BRCA1-IRIS promotes human tumor progression through PTEN blockade and HIF-1α activation.

BRCA1 is an established breast and ovarian tumor suppressor gene that encodes multiple protein products whose individual contributions to human cancer suppression are poorly understood. BRCA1-IRIS (also known as "IRIS"), an alternatively spliced BRCA1 product and a chromatin-bound replication and transcription regulator, is overexpressed in various primary human cancers, including breast cancer, lung cancer, acute myeloid leukemia, and certain other carcinomas. Its naturally occurring overexpression can promote the metastasis of patient-derived xenograft (PDX) cells and other human cancer cells in mouse models. The IRIS-driven metastatic mechanism results from IRIS-dependent suppression of phosphatase and tensin homolog (PTEN) transcription, which in turn perturbs the PI3K/AKT/GSK-3ß pathway leading to prolyl hydroxylase-independent HIF-1α stabilization and activation in a normoxic environment. Thus, despite the tumor-suppressing genetic origin of IRIS, its properties more closely resemble those of an oncoprotein that, when spontaneously overexpressed, can, paradoxically, drive human tumor progression.

The pro- and anti-tumor roles of mesenchymal stem cells toward BRCA1-IRIS-overexpressing TNBC cells.

BACKGROUND: To evaluate the cross-talk between BRCA1-IRIS (IRIS)-overexpressing (IRISOE) TNBC cells and tumor-resident mesenchymal stem cells (MSCs) that triggers the aggressiveness or elimination of IRISOE TNBC tumors. METHODS: We analyzed the effect of silencing or inactivating IRIS on the bi-directional interaction between IRISOE TNBC cells and MSCs on tumor formation and progression. We analyzed the downstream signaling in MSCs induced by IL-6 secreted from IRISOE TNBC cells. We compared the effect of MSCs on the formation and progression of IRIS-proficient and deficient-TNBC cells/tumors using in vitro and in vivo models. Finally, we analyzed the association between IL-6, PTGER2, and PTGER4 overexpression and breast cancer subtype; hormone receptor status; and distant metastasis-free or overall survival. RESULTS: We show high-level IL-6 secreted from IRISOE TNBC cells that enhances expression of its receptor (IL-6R) in MSCs, their proliferation, and migration toward IRISOE, in vitro, and recruitment into IRISOE TNBC tumors, in vivo. In serum-free medium, recombinant IL-6 and the IL-6-rich IRISOE TNBC cell condition media (CM) decreased STAT3Y705 phosphorylation (p-STAT3Y705) in MSCs. Inhibiting IRIS expression or activity prolonged STAT3Y705 phosphorylation in MSCs. The interaction with IRISOE TNBC cells skewed MSC differentiation toward prostaglandin E2 (PGE2)-secreting pro-aggressiveness cancer-associated fibroblasts (CAFs). Accordingly, co-injecting human or mouse MSCs with IRISOE TNBC tumor cells promoted the formation of aggressive mammary tumors, high circulating IL-6 and PGE2 levels, and reduced overall survival. In contrast, IRIS-silenced or inactivated cells showed reduced tumor formation ability, limited MSC recruitment into tumors, reduced circulating IL-6 and PGE2 levels, and prolonged overall survival. A positive correlation between IL-6, PTGER2, and PTGER4 expression and basal phenotype; ER-negativity; distant metastasis-free and overall survival in basal; or BRCAmutant carriers was observed. Finally, the bi-directional interaction with MSCs triggered death rather than growth of IRIS-silenced TNBC cells, in vitro and in vivo. CONCLUSIONS: The IL-6/PGE2-positive feedback loop between IRISOE TNBC tumor cells and MSCs enhances tumor aggressiveness. Inhibiting IRIS expression limits TNBC tumor growth and progression through an MSC-induced death of IRIS-silenced/inactivated TNBC cells.

The role of BRCA1-IRIS in the development and progression of triple negative breast cancers in Egypt: possible link to disease early lesion.

BACKGROUND: Breast cancer is the most globally diagnosed female cancer, with the triple negative breast cancer (TNBC) being the most aggressive subtype of the disease. In this study we aimed at comparing the effect of BRCA1-IRIS overexpression on the clinico-pathological characteristics in breast cancer patients with TNBC or non-TNBC in the largest comprehensive cancer center in Egypt. METHODS: To reach this goal, we conducted an observational study at the National Cancer Institute (NCI), Cairo University (Cairo, Egypt). The data on all diagnosed breast cancer patients, between 2009 and 2012, were reviewed. BRCA1-IRIS expression measured using real time RT/PCR in these patients' tumor samples was correlated to tumor characteristics, such as to clinico-pathological features, therapeutic responses, and survival outcomes. RESULTS: 96 patients were enrolled and of these 45% were TNBC, and 55% were of other subtypes (hereafter, non-TNBC). All patients presented with invasive ductal carcinomas. No significant difference was observed for risk factors, such as age and menopausal status between the TNBC and the non-TNBC groups except after BRCA1-IRIS expression was factored in. The majority of the tumors in both groups were ≤5 cm at surgery (p = 0.013). However, in the TNBC group, ≤5 cm tumors were BRCA1-IRIS-overexpressing, whereas in the non-TNBC group they were BRCA1-IRIS-negative (p = 0.00007). Most of the TNBC patients diagnosed with grade 1 or 2 were BRCA1-IRIS-overexpressing, whereas non-TNBCs were IRIS-negative (p = 0.00035). No statistical significance was measured in patients diagnosed with grade 3 tumors. Statistically significant difference between TNBCs and non-TNBCs and tumor stage with regard to BRCA1-IRIS-overexpression was observed. Presence of axillary lymph node metastases was positively associated with BRCA1-IRIS overexpression in TNBC group, and with BRCA1-IRIS-negative status in the non-TNBC group (p = 0.00009). Relapse after chemotherapy (p < 0.00001), and local recurrence/distant metastasis after surgery (p = 0.0028) were more pronounced in TNBC patients with BRCA1-IRIS-overexpressing tumors compared to non-TNBC patients. Finally, decreased disease-free survival in TNBC/BRCA1-IRIS-overexpressing patients compared to TNBC/BRCA1-IRIS-negative patients, and decreased overall survival in TNBC as well as non-TNBC patients was driven by BRCA1-IRIS overexpression. CONCLUSIONS: TNBC/BRCA1-IRIS-overexpressing tumors are more aggressive than TNBC/BRCA1-IRIS-negative or non-TNBC/BRCA1-IRIS-overexpressing or both negative tumors. Further studies are warranted to define whether BRCA1-IRIS drives the early TNBC lesions growth and dissemination and whether it could be used as a diagnostic biomarker and/or therapeutic target for these lesions at an early stage setting.

Novel insights linking BRCA1-IRIS role in mammary gland development to formation of aggressive PABCs: the case for longer breastfeeding.

Pregnancy-associated breast cancer (PABC) is diagnosed during or shortly after pregnancy. Although rare, PABC is a serious occurrence often of the triple negative (TNBC) subtype. Here we show progesterone, prolactin, and RANKL upregulate BRCA1-IRIS (IRIS) in separate and overlapping subpopulations of human mammary epithelial cell lines, which exacerbates the proliferation, survival, and the TNBC-like phenotype in them. Conversely, vitamin D3 reduces IRIS expression in TNBC cell lines, which attenuates growth, survival, and the TNBC-like phenotype in them. In the mouse, Brca1-Iris (Iris, mouse IRIS homolog) is expressed at low-level in nulliparous mice, increases ~10-fold in pregnant/lactating mice, to completely disappear in involuting mice, and reappears at low-level in regressed glands. Mice underwent 3 constitutive pregnancies followed by a forced involution (after 5 days of lactation) contained ~10-fold higher Iris in their mammary glands compared to those underwent physiological involution (after 21 days of lactation). While protein extracts from lactating glands promote proliferation in IRISlow and IRIS overexpressing (IRISOE) cells, extracts from involuting glands promote apoptosis in IRISlow, and aneuploidy in IRISOE cells. In a cohort of breast cancer patients, lack of breastfeeding was associated with formation of chemotherapy resistant, metastatic IRISOE breast cancers. We propose that terminal differentiation triggered by long-term breastfeeding reduces IRIS expression in mammary cells allowing their elimination by the inflammatory microenvironment during physiological involution. No/short-term breastfeeding retains in the mammary gland IRISOE cells that thrive in the inflammatory microenvironment during forced involution to become precursors for aggressive breast cancers shortly after pregnancy.

BRCA1-IRIS overexpression promotes and maintains the tumor initiating phenotype: implications for triple negative breast cancer early lesions.

Tumor-initiating cells (TICs) are cancer cells endowed with self-renewal, multi-lineage differentiation, increased chemo-resistance, and in breast cancers the CD44+/CD24-/ALDH1+ phenotype. Triple negative breast cancers show lack of BRCA1 expression in addition to enhanced basal, epithelial-to-mesenchymal transition (EMT), and TIC phenotypes. BRCA1-IRIS (hereafter IRIS) is an oncogene produced by the alternative usage of the BRCA1 locus. IRIS is involved in induction of replication, transcription of selected oncogenes, and promoting breast cancer cells aggressiveness. Here, we demonstrate that IRIS overexpression (IRISOE) promotes TNBCs through suppressing BRCA1 expression, enhancing basal-biomarkers, EMT-inducers, and stemness-enforcers expression. IRISOE also activates the TIC phenotype in TNBC cells through elevating CD44 and ALDH1 expression/activity and preventing CD24 surface presentation by activating the internalization pathway EGFR→c-Src→cortactin. We show that the intrinsic sensitivity to an anti-CD24 cross-linking antibody-induced cell death in membranous CD24 expressing/luminal A cells could be acquired in cytoplasmic CD24 expressing IRISOE TNBC/TIC cells through IRIS silencing or inactivation. We show that fewer IRISOE TNBC/TICs cells form large tumors composed of TICs, resembling TNBCs early lesions in patients that contain metastatic precursors capable of disseminating and metastasizing at an early stage of the disease. IRIS-inhibitory peptide killed these IRISOE TNBC/TICs, in vivo and prevented their dissemination and metastasis. We propose IRIS inactivation could be pursued to prevent dissemination and metastasis from early TNBC tumor lesions in patients.

A niche that triggers aggressiveness within BRCA1-IRIS overexpressing triple negative tumors is supported by reciprocal interactions with the microenvironment.

Production of metastasis capable precursors begins within the primary tumor. Here, we define the bidirectional interactions with stromal cells involved in promoting these precursors within BRCA1-IRIS (hereafter IRIS) overexpressing (IRISOE) TNBC tumors. We define an aggressiveness niche, functionally defined as the necrotic/hypoxic core of the tumor, in which metabolically stressed, hypoxic, and inflamed IRISOE TNBC cells secrete higher levels of cytokines, chemokines and growth factors. One cytokine; IL-1ß attracts mesenchymal stem cells (MSCs) to the niche and activates them to secrete CXCL1 that entrains IRISOE cells to secrete higher levels of CCL2 and VEGF. CCL2 attracts macrophages (TAMs) to the niche and activates them to secrete S100A8, and VEGF attracts endothelial cells (ECs) and activates them to secrete IL-8. In concert, CXCL1, S100A8 and IL-8 entrain aggressiveness in IRISOE TNBC cells within the niche. Indeed, compared to IRISOE cells alone, tumors developed by co-injecting IRISOE cells admixed with MSCs (10:1) in athymic mice were bigger and more aggressive. They contained more TAMs and ECs, expressed higher-levels of basal, epithelial to mesenchymal transition, and stemness biomarkers, quickly progressed to lymph-node or visceral metastases, and were highly sensitive to the IL-1ß inhibitor "Anakinra". Our findings supported by human data show that breast cancer patients with high-levels of IL-1ß, CXCL1, CCL2, S100A8, VEGF, and IL-8 would show worse clinical outcomes. Our findings argue that this cytokine set is a diagnostic biomarker for patients who may benefit from an IRIS inhibitor-based therapy, and is a blue print for translation of approaches to combining that therapy with inhibitors of these bidirectional interactions to overcome TNBC metastasis.