Hyperactive mTOR and MNK1 phosphorylation of eIF4E confer tamoxifen resistance and estrogen independence through selective mRNA translation reprogramming.

The majority of breast cancers expresses the estrogen receptor (ER+) and is treated with anti-estrogen therapies, particularly tamoxifen in premenopausal women. However, tamoxifen resistance is responsible for a large proportion of breast cancer deaths. Using small molecule inhibitors, phospho-mimetic proteins, tamoxifen-sensitive and tamoxifen-resistant breast cancer cells, a tamoxifen-resistant patient-derived xenograft model, patient tumor tissues, and genome-wide transcription and translation studies, we show that tamoxifen resistance involves selective mRNA translational reprogramming to an anti-estrogen state by Runx2 and other mRNAs. Tamoxifen-resistant translational reprogramming is shown to be mediated by increased expression of eIF4E and its increased availability by hyperactive mTOR and to require phosphorylation of eIF4E at Ser209 by increased MNK activity. Resensitization to tamoxifen is restored only by reducing eIF4E expression or mTOR activity and also blocking MNK1 phosphorylation of eIF4E. mRNAs specifically translationally up-regulated with tamoxifen resistance include Runx2, which inhibits ER signaling and estrogen responses and promotes breast cancer metastasis. Silencing Runx2 significantly restores tamoxifen sensitivity. Tamoxifen-resistant but not tamoxifen-sensitive patient ER+ breast cancer specimens also demonstrate strongly increased MNK phosphorylation of eIF4E. eIF4E levels, availability, and phosphorylation therefore promote tamoxifen resistance in ER+ breast cancer through selective mRNA translational reprogramming.

mTORC1/2 inhibition preserves ovarian function and fertility during genotoxic chemotherapy.

The ovary contains oocytes within immature (primordial) follicles that are fixed in number at birth. Activation of follicles within this fixed pool causes an irreversible decline in reproductive capacity, known as the ovarian reserve, until menopause. Premenopausal women undergoing commonly used genotoxic (DNA-damaging) chemotherapy experience an accelerated loss of the ovarian reserve, leading to subfertility and infertility. Therefore, there is considerable interest but little effective progress in preserving ovarian function during chemotherapy. Here we show that blocking the kinase mammalian/mechanistic target of rapamycin (mTOR) with clinically available small-molecule inhibitors preserves ovarian function and fertility during chemotherapy. Using a clinically relevant mouse model of chemotherapy-induced gonadotoxicity by cyclophosphamide, and inhibition of mTOR complex 1 (mTORC1) with the clinically approved drug everolimus (RAD001) or inhibition of mTORC1/2 with the experimental drug INK128, we show that mTOR inhibition preserves the ovarian reserve, primordial follicle counts, serum anti-Mullerian hormone levels (a rigorous measure of the ovarian reserve), and fertility. Chemotherapy-treated animals had significantly fewer offspring compared with all other treatment groups, whereas cotreatment with mTOR inhibitors preserved normal fertility. Inhibition of mTORC1 or mTORC1/2 within ovaries was achieved during chemotherapy cotreatment, concomitant with preservation of primordial follicle counts. Importantly, our findings indicate that as little as a two- to fourfold reduction in mTOR activity preserves ovarian function and normal birth numbers. As everolimus is approved for tamoxifen-resistant or relapsing estrogen receptor-positive breast cancer, these findings represent a potentially effective and readily accessible pharmacologic approach to fertility preservation during conventional chemotherapy.

Atypical ezrin localization as a marker of locally advanced breast cancer.

Locally advanced breast cancer (LABC) was initially characterized as a large primary tumor (≥5 cm), associated with or without skin or chest-wall involvement, fixed axillary lymph nodes, or disease spread to the ipsilateral internal mammary or supraclavicular nodes. Since 2002, LABC has been reclassified to include smaller stage IIB tumors (2 to <5 cm) with lymph node involvement, or stages IIIA-IIIB (≥5 cm) with or without nodal involvement. Despite the rather common presentation of LABC, it remains a poorly understood and highly variable clinical presentation of breast cancer that is a challenge to treatment. Here, we characterized a panel of breast tumors of known stage, grade, and key clinical-pathological parameters for the expression of the protein ezrin, which is involved in promoting signaling of the PI3K-Akt-mTOR pathway in response to extracellular and tumor micro-environmental signals, and is involved in breast cancer invasion and metastasis. We show that ezrin, which resides primarily in the apical membrane in normal breast epithelium, relocalizes primarily to the cytoplasm in >80 % of traditional (T3) invasive ductal LABC tumors (≥5 cm). Cytoplasmic ezrin is very strongly associated with a single characteristic in breast cancer-large tumor size. In contrast, in large non-malignant fibroadenomas, ezrin staining was similar to that of normal breast epithelium. Small (T1, 1 cm) invasive ductal carcinomas displayed largely apical membrane and perinuclear ezrin localization with weak cytoplasmic staining. Cytoplasmic ezrin localization was also associated with positive lymph node status, but no other clinical-pathological features, including hormone receptor status, histological or nuclear grade of tumor cell. The cytoplasmic relocalization of ezrin may therefore represent a novel marker for large malignant tumor size, reflecting the unique biology of LABC.

Inflammatory Breast Cancer Promotes Development of M2 Tumor-Associated Macrophages and Cancer Mesenchymal Cells through a Complex Chemokine Network.

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine-paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer. SIGNIFICANCE: This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3360/F1.large.jpg.

mTORC1 and -2 Coordinate Transcriptional and Translational Reprogramming in Resistance to DNA Damage and Replicative Stress in Breast Cancer Cells.

mTOR coordinates growth signals with metabolic pathways and protein synthesis and is hyperactivated in many human cancers. mTOR exists in two complexes: mTORC1, which stimulates protein, lipid, and ribosome biosynthesis, and mTORC2, which regulates cytoskeleton functions. While mTOR is known to be involved in the DNA damage response, little is actually known regarding the functions of mTORC1 compared to mTORC2 in this regard or the respective impacts on transcriptional versus translational regulation. We show that mTORC1 and mTORC2 are both required to enact DNA damage repair and cell survival, resulting in increased cancer cell survival during DNA damage. Together mTORC1 and -2 enact coordinated transcription and translation of protective cell cycle and DNA replication, recombination, and repair genes. This coordinated transcriptional-translational response to DNA damage was not impaired by rapalog inhibition of mTORC1 or independent inhibition of mTORC1 or mTORC2 but was blocked by inhibition of mTORC1/2. Only mTORC1/2 inhibition reversed cancer cell resistance to DNA damage and replicative stress and increased tumor cell killing and tumor control by DNA damage therapies in animal models. When combined with DNA damage, inhibition of mTORC1/2 blocked transcriptional induction more strongly than translation of DNA replication, survival, and DNA damage response mRNAs.

Gastric siderosis: An under-recognized and rare clinical entity.

The increased deposition of iron in gastric mucosa is known as gastric siderosis. It is believed that the only regulated step of the iron metabolism cycle occurs during absorption in the small intestine. Once this system becomes overwhelmed due to either local or widespread iron levels, then iron can be absorbed very quickly by a passive concentration-dependent mechanism. This excess iron is initially stored in the liver but later can be found in the pancreas, heart and joints. Excess iron is not expected to deposit in the gastric mucosa. This gastric deposition has been found in association with hemochromatosis, oral iron medications, alcohol abuse, blood transfusions, hepatic cirrhosis and spontaneous portacaval shunt with esophageal varices. The precise mechanism of this iron deposition in gastric epithelial and stromal cells is still not well understood; thus, identification of iron in gastric mucosa raises many questions. On histology, the pattern of deposition is variable, and recognition of the pattern is often useful to choose the appropriate workup for the patient and to diagnose and possibly treat the cause of iron overload. In this article, we have described a well-referenced review of this rare clinical entity with different histological patterns, diagnostic tests and the clinical significance of the different patterns of iron deposition.

The proteome signature of the inflammatory breast cancer plasma membrane identifies novel molecular markers of disease.

Inflammatory Breast Cancer (IBC) is the most lethal form of breast cancer with a 35% 5-year survival rate. The accurate and early diagnosis of IBC and the development of targeted therapy against this deadly disease remain a great medical challenge. Plasma membrane proteins (PMPs) such as E-cadherin and EGFR, play an important role in the progression of IBC. Because the critical role of PMPs in the oncogenic processes they are the perfect candidates as molecular markers and targets for cancer therapies. In the present study, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) followed by mass spectrometry analysis was used to compare the relative expression levels of membrane proteins (MP) between non-cancerous mammary epithelial and IBC cells, MCF-10A and SUM-149, respectively. Six of the identified PMPs were validated by immunoblotting using the membrane fractions of non-IBC and IBC cell lines, compared with MCF-10A cells. Immunohistochemical analysis using IBC, invasive ductal carcinoma or normal mammary tissue samples was carried out to complete the validation method in nine of the PMPs. We identified and quantified 278 MPs, 76% of which classified as PMPs with 1.3-fold or higher change. We identified for the first time the overexpression of the novel plasminogen receptor, PLGRKT in IBC and of the carrier protein, SCAMP3. Furthermore, we describe the positive relationship between L1CAM expression and metastasis in IBC patients and the role of SCAMP3 as a tumor-related protein. Overall, the membrane proteomic signature of IBC reflects a global change in cellular organization and suggests additional strategies for cancer progression. Together, this study provides insight into the specialized IBC plasma membrane proteome with the potential to identify a number of novel therapeutic targets for IBC.

Hyperactivated mTOR and JAK2/STAT3 Pathways: Molecular Drivers and Potential Therapeutic Targets of Inflammatory and Invasive Ductal Breast Cancers After Neoadjuvant Chemotherapy.

INTRODUCTION: Inflammatory breast cancer (IBC) is an aggressive and rare cancer with a poor prognosis and a need for novel targeted therapeutic strategies. Preclinical IBC data showed strong activation of the phosphatidylinositide-3-kinase/mammalian target of rapamycin (mTOR) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways, and expression of inflammatory cytokines and tumor-associated macrophages (TAMs). PATIENTS AND METHODS: Archival tumor tissue from 3 disease types (IBC treated with neoadjuvant chemotherapy [NAC], n = 45; invasive ductal carcinoma [IDC] treated with NAC [n = 24; 'treated IDC'; and untreated IDC [n = 27; 'untreated IDC']) was analyzed for the expression of biomarkers phospho-S6 (pS6) (mTOR), phospho-JAK2 (pJAK2), pSTAT3, interleukin (IL)-6, CD68 (monocytes, macrophages), and CD163 (TAMs). Surrounding nontumor tissue was also analyzed. RESULTS: Biomarker levels and surrogate activity according to site-specific phosphorylation were shown in the tumor tissue of all 3 disease types but were greatest in IBC and treated IDC and least in untreated IDC for pS6, pJAK2, pSTAT3, and IL-6. Of 37 IBC patients with complete biomarker data available, 100% were pS6-positive and 95% were pJAK2-positive. In nontumor tissue, biomarker levels were observed in all groups but were generally greatest in untreated IDC and least in IBC, except for JAK2. CONCLUSION: IBC and treated IDC display similar levels of mTOR and JAK2 biomarker activation, which suggests a potential mechanism of resistance after NAC. Biomarker levels in surrounding nontumor tissue suggested that the stroma might be activated by chemotherapy and resembles the oncogenic tumor-promoting environment. Activation of pS6 and pJAK2 in IBC might support dual targeting of the mTOR and JAK/STAT pathways, and the need for prospective studies to investigate combined targeted therapies in IBC.

Metastatic colonic adenocarcinoma in breast: report of two cases and review of the literature.

Metastatic adenocarcinoma to the breast from an extramammary site is extremely rare. In the literature, the most current estimate is that extramammary metastases account for only 0.43% of all breast malignancies and that, of these extramammary sites, colon cancer metastases form a very small subset. Most commonly seen metastasis in breast is from a contralateral breast carcinoma, followed by metastasis from hematopoietic neoplasms, malignant melanoma, sarcoma, lung, prostate, and ovary and gastric neoplasms. Here we present two rare cases, in which colonic adenocarcinomas were found to metastasize to the breast. In both cases, core biopsies were obtained from the suspicious areas identified on mammogram. Histopathology revealed neoplastic proliferation of atypical glandular components within benign breast parenchyma which were morphologically consistent with metastatic adenocarcinoma. By immunohistochemical staining, it was confirmed that the neoplastic components were immunoreactive to colonic markers and nonreactive to breast markers, thus further supporting the morphologic findings. It is extremely important to make this distinction between primary breast cancer and a metastatic process, in order to provide the most effective and appropriate treatment for the patient and to avoid any harmful or unnecessary surgical procedures.

Jorge Lobo's disease: a case of keloidal blastomycosis (lobomycosis) in a nonendemic area.

Lobomycosis or lacaziosis is a chronic subcutaneous fungal infection, caused by the fungus Lacazia loboi, which is phylogenetically related to Coccidioides, Blastomyces, Histoplasma, and Paracoccidioides. The disease was first recognized in 1931 by Jorge Lobo, who found the disease to be a keloidal blastomycosis and named it Jorge Lobo's disease. This case was perplexing initially as this fungal infection is very uncommon in the USA. However, with the ever-increasing frequency of international travel, many more cases of lobomycosis have been diagnosed in areas of nonendemicity, such as the USA, Europe, and South Africa. The clinical histories of such imported fungal infections often illustrate their long latency periods. In lobomycosis, the onset of the disease is usually insidious and often difficult to document. We describe a case of a New York resident who presented with multiple skin nodules over both his arms and forearms, and was subsequently diagnosed with Jorge Lobo's disease. The case, diagnosis, histopathologic findings, complication, and management of this rare clinical disease are discussed.

High levels of Hsp90 cochaperone p23 promote tumor progression and poor prognosis in breast cancer by increasing lymph node metastases and drug resistance.

p23 is a heat shock protein 90 (Hsp90) cochaperone located in both the cytoplasm and nucleus that stabilizes unliganded steroid receptors, controls the catalytic activity of certain kinases, regulates protein-DNA dynamics, and is upregulated in several cancers. We had previously shown that p23-overexpressing MCF-7 cells (MCF-7+p23) exhibit increased invasion without affecting the estrogen-dependent proliferative response, which suggests that p23 differentially regulates genes controlling processes linked to breast tumor metastasis. To gain a comprehensive view of the effects of p23 on estrogen receptor (ER)-dependent and -independent gene expression, we profiled mRNA expression from control versus MCF-7+p23 cells in the absence and presence of estrogen. A number of p23-sensitive target genes involved in metastasis and drug resistance were identified. Most striking is that many of these genes are also misregulated in invasive breast cancers, including PMP22, ABCC3, AGR2, Sox3, TM4SF1, and p8 (NUPR1). Upregulation of the ATP-dependent transporter ABCC3 by p23 conferred resistance to the chemotherapeutic agents etoposide and doxorubicin in MCF-7+p23 cells. MCF-7+p23 cells also displayed higher levels of activated Akt and an expanded phosphoproteome relative to control cells, suggesting that elevated p23 also enhances cytoplasmic signaling pathways. For breast cancer patients, tumor stage together with high cytoplasmic p23 expression more accurately predicted disease recurrence and mortality than did stage alone. High nuclear p23 was found to be associated with high cytoplasmic p23, therefore both may promote tumor progression and poor prognosis by increasing metastatic potential and drug resistance in breast cancer patients.

Essential role for eIF4GI overexpression in the pathogenesis of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer. IBC lethality derives from generation of tumour emboli, which are non-adherent cell clusters that rapidly spread by a form of continuous invasion known as passive metastasis. In most cancers, expression of E-cadherin, an epithelial marker, is indicative of low metastatic potential. In IBC, E-cadherin is overexpressed and supports formation of tumour emboli by promoting tumour cell interactions rather than adherence to stroma. E-cadherin, a surface component of adherens junctions, is anchored by interaction with p120 catenin (p120). We show that the unique pathogenic properties of IBC result in part from overexpression of the translation initiation factor eIF4GI in most IBCs. eIF4GI reprograms the protein synthetic machinery for increased translation of mRNAs with internal ribosome entry sites (IRESs) that promote IBC tumour cell survival and formation of tumour emboli. Overexpression of eIF4GI promotes formation of IBC tumour emboli by enhancing translation of IRES-containing p120 mRNAs. These findings provide a new understanding of translational control in the development of advanced breast cancer.

A hypoxia-controlled cap-dependent to cap-independent translation switch in breast cancer.

Translational regulation is critical in cancer development and progression. Translation sustains tumor growth and development of a tumor vasculature, a process known as angiogenesis, which is activated by hypoxia. Here we first demonstrate that a majority of large advanced breast cancers overexpress translation regulatory protein 4E-BP1 and initiation factor eIF4G. Using model animal and cell studies, we then show that overexpressed 4E-BP1 and eIF4G orchestrate a hypoxia-activated switch from cap-dependent to cap-independent mRNA translation that promotes increased tumor angiogenesis and growth at the level of selective mRNA translation. Elevated levels of 4E-BP1 trigger hypoxia inhibition of cap-dependent mRNA translation at high-oxygen levels and, with eIF4G, increase selective translation of mRNAs containing internal ribosome entry sites (IRESs) that include key proangiogenic, hypoxia, and survival mRNAs. The switch from cap-dependent to cap-independent mRNA translation facilitates tumor angiogenesis and hypoxia responses in animal models.