CRM1 inhibitors have demonstrated antitumor effects in ovarian and other cancers; however, rational combinations are largely unexplored. We performed a high-throughput drug library screen to identify drugs that might combine well with selinexor in ovarian cancer. Next, we tested the combination of selinexor with the top hit from the drug screen in vitro and in vivo. Finally, we assessed for mechanisms underlying the identified synergy using reverse phase protein arrays (RPPA). The drug library screen assessing 688 drugs identified olaparib (a PARP inhibitor) as the most synergistic combination with selinexor. Synergy was further demonstrated by MTT assays. In the A2780luc ip1 mouse model, the combination of selinexor and olaparib yielded significantly lower tumor weight and fewer tumor nodules compared with the control group (P < 0.04 and P < 0.03). In the OVCAR5 mouse model, the combination yielded significantly fewer nodules (P = 0.006) and markedly lower tumor weight compared with the control group (P = 0.059). RPPA analysis indicated decreased expression of DNA damage repair proteins and increased expression of tumor suppressor proteins in the combination treatment group. Collectively, our preclinical findings indicate that combination with selinexor to expand the utility and efficacy of PARP inhibitors in ovarian cancer warrants further exploration.

Investigations into the function of nonpromoter DNA methylation have yielded new insights into epigenetic regulation of gene expression. Previous studies have highlighted the importance of distinguishing between DNA methylation in discrete functional regions; however, integrated nonpromoter DNA methylation and gene expression analyses across a wide number of tumor types and corresponding normal tissues have not been performed. Through integrated analysis of gene expression and DNA methylation profiles, we examined 32 tumor types and identified 57 tumor suppressors and oncogenes out of 260 genes exhibiting a correlation of > 0.5 between gene body methylation and gene expression in at least one tumor type. The lymphocyte-specific gene CARD11 exhibits robust association between gene body methylation and expression across 19 of 32 tumor types examined. It is significantly overexpressed in kidney renal cell carcinoma (KIRC) and lung adenocarcinoma (LUAD) tumor tissues in comparison with respective control samples; and is significantly associated with lower overall survival in KIRC. Contrary to its canonical function in lymphocyte NFκB activation, CARD11 activates the mTOR pathway in KIRC and LUAD, resulting in suppressed autophagy. Furthermore, demethylation of a CpG island within the gene body of CARD11 decreases gene expression. Collectively, our study highlights how DNA methylation outside the promoter region can impact tumor progression.Implications:Our study describes a novel regulatory role of gene body DNA methylation–dependent CARD11 expression on mTOR signaling and its impact on tumor progression.

Supplementary Table S1. The IC50s of EP-100 against ovarian cancer cells after 4-, 24-, and 72-h treatment.

Revised Supplementary Data Demonstrating All Supplementary Figures 1-6 and Supplementary Table 1

Full report from Ingenuity Pathway Analysis of the RPPA results.

Supplementary Video 2. 3-D SKOV3ip1 spheroids expressing pGFP-RFP-LC3 were treated with MORAB-003

Supplementary Data from Proapoptotic Function of Integrin β3 in Human Hepatocellular Carcinoma Cells

PDF file 196K, Figure S2. Effects of KX-01 and oxaliplatin on cell proliferation (Ki-67; A), angiogenesis (CD31; B), apoptosis (Cleaved caspase-3; C) RMUG-L model. Original magnification 200x . Bars in the graphs correspond sequentially to the labeled columns of images on the left. Data are shown as mean {plus-minus} standard deviation (error bars). *p

PDF file - 89K, Enhanced gap filling rates in HER2+ versus HER2- BCSCs

Purpose: This study evaluates the proapoptotic function of integrin β3 in human hepatocellular carcinoma (HCC).Experimental Design: The expression of integrin β3 in 67 HCC specimens paired with corresponding neighboring nontumorous tissue was studied by quantitative real-time PCR and Western blot. The proapoptotic function of integrin β3 in SMMC-7721 human hepatoma cells overexpressing ITGB3 (gene coding integrin β3) was determined through colony formation, serum starvation, and anoikis assay.Results: Compared with neighboring pathologically normal liver tissue, ∼60% of the HCC specimens showed a significant down-regulated level of integrin β3 expression. Transient expression of integrin β3 in SMMC-7721 resulted in an enhanced level of apoptosis and suppression of colony formation. Cell growth inhibition on serum/ligand deprivation and incidences of anoikis were remarkably increased in SMMC-7721 with stable expression of integrin β3 in comparison with vector control transfectants. In addition, expression of fibrinogen and vitronectin, two native ligands for integrin αvβ3 in liver, was inhibited, which was correlated with the decreased integrin β3 expression. Replenishing these ligands to the starved SMMC-7721 stable transfectants effectively restored the proapoptotic function of integrin β3.Conclusions: Down-regulation of integrin β3 and its ligands in liver is related to the aggressive growth of HCC. Thus, reconstitution of integrin β3 in HCC may be a potential therapeutic approach to inhibit aggressive growth of liver cancer.

PDF file - 540K, Protein profiling analysis of HER2+ versus HER2- BCSCs

Purpose: Cancer cells are highly dependent on folate metabolism, making them susceptible to drugs that inhibit folate receptor activities. Targeting overexpressed folate receptor alpha (FRα) in cancer cells offers a therapeutic opportunity. We investigated the functional mechanisms of MORAB-003 (farletuzumab), a humanized mAb against FRα, in ovarian cancer models.Experimental Design: We first examined FRα expression in an array of human ovarian cancer cell lines and then assessed the in vivo effect of MORAB-003 on tumor growth and progression in several orthotopic mouse models of ovarian cancer derived from these cell lines. Molecular mechanisms of tumor cell death induced by MORAB-003 were investigated by cDNA and protein expression profiling analysis. Mechanistic studies were performed to determine the role of autophagy in MORAB-003–induced cell death.Results: MORAB-003 significantly decreased tumor growth in the high-FRα IGROV1 and SKOV3ip1 models but not in the low-FRα A2780 model. MORAB-003 reduced proliferation, but had no significant effect on apoptosis. Protein expression and cDNA microarray analyses showed that MORAB-003 regulated an array of autophagy-related genes. It also significantly increased expression of LC3 isoform II and enriched autophagic vacuolization. Blocking autophagy with hydroxychloroquine or bafilomycin A1 reversed the growth inhibition induced by MORAB-003. In addition, alteration of FOLR1 gene copy number significantly correlated with shorter disease-free survival in patients with ovarian serous cancer.Conclusions: MORAB-003 displays prominent antitumor activity in ovarian cancer models expressing FRα at high levels. Blockade of folate receptor by MORAB-003 induced sustained autophagy and suppressed cell proliferation. Clin Cancer Res; 21(2); 448–59. ©2014 AACR.

PDF file - 513K, IHS of co-activation of HER2/CD44 cells in tumor tissues

Purpose: To understand the role of HER2-associated signaling network in breast cancer stem cells (BCSC) using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted therapy as a tumor eliminating strategy for recurrent HER2−/low breast cancers.Experimental Design: HER2-expressing BCSCs (HER2+/CD44+/CD24−/low) were isolated from radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggressiveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer specimens. Protein expression profiling in HER2+/CD44+/CD24−/low versus HER2−/CD44+/CD24−/low BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated signaling network was generated by MetaCore program.Results: Compared with HER2-negative BCSCs, HER2+/CD44+/CD24−/low cells showed elevated aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the HER2+/CD44+/CD24−/low cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/MS-MS of HER2+/CD44+/CD24−/low versus HER2−/CD44+/CD24−/low BCSCs reported a unique HER2-associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial function, and DNA repair. A specific feature of HER2–STAT3 network was identified.Conclusion: This study provides the evidence that HER2-mediated prosurvival signaling network is responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant HER2−/low breast cancer. Clin Cancer Res; 18(24); 6634–47. ©2012 AACR.

Supplementary Video 3. 3-D SKOV3ip1 spheroids expressing pGFP-LC3 only were treated with MORAB-003

PDF file - 187K, Enhanced aggressiveness of C6 and HER2 cells

PDF file - 112K

PDF file - 79K, Peptide and protein HPLC-MS/MS identification data for all proteins included in Table S1

PDF file - 36K, Mitochondrial membrane potential in HER2 siRNA treated cells

PDF file 106K, Table S1. Molecules whose expression significantly differed between cells treated with KX-01 and control cells in RMUG-S cells Table S2. Molecules whose expression significantly differed between cells treated with KX-01 and control cells in RMUG-L cells

Purpose: To investigate the antitumor effects of targeting Src and tubulin in mucinous ovarian carcinoma.Experimental Design: The in vitro and in vivo effects and molecular mechanisms of KX-01, which inhibits Src pathway and tubulin polymerization, were examined in mucinous ovarian cancer models.Results:In vitro studies using RMUG-S and RMUG-L cell lines showed that KX-01 inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G2–M phase, and enhanced the cytotoxicity of oxaliplatin in the KX-01–sensitive cell line, RMUG-S. In vivo studies showed that KX-01 significantly decreased tumor burden in RMUG-S and RMUG-L mouse models relative to untreated controls, and the effects were greater when KX-01 was combined with oxaliplatin. KX-01 alone and in combination with oxaliplatin significantly inhibited tumor growth by reducing cell proliferation and inducing apoptosis in vivo. PTEN knock-in experiments in RMUG-L cells showed improved response to KX-01. Reverse phase protein array analysis showed that in addition to blocking downstream molecules of Src family kinases, KX-01 also activated acute stress-inducing molecules.Conclusion: Our results showed that targeting both the Src pathway and tubulin with KX-01 significantly inhibited tumor growth in preclinical mucinous ovarian cancer models, suggesting that this may be a promising therapeutic approach for patients with mucinous ovarian carcinoma. Clin Cancer Res; 19(23); 6532–43. ©2013 AACR.

PDF file - 80K, Proteins identified by HPLC-MS/MS analysis are categorized into functional groups

Supplementary Video 1. 3-D SKOV3ip1 spheroids expressing pGFP-RFP-LC3 were treated with control IgG

Supplementary Figures S1-5, Tables S1-5. Figure S1 The Cytotoxic Effect of KPT-185 is Tumor Suppressor-independent. Figure S2 Combination Index of KPT-185 with Chemotherapeutic Agents. Figure S3 1D Gel Analysis of Cytoplasmic Protein. Figure S4 Effects of Selinexor (KPT-330) in an Ovarian Cancer Mouse Model. Figure S5 The Efficacy of Selinexor (KPT-330) on Tumor Regression and Metastasis Table S1 p53 status and IC50 concentration of KPT-185 after 72 hours of incubation in human cancer cell lines Table S2 Combination index (CI) of KPT-185 with cytotoxic agents Table S3 Proteins showing altered mitochondrial localization after treatment with KPT-185 Table S4 1D Proteomic analysis of cytoplasmic proteins immunoprecipitated with eIF5A Table S5 siRNA Sequences

MOV file - 2609K, Motility S2-013.M.1A

MOV file - 2609K, Motility S2-013.4A

PDF file - 45K, Evaluation of metastasis properties of S2-013 cells following orthotopic implantation of cell lines re-expressing different p120 catenin isoforms with and without overexpression of MUC1.

MOV file - 2670K, Motility S2-013.Neo

PDF file - 126K, Expression levels of E-cadherin upon re-expression of different p120 catenin isoforms in the context of with/without MUC1 overexpression. Protein lysates for western blots were derived from S2-013.Neo (deficient in p120 catenin) and p120 catenin isoform re-expressing cells with/without MUC1 overexpression.

MOV file - 2625K, Motility S2-013.1A

MOV file - 2626K, Motility S2-013.M.3A

PDF file - 209K, Graphic representation of mp120 constructs used in this study. (A) Full-length, N-terminal and C terminal deleted mp 120. (B) ARM domain deleted mp120. Each of the 10 ARM domains was deleted individually by mutagenesis, and were designated mp120 deltaARM 1-10.

The mechanisms by which MUC1 and p120 catenin contribute to progression of cancers from early transformation to metastasis are poorly understood. Here we show that p120 catenin ARM domains 1, 3–5, and 8 mediate interactions between p120 catenin and MUC1, and that these interactions modulate dynamic properties of cell adhesion, motility, and metastasis of pancreatic cancer cells. We also show that different isoforms of p120 catenin, when coexpressed with MUC1, create cells that exhibit distinct patterns of motility in culture (motility independent of cell adhesion, motility within a monolayer while exchanging contacts with other cells, and unified motility while maintaining static epithelial contacts) and patterns of metastasis. The results provide new insight into the dynamic interplay between cell adhesion and motility and the relationship of these to the metastatic process. Cancer Res; 74(5); 1609–20. ©2013 AACR.