Alteration of protein trafficking and localization is associated with several diseases, including cystic fibrosis, breast cancer, colorectal cancer, leukemia and diabetes. Specifically, aberrant nuclear localization of the epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, is a poor prognostic indicator in several epithelial carcinomas. It is now appreciated that in addition to signaling from the plasma membrane, EGFR also trafficks to the nucleus, and can directly bind the promoter regions of genes encoding cyclin D1 (CCND1) and B-Myb (MYBL2). We have previously established that loss of MUC1 in an EGFR-dependent transgenic mouse model of breast cancer correlates with the loss of cyclin D1 expression. Here, we provide evidence for a novel regulatory function of MUC1 in the trafficking and nuclear activity of EGFR. We found that MUC1 and EGFR interact in the nucleus of breast cancer cells, which promotes the accumulation of chromatin-bound EGFR. Additionally, the presence of MUC1 results in significant colocalization of EGFR and phosphorylated RNA polymerase II, indicating that MUC1 influences the association of EGFR with transcriptionally active promoter regions. Importantly, we found that the loss of MUC1 expression resulted in a decrease in the interaction between EGFR and the CCND1 promoter, which translated to a significant decrease in cyclin D1 protein expression. This data offers insights into a novel regulatory mechanism of EGFR nuclear function and could have important implications for evaluating nuclear localization in cancer.

Introduction: Breast cancer is the second-leading cause of oncology-related death in US women. Despite extensive research, over 30% of breast cancer patients develop metastatic disease, and metastases account for majority of breast cancer-associated morbidity and mortality. Of all invasive breast cancers, patients with tumors lacking expression of the Estrogen and Progesterone hormone Receptors and Human Epidermal growth factor Receptor 2 have the poorest clinical prognosis. These triple-negative tumors (TNBC) represent an aggressive form of the disease that is marked by an early-onset metastasis, a high tumor recurrence rate, and a low overall survival during the first three years post-diagnosis. However, few TNBC mouse models of metastasis currently exist. Results: We noticed that a well-established MDA-MB-231 TNBC cell line produces rapid and extremely lytic bone lesions in lumbar, sacral and caudal vertebrae, and hind limbs in about 10% of animals injected intravenously. We biopsied one of these bone metastases and established a new metastatic MDA-MB-231-1566 cell line. Following an intravenous injection, MDA-MB-231-1566 cells produce early-onset metastasis to bone in up to 70% of animals with concurrent metastases to lungs, liver, and soft tissues. We demonstrate that 100% of animals injected with MDA-MB-231-1566 cells developed metastasis and had median survival of 60 days vs. 80 days in mice injected with the parental cell line. We also demonstrate that ribosomal protein S6 is highly phosphorylated on Ser235/236 in metastatic TNBC tumors, and that this phosphorylation is indicative of upstream S6 kinase (S6K) activity. Lastly, we provide evidence that targeting S6K with a highly specific inhibitor, PF-4708671, at sub-lethal doses inhibits cell migration without inducing cell death, and thus may provide a potent anti-metastatic adjuvant therapy approach. Conclusion: We established a new model of rapid TNBC metastases to multiple organs following a simple intravenous injection. We believe this model provides a valuable tool for screening new therapeutics aimed to stop growth of metastases. Citation Format: Yekaterina B. Khotskaya, Aarthi Goverdhan, Jia Shen, Mariano Ponz Sarvice, Shih-Shin Chang, Ming-Chuan Hsu, Yongkun Wei, Weiya Xia, Patricia Steeg, Dihua Yu, Mien-Chie Hung. S6K1 promotes invasiveness of breast cancer cells in a novel model of triple-negative breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A49.

The ERBB receptors are a family of heterodimerization partners capable of driving transformation and metastasis. While the therapeutic targeting of single receptors has proven efficacious, optimal targeting of this receptor family should target all oncogenic members simultaneously. The juxtamembrane domains of ERBB1, ERBB2, and ERBB3 are highly conserved and control various aspects of ERBB-dependent biology. In an effort to block those functions, we have targeted this domain with decoy peptides synthesized in tandem with a cell-penetrating peptide, termed EJ1. Treatment with EJ1 induces cell death, promotes the formation of inactive ERBB multimers, and results in simultaneous reduction of ERBB1, ERBB2, and ERBB3 activation. Treatment also results in the activation of myosin light chain-dependent cell blebbing while inactivating CaMKII signaling, coincident with the induction of cell death. EJ1 also directly translocates to mitochondria, correlating with a loss of mitochondrial membrane potential and production of reactive oxygen species. Finally, treatment of a mouse model of breast cancer with EJ1 results in the inhibition of tumor growth and metastasis without associated toxicities in normal cells. Overall, these data demonstrate that a portion of the ERBB jxm domain, when used as an intracellular decoy, can inhibit tumor growth and metastasis, representing a novel anticancer therapeutic.

Diffuse large B-cell lymphomas (DLBCL) harbor recurrent abnormalities in epigenetic regulators, such as loss-of-function mutations in EP300 and CREBBP. These include gain-of-function mutations in EZH2, largely restricted to the germinal center B-Cell (GCB) subtype. An EZH2 inhibitor EPZ-6438 showed efficacy as a single agent in phase I clinical trials in relapsed/refractory GCB-DLBCL. We examined the effect of EZH2 inhibition in GCB-DLBCL cell lines, and found an increase in histone arginine methylation in surviving cells, which was attributable to increased Type I PRMT activity. Specifically, we discovered that EZH2 inhibitors activated PRMT1, which mediated protective effects through transcriptional changes at survival genes such as BCL2, and through modulation of B-cell receptor signaling. This exciting observation suggested an opportunity for combination therapy with PRMT1 inhibitors. Since PRMT1 is poorly studied in DLBCL, we performed single-agent studies in lymphoma cell lines and discovered that inhibition of PRMT1 elicited remarkable suppression of lymphoma cell growth and viability. Analysis of gene expression data from patients indicates that PRMT1 is overexpressed in untreated DLBCL tumor samples, compared to normal B-lymphocytes. Additionally, survival analysis revealed that patients with high PRMT1 expression showed poor overall survival. These observations support the potential for combinatorial treatment with EZH2 and PRMT1 inhibitors in patients. To summarize, our studies uncover a novel epigenetic target in DLBCL, and offer avenues for improving the antitumor efficacy of EZH2 inhibitors. Citation Format: Aarthi Goverdhan, Heng-Huan Lee, Ondrej Havranek, Richard Eric Davis, Mien-Chie Hung. PRMT1 as a therapeutic target in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 13.

The triple negative (ER-, PR-, Her2-) or basal-like breast cancers are the most difficult forms of breast cancer to treat, because the cancer is often chemoresistant and highly metastatic. As these cancers progress from early to late stage the 5-year survival rate drops dramatically. Many proteins are responsible for this progression, including MUC1 and the Epidermal Growth Factor Receptor (EGFR). MUC1 is a heavily O-glycosylated transmembrane protein that is over expressed in 90% of all breast and ovarian cancers. In normal cells, MUC1 is only found on the apical surface of epithelium, but in cancer, MUC1 is found to be mislocalized. As a result, MUC1 plays a role in promoting tumorigenesis and metastases, by interacting with other oncogenes such as EGFR. EGFR is overexpressed in about 60% of the most aggressive form of breast cancer (triple negative). In an EGFR-dependent mouse model of breast cancer, the loss of Muc1 directly correlates to the loss of cyclin D1. Additionally, blocking MUC1 and EGFR interactions significantly inhibited tumor progression in a xenograft and s spontaneous mouse model of breast cancer. Recently, a novel trafficking pathway of EGFR has been discovered, which was described as EGFR trafficking to the nuclear compartment. Following entry into the nucleus EGFR can act in an oncogenic manner by aberrantly activating transcription of genes such as, cyclin D1 and v-myb. In this study we discovered a novel regulator function of MUC1 in EGFR trafficking and transcriptional activity. We found that MUC1 expression in basal-like breast cancer promotes the accumulation of nuclear EGFR and mediates EGFR's interaction with chromatin. Additionally, we determined that EGFR's interaction with the cyclin D1 promoter and upregulation of the cyclin D1 protein was MUC1-dependent. The main focus of anti-EGFR therapeutics have been its tyrosine kinase activity, however this study reveals that EGFR's subcellular localization should also be considered in the development of future cancer therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 270.