The AKT pathway in human breast cancer: a tissue-array-based analysis

Department of Pathology, Cedars Sinai Medical Center, Los Angeles, CA 90048, USA.
Modern Pathology (Impact Factor: 6.19). 03/2006; 19(2):238-45. DOI: 10.1038/modpathol.3800525
Source: PubMed


The Akt pathway, an important regulator of cell proliferation and survival, is deregulated in many cancers. The pathway has achieved considerable importance due to the development of kinase inhibitors that are able to successfully reduce tumor growth. This study was conducted to determine the status of the Akt pathway in human breast cancers and to study the relationship between the different component proteins. Expression levels of PTEN, phosphorylated forms of the constituent proteins (Akt, FKHR, mTOR, and S6) and cyclin D1 were evaluated by immunohistochemistry, on consecutive sections from a tissue microarray containing 145 invasive breast cancers and 140 pure ductal carcinomas in-situ. Aberrant expression was correlated statistically with tumor characteristics and disease outcome. The Akt pathway was found to be activated early in breast cancer, in the in-situ stage. In all, 33, 15, 32, and 60% of ductal carcinoma in-situ showed overexpression of Akt, FKHR, mTOR, and cyclin D1. PTEN loss did not correlate statistically with expression of AKT or any of the other proteins with the exception of S6, indicating that Akt activation was not a result of PTEN loss. Expression levels of PTEN and S6 were significantly different in in-situ and invasive cancers, indicating association with disease progression. Loss of PTEN was noted in 11% of in-situ as compared to 26% of invasive cancers, while S6 overexpression was seen in 47% in-situ and in 72% invasive cancers. High-grade carcinomas were associated with PTEN loss, while low-grade carcinomas with good prognostic features showed cyclin D1 overexpression and were associated with longer disease free survival. Additionally, cancers with mTOR overexpression showed a three times greater risk for disease recurrence. Overall, a large proportion of in-situ and invasive breast cancers overexpressed cyclinD1 and S6. Our results may have significant implications in the development and application of targeted therapy.

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    • "Also, due to the nature of the tumor collection at the MBTB, the cases are biased to larger sized tumors [15,16]. Previously reported studies included cohorts which: were ERα negative with the majority being triple negative breast cancers [21], contained both ERα + and ERα- cases with the majority being defined as low risk (small tumor size and node negative) [32], had no information available concerning therapies [30], consisted of mainly familial breast cancer cases where few are ERα + [29] or consisted of a cohort in which only 50% of tumors were ERα+, or more than 60% of the women were under 50 years of age [31]. "
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    ABSTRACT: Introduction A phosphorylation score for estrogen receptor-alpha (ERα), called P7 score, was shown previously to be an independent prognostic factor in breast cancer patients treated with tamoxifen. Since mechanistic target of rapamycin (mTOR) activation is implicated in resistance to endocrine therapy in breast cancer we determined whether mechanistic target of rapamycin complex 1 (mTORC1) activation, measured by phosphorylation on S2448 (p-mTOR), was associated with the P7-score and/or clinical outcome in the same cohort. Methods mTOR phosphorylation status was determined at S2448 residue in vivo by immunohistochemistry in a cohort of more than 400 well-characterized ERα positive breast tumors. MCF7 cells were treated with estrogen and activation of mTOR pathway was determined by Western blotting. Results Contrary to earlier reports, p-mTOR expression, measured by immunohistochemistry, was negatively associated with size and nodal status. Additionally, p-S2448 mTOR expression was positively correlated with p-S118- ERα, p-S167-ERα and p-S282-ERα but negatively correlated with p-T311- ERα. Consistent with these, p-S2448 mTOR was negatively associated with P7-score and was significantly associated with overall survival (OS) (hazard ratio (HR) = 0.61, P = 0.028, 95% confidence interval (CI) 0.39 to 0.95, n = 337) and relapse-free survival (HR = 0.58, P = 0.0032, 95% CI 0.41 to 0.83, n = 337) following univariate but not multivariate analysis. Furthermore, we show that estrogen can regulate phosphorylation of mTOR and its down stream target p70S6 kinase. Additionally, recombinant mTOR can phosphorylate ERα in vitro. Conclusions These data suggest that in breast tumors where there is intact estrogen regulated signaling, mTOR is regulated by estrogen and therefore associated with an increased likelihood of responsiveness to endocrine therapy.
    Breast cancer research: BCR 05/2014; 16(3):R49. DOI:10.1186/bcr3660 · 5.49 Impact Factor
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    • "In breast cancer, 44.9% of tumor tissues had increased levels of mTOR, while 71.9% of invasive breast cancer tissues expressed high level of phosphorylated S6K1 protein[32]. Other studies reported overexpression of mTOR and its substrate in breast cancer tissue and cell lines[33], [34]. In our study, we found that knockdown of mTOR expression using mTOR siRNA decreased breast cancer cell viability and induced apoptosis, a similar outcome to that of tumor cells transfected with miR-99a mimics. "
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    ABSTRACT: MicroRNAs (miRNAs) play an important role in human tumorigenesis as oncogenes or tumor suppressors. miR-99a has been reported as a tumor suppressor gene in various cancers in humans. However, only limited information about the function of miR-99a in human breast cancers is available. Here we investigated the expression of miR-99a in breast cancer tissue specimens and its antitumor activity in breast cancer cells. We initially identified that the expression of miR-99a was significantly reduced in four breast cancer cell lines. More importantly, we found downregulation of miR-99a in breast cancer specimens from ten different patients. We then analyzed the mechanism of miR-99a in inhibiting tumorigenesis. Cell-based assays that showed overexpression of miR-99a not only reduced breast cancer cell viability by inducing accumulation of cells at sub-G1 phase and cell apoptosis, but also inhibited tumorigenicity in vivo. As a critical miR-99a target, we have shown that the function of mammalian target of rapamycin (mTOR) was greatly inhibited by miR-99a-based Luciferase report assay; overexpression of miR-99a reduced the expression of mTOR and its downstream phosphorylated proteins (p-4E-BP1 and p-S6K1). Similar to restoring miR-99a expression, mTOR downregulation suppressed cell viability and increased cell apoptosis, whereas restoration of mTOR expression significantly reversed the inhibitory effects of miR-99a on the mTOR/p-4E-BP1/p-S6K1 signal pathway and the miR-99a antitumor activity. In clinical specimens and cell lines, mTOR was commonly overexpressed and its protein levels were statistically inversely correlated with miR-99a expression. Taken together, these results have demonstrated that miR-99a antitumor activity is achieved by targeting the mTOR/p-4E-BP1/p-S6K1 pathway in human breast cancer cells. This study suggests a potential therapeutic strategy to effectively control breast cancer development.
    PLoS ONE 03/2014; 9(3):e92099. DOI:10.1371/journal.pone.0092099 · 3.23 Impact Factor
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    • "We sought to investigate the effect of radiation and its underlying molecular mechanisms in DCIS. As human DCIS lesions were found to up-regulate p-Akt, we modeled DCIS by propagating MCF10A human mammary epithelial cells with inducible active ER-Akt [18,24]. Typically, MCF10A cells form a hollow lumen in three-dimensional lrECM [20], recapitulating normal in vivo ductal structures. "
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    ABSTRACT: Ductal carcinoma in situ (DCIS) is characterized by non-invasive cancerous cell growth within the breast ducts. Although radiotherapy is commonly used in the treatment of DCIS, the effect and molecular mechanism of ionizing radiation (IR) on DCIS are not well understood, and invasive recurrence following radiotherapy remains a significant clinical problem. This study investigated the effects of IR on a clinically relevant model of Akt-driven DCIS, and identified possible molecular mechanisms underlying invasive progression in surviving cells. We measured the level of phosphorylated-Akt (p-Akt) in a cohort of human DCIS specimens by immunohistochemistry (IHC) and correlated it with recurrence risk. To model human DCIS, we used Akt overexpressing human mammary epithelial cells (MCF10A-Akt), which, in 3-dimensional laminin-rich extracellular matrix (3D lrECM) and in vivo form organotypic DCIS-like lesions with lumena expanded by pleiomorphic cells contained within an intact basement membrane. In a population of cells that survived significant IR doses in 3D lrECM, a malignant phenotype emerged creating a model for invasive recurrence. P-Akt was up-regulated in clinical DCIS specimens, and was associated with recurrent disease. MCF10A-Akt cells that formed DCIS-like structures in 3D lrECM showed significant apoptosis after IR, preferentially in the luminal compartment. Strikingly, when cells that survived IR were repropagated in 3D lrECM, a malignant phenotype emerged, characterized by enhanced invasive activity, up-regulation of fibronectin, alpha5beta1-integrin, MMP-9 and loss of E-cadherin. In addition, IR induced nuclear translocation and binding of NF-kappaB to the beta1-integrin promoter region, associated with up-regulation of alpha5beta1-integrins. Inhibition of NF-kappaB or beta1-integrin signaling abrogated emergence of the invasive activity. P-Akt is up-regulated in some human DCIS lesions, and is possibly associated with recurrence. MCF10A-Akt cells form organotypic DCIS-like lesions in 3D lrECM and in vivo, and are a plausible model for some forms of human DCIS. A population of Akt-driven DCIS-like spheroids that survive IR progresses to an invasive phenotype in 3D lrECM mediated by beta1-integrin and NF-kappaB signaling.
    Breast cancer research: BCR 07/2013; 15(4):R60. DOI:10.1186/bcr3454 · 5.49 Impact Factor
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