Purpose: During cancer progression, the oncoprotein MUC1 binds h-catenin while simultaneously inhibiting the degradation of the epidermal growth factor receptor (EGFR), resulting in enhanced transformation and metastasis. The purpose of this study was to design a peptidebased therapy that would block these intracellular protein-protein interactions as a treatment for metastatic breast cancer. Experimental Design: The amino acid residues responsible for these interactions lie in tandem in the cytoplasmic domain of MUC1, and we have targeted this sequence to produce a MUC1 peptide that blocks the protumorigenic functions of MUC1. We designed the MUC1 inhibitory peptide (MIP) to block the intracellular interactions between MUC1/h-catenin and MUC1/EGFR. To allow for cellular uptake we synthesized MIP adjacent to the protein transduction domain, PTD4 (PMIP). Results: We have found that PMIP acts in a dominant-negative fashion, blocking both MUC1/hcatenin and MUC1/EGFR interactions. In addition, PMIP induces ligand-dependent reduction of EGFR levels. These effects correspond to a significant reduction in proliferation, migration, and invasion of metastatic breast cancer cells in vitro, and inhibition of tumor growth and recurrence in an established MDA-MB-231 immunocompromised (SCID) mouse model. Importantly, PMIP also inhibits genetically driven breast cancer progression, as injection of tumor-bearing MMTV-pyV mT transgenic mice with PMIP results in tumor regression and a significant inhibition of tumor growth rate. Conclusions: These data show that intracellular MUC1 peptides possess significant antitumor activity and have important clinical applications in the treatment of cancer.MUC1 (DF3, CD227, episialin, PEM) is a heavily Oglycosylated heterodimeric protein of >300 kDa, normally expressed abundantly on the apical surface of glandular epithelia. In more than 90% of human breast carcinomas and metastases, apical localization is lost and MUC1 is overexpressed (by >10-fold) and underglycosylated (1, 2). Deregulated expression of MUC1 is found in many other types of adenocarcinomas as well, including cancers of the lung, pancreas, ovary, and prostate, in addition to being highly expressed in leukemias, myelomas, and lymphomas (3 -5). Studies in both genetic mouse models and cell line models have shown that MUC1 is an oncogene. A transgenic mouse model driving MUC1 (human) overexpression to the mouse mammary gland (MMTV-MUC1) results in the development of breast cancer and is accompanied by a failure of the mammary gland to undergo complete postlactational regression via apoptosis (6). Transfection of MUC1 into 3Y1 fibroblasts induces their transformation, and transfection of MUC1 into colon cancer cells shows that MUC1 overexpression inhibits drug-induced apoptosis (7).The cytoplasmic domain of MUC1 contains sites for multiple protein interactions, although these interactions go largely unformed in the polarized epithelium of the normal breast, as the binding partners of MUC1 are typically found on the baso...

Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2 α ; P-EIF2 α ), active DNA damage checkpoints, and cell cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a ‘stress-dependent’ translation profile. This includes further elevated P-eIF2 α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

The role of β-catenin/TCF transcriptional activity in endometrial cancer (EC) recurrence is not well understood. We assessed the impact of Wnt/β-catenin inhibition in EC models. In an analysis of the Cancer Genome Atlas, we confirmed that CTNNB1 mutations are enriched in recurrent low-risk EC and showed that aberrant Wnt/β-catenin pathway activation is associated with recurrence. We studied CTNNB1-wildtype (HEC1B, Ishikawa) and CTNNB1-mutant (HEC108, HEC265, HEC1B-S33Y, Ishikawa-S33Y) EC cell lines. Dose response curves were determined for 5 Wnt/β-catenin pathway inhibitors (Wnt-C59, XAV-939, PyrPam, PRI-724, SM04690). XAV939, Wnt-C59 and PyrPam inhibited function upstream of β-catenin transcriptional activity and were ineffective at inhibiting cell viability. In contrast, PRI724 and SM04690 indirectly inhibited β-catenin transcriptional activity and significantly reduced cell viability in CTNNB1-mutant cell lines. Treatment with SM04690 reduced cell viability (Licor Cell stain) in all EC cell lines, but viability was significantly lower in CTNNB1-mutant cell lines (p < 0.01). Mechanistically, SM04690 significantly inhibited proliferation measured via 5′-bromo-2′-deoxyuridine incorporation and reduced T cell factor (TCF) transcriptional activity. HEC1B, HEC1B-S33Y and HEC265 tumor-bearing mice were treated with vehicle or SM04690.Tumors treated with SM04690 had smaller mean volumes than those treated with vehicle (p < 0.001, p = 0.014, p = 0.06). In HEC1B-S33Y and HEC265 tumors, SM04690 treatment significantly reduced Ki67 H-scores compared to vehicle (p = 0.035, p = 0.024). Targeting the Wnt/β-catenin pathway in CTNNB1-mutant EC effectively inhibited proliferation and β-catenin/TCF transcriptional activity and blunted tumor progression in in vivo models. These studies suggest β-catenin transcriptional inhibitors are effective in EC and particularly in CTNNB1-mutant EC, highlighting a potential therapeutic vulnerability for treatment of CTNNB1mutant EC.

Objective Multiplex immunohistochemistry (mIHC) and multiplexed ion beam imaging (MIBI) images are usually phenotyped using a manual thresholding process. The thresholding is prone to biases, especially when examining multiple images with high cellularity. Results Unsupervised cell-phenotyping methods including PhenoGraph, flowMeans, and SamSPECTRAL, primarily used in flow cytometry data, often perform poorly or need elaborate tuning to perform well in the context of mIHC and MIBI data. We show that, instead, semi-supervised cell clustering using Random Forests, linear and quadratic discriminant analysis are superior. We test the performance of the methods on two mIHC datasets from the University of Colorado School of Medicine and a publicly available MIBI dataset. Each dataset contains a bunch of highly complex images.