<p>Supplementary Figure S4. Schematic representation of the treatment regime of xenograft mice.</p>

<p>Supplementary Figure S1. ORM inhibits proliferation and invasion of PDAC cells.</p>

Supplementary Figure 1 from Expression and Functions of Transmembrane Mucin MUC13 in Ovarian Cancer

Legends for Supplementary Figures S1-S4.

Supplementary Figure S3. ORM targets Sonic hedgehog signaling and inhibits NFĸB and Gli-1 transcriptional activity in PDAC cells.

Abstract The high death rate of pancreatic cancer is attributed to the lack of reliable methods for early detection and underlying molecular mechanisms of its aggressive pathogenesis. Although MUC13, a newly identified transmembrane mucin, is known to be aberrantly expressed in ovarian and gastro-intestinal cancers, its role in pancreatic cancer is unknown. Herein, we investigated the expression profile and functions of MUC13 in pancreatic cancer progression. The expression profile of MUC13 in pancreatic cancer was investigated using a recently generated monoclonal antibody (clone PPZ0020) and pancreatic tissue microarrays. The expression of MUC13 was significantly (P < 0.005) higher in cancer samples compared with normal/nonneoplastic pancreatic tissues. For functional analyses, full-length MUC13 was expressed in MUC13 null pancreatic cancer cell lines, MiaPaca and Panc1. MUC13 overexpression caused a significant (P < 0.05) increase in cell motility, invasion, proliferation, and anchorage-dependent or -independent clonogenicity while decreasing cell–cell and cell-substratum adhesion. Exogenous MUC13 expression significantly (P < 0.05) enhanced pancreatic tumor growth and reduced animal survival in a xenograft mouse model. These tumorigenic characteristics correlated with the upregulation/phosphorylation of HER2, p21-activated kinase 1 (PAK1), extracellular signal-regulated kinase (ERK), Akt, and metastasin (S100A4), and the suppression of p53. Conversely, suppression of MUC13 in HPAFII pancreatic cancer cells by short hairpin RNA resulted in suppression of tumorigenic characteristics, repression of HER2, PAK1, ERK, and S100A4, and upregulation of p53. MUC13 suppression also significantly (P < 0.05) reduced tumor growth and increased animal survival. These results imply a role of MUC13 in pancreatic cancer and suggest its potential use as a diagnostic and therapeutic target. Mol Cancer Ther; 11(1); 24–33. ©2011 AACR.

PDF file - 153K, Figure S2. Serum proteins adsorption of MNP-CUR formulation.

PDF file - 126K, MNP-CUR nanoparticles efficiently internalize in cancer cells.

Supplementary Figure S2. Effect of GDC-0449 and ORM on proliferation, clonogenicity and invasion of PDAC cells.

Supplementary Figure Legends 1-4 from Expression and Functions of Transmembrane Mucin MUC13 in Ovarian Cancer

<p>Supplementary Figure S1. ORM inhibits proliferation and invasion of PDAC cells.</p>

Fidelity in pluripotent stem cell differentiation protocols is necessary for the therapeutic and commercial use of cells derived from embryonic and induced pluripotent stem cells. Recent advances in stem cell technology, especially the widespread availability of a range of chemically defined media, substrates and differentiation components, now allow the design and implementation of fully defined derivation and differentiation protocols intended for replication across multiple research and manufacturing locations. In this report we present an application of these criteria to the generation of retinal pigmented epithelium from iPSCs derived from the conjunctiva of donors with and without age related macular degeneration. Primary conjunctival cells from human donors aged 70-85 years were reprogrammed to derive multiple iPSC lines that were differentiated into functional RPE using a rapid and defined differentiation protocol. The combination of defined iPSC derivation and culture with a defined RPE differentiation protocol, reproducibly generated functional RPE from each donor without requiring protocol adjustments for each individual. This successful validation of a standardized, iPSC derivation and RPE differentiation process demonstrates a practical approach for applications requiring the cost-effective generation of RPE from multiple individuals such as drug testing, population studies or for therapies requiring patient-specific RPE derivations. In addition, conjunctival cells are identified as a practical source of somatic cells for deriving iPSCs from elderly individuals.

Although MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic ductal adenocarcinoma (PDAC) and generally correlates with increased expression of HER2, the underlying mechanism remains poorly understood. Herein, we found that MUC13 co-localizes and interacts with HER2 in PDAC cells (reciprocal co-immunoprecipitation, immunofluorescence, proximity ligation, co-capping assays) and tissues (immunohistofluorescence). The results from this study demonstrate that MUC13 functionally interacts and activates HER2 at p1248 in PDAC cells, leading to stimulation of HER2 signaling cascade, including ERK1/2, FAK, AKT and PAK1 as well as regulation of the growth, cytoskeleton remodeling and motility, invasion of PDAC cells—all collectively contributing to PDAC progression. Interestingly, all of these phenotypic effects of MUC13–HER2 co-localization could be effectively compromised by depleting MUC13 and mediated by the first and second EGF-like domains of MUC13. Further, MUC13–HER2 co-localization also holds true in PDAC tissues with a strong functional correlation with events contributing to increased degree of disorder and cancer aggressiveness. In brief, findings presented here provide compelling evidence of a functional ramification of MUC13–HER2: this interaction could be potentially exploited for targeted therapeutics in a subset of patients harboring an aggressive form of PDAC.

Pancreatic cancer (PanCa) is the fourth leading cancer with 85% mortality rate in the United States. Therefore, there is an utmost need to discover new modalities that can result in enhanced therapeutic efficacy with low to minimal side effects. Ormeloxifene (ORM) is a synthetic molecule which is widely used as an oral contraceptive in humans. Numerous studies indicate that ORM exhibits potent anti-cancer effects through inhibition of important oncogenic signaling events in several cancers. Our studies also demonstrate the anticancer effects of ORM in various cancer cells. However, the ORM effects can be improved by encapsulating in a nanoformulation which provides targeted delivery of ORM to the tumors. Therefore, in order to increase the therapeutic efficacy of ORM, we have developed a novel ORM encapsulated poly(lactic-co-glycolic acid) formulation (PLGA-ORM NPs). This formulation has been characterized for particle size, zeta potential, chemical composition, drug loading efficiency using various physico-chemical methods such as DLS, TEM, FT-IR, DSC, and TGA. Because of its facile composition (PLGA core, PVA, PLL and PEG-linker) this novel formulation is compatible for antibody/aptamer conjugation to achieve tumor specific targeting. The PLGA-ORM formulations indicate efficient encapsulation of ORM. The particle size of PLGA-ORM formulation (∼ 100 nm) indicates that this formulation can preferentially reach and accumulate in tumors by the Enhanced Permeation and Retention (EPR) effect. The uptake and internalization studies demonstrate that PLGA-ORM NPs escape lysosomal degradation providing its efficient endosomal release to cytosol in PanCa cells. PLGA-ORM NPs showed superior anti-cancer potential in various pancreatic cancer cells (HPAF-II, BXPC-3, Panc-1, MiaPaca) and in BXPC-3 xenograft mice. PLGA-ORM NPs suppressed pancreatic xenograft tumor growth and improved the mice survival. In addition, PLGA-ORM NPs also reduce the metastasis potential. PLGA-ORM NPs inhibit tumorigenic and metastatic phenotypes via suppression of AKT phosphorylation and inhibition of key oncogenes involved in pancreatic progression such as MUC1, HER2 and CD31. Additionally, PLGA-ORM NPs treated xenograft tumors showed reduced staining of the proliferating cell nuclear antigen (PCNA), cytokeratin-19 (CK19), MUC1, HER2 and CD31 in immunohistochemical analysis. In conclusion, this study suggests that PLGA-ORM formulation is highly efficient for the inhibition of pancreatic tumor growth and can be valuable for the treatment of pancreatic cancer in future. Citation Format: Sheema Khan, Neeraj Chauhan, Murali M. Yallapu, Mara C. Ebeling, Swathi Balakrishna, Robert T. Ellis, Paul A. Thompson, Stephen W. Behrman, Nadeem Zafar, Man M. Singh, Fathi T. Halaweish, Meena Jaggi, Subhash C. Chauhan. Generation of a novel ormeloxifene nanoparticle formulation for pancreatic cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4392. doi:10.1158/1538-7445.AM2015-4392

Abstract The management of pancreatic ductal adenocarcinoma (PDAC) is extremely poor due to lack of an efficient therapy and development of chemoresistance to the current standard therapy, gemcitabine. Recent studies implicate the intimate reciprocal interactions between epithelia and underlying stroma due to paracrine Sonic hedgehog (SHH) signaling in producing desmoplasia and chemoresistance in PDAC. Herein, we report for the first time that a nonsteroidal drug, ormeloxifene, has potent anticancer properties and depletes tumor-associated stromal tissue by inhibiting the SHH signaling pathway in PDAC. We found that ormeloxifene inhibited cell proliferation and induced death in PDAC cells, which provoked us to investigate the combinatorial effects of ormeloxifene with gemcitabine at the molecular level. Ormeloxifene caused potent inhibition of the SHH signaling pathway via downregulation of SHH and its related important downstream targets such as Gli-1, SMO, PTCH1/2, NF-κB, p-AKT, and cyclin D1. Ormeloxifene potentiated the antitumorigenic effect of gemcitabine by 75% in PDAC xenograft mice. Furthermore, ormeloxifene depleted tumor-associated stroma in xenograft tumor tissues by inhibiting the SHH cellular signaling pathway and mouse/human collagen I expression. Xenograft tumors treated with ormeloxifene in combination with gemcitabine restored the tumor-suppressor miR-132 and inhibited stromal cell infiltration into the tumor tissues. In addition, invasiveness of tumor cells cocultivated with TGFβ-stimulated human pancreatic stromal cells was effectively inhibited by ormeloxifene treatment alone or in combination with gemcitabine. We propose that ormeloxifene has high therapeutic index and in a combination therapy with gemcitabine, it possesses great promise as a treatment of choice for PDAC/pancreatic cancer. Cancer Res; 75(11); 2292–304. ©2015 AACR.