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.

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.

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.

Translational control in cancer.

Remarkable progress has been made in defining a new understanding of the role of mRNA translation and protein synthesis in human cancer. Translational control is a crucial component of cancer development and progression, directing both global control of protein synthesis and selective translation of specific mRNAs that promote tumour cell survival, angiogenesis, transformation, invasion and metastasis. Translational control of cancer is multifaceted, involving alterations in translation factor levels and activities unique to different types of cancers, disease stages and the tumour microenvironment. Several clinical efforts are underway to target specific components of the translation apparatus or unique mRNA translation elements for cancer therapeutics.

Inflammatory breast cancer cells are constitutively adapted to hypoxia.

We recently showed that overexpression of translation initiation factor eIF4G partially drives the unusual pathological features of Inflammatory Breast Cancer (IBC), the most lethal form of primary breast cancer. IBC has the peculiar feature that, rather than develop as a solid tumor, it typically generates rapidly metastasizing tight clusters of cancer cells referred to as tumor cell emboli, consisting of cancer cells held together by increased membrane expression of E-cadherin. Overexpression of eIF4GI in IBC leads to a specific increase in the translation of internal ribosomal entry site (IRES) containing mRNAs, of which two encode key proteins involved in the pathological features of IBC. One of these mRNAs encodes p120 catenin, which mediates E-cadherin retention at the cell surface, and the other encodes VEGF, which accounts for high levels of IBC angiogenesis and resistance to hypoxia. Here we show that IBC cells have adapted to the persistent hypoxia they experience as tumor emboli, by reprogramming the protein synthesis machinery to constitutively translate mRNAs required for IBC cell survival during hypoxia, even under normal oxygen (normoxic) conditions. Thus, IBC cells have been able to behave as if they are continuously hypoxic even when they are not.

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.

Effect of Ku proteins on IRES-mediated translation.

BACKGROUND INFORMATION: Ku is an abundant nuclear heterodimeric protein composed of 70 and 86 kDa subunits. As an activator of the catalytic subunit of DNA-PK (DNA-dependent protein kinase), Ku plays an important role in DNA repair and recombination. Ku is also involved in actions independent of DNA-PK, such as transcription regulation and telomere maintenance. Although Ku is localized in the cytoplasm under specific cellular conditions, no functions for Ku outside of the nucleus have as yet been reported. In addition to DNA binding, Ku binds specific RNA sequences with high affinity. However, no specific cellular mRNA targets for Ku have been identified. RESULTS: In a yeast three-hybrid system, Ku70 bound to an RNA bait that contained an IRES (internal ribosomal entry site) element. A single band with migration properties similar to those of Ku70 was immunoprecipitated with anti-Ku antibody, using UV cross-linked complexes formed by HeLa cell nuclear extracts and an IRES-containing RNA probe. IRES activity was reduced in Ku80(-/-) cells. Overexpression of Ku proteins stimulated IRES-dependent translation. CONCLUSIONS: The present study suggests that Ku binds IRES elements within RNA molecules, and that Ku plays a role in the modulation of IRES-mediated mRNA translation.