The Mechanisms Of Metadherin Modulating Breast Cancer Metastasis And Trail-Resistance

Breast cancer, as one of the most common malignant tumors, is a serious threat to women’s lives and health. Worldwide, an estimated1.3million new cases of invasive breast cancer are expected to occur among women each year, and over500,000women die of breast cancer.In China, the incidence and mortality of female breast cancer were increasing in recent years. According to the latest Ministry of Health statistics for2011, mainland China has more than126,000women suffer from breast cancer each year, among which37,000deaths were recored due to breast cancer relapse and metastasis. Currently, comprehensive treatment strategy based on surgery has achieved good efficiency; however, relapse and metastasis are still the major factors affecting survival rate and mortality rate of breast cancer. Therefore, exploring the molecular mechanisms of breast cancer development and progression as well as finding the important molecular targets have become the hot topic in translational medicine.Breast cancer is now considered a systemic disease, the initiation, progression and metastasis is the multigenic and multifactorial consequence. Inthepastfew decades, massive efforts in cancer research have led to the identification of a seemingly exhaustive list of oncogenes and tumor suppressor genes that are potential targets for anticancer therapeutics. Using computational algorithms, weand colleagues found the human MTDH gene was localized central at chromosome8q22, whichregion has been reported to be amplified in a number of malignancies. Metadherin （MTDH, also known as AEG-1,and Lyric）, was first reported in2002as a novel late response gene following HIV-1infection or treatment with recombinant HIV-1envelope glycoprotein （gp120）. The full-length cDNA has subsequently been independently cloned by four different groups of investigators. A "lung homing domain", which mediates lung metastasis in the4T1mouse mammary tumor cell, was identified in MTDH in2004with phage display screening and was subsequently named metadherin. Kang and colleagues first cloned and characterized the human full-length MTDH gene; it was found to encode a single-pass transmembrane protein with a calculated molecular mass of64KDa, containing12exons and11introns with a full-length of86,082bp, and a cDNA of3611bp （excluding the poly-A tail）.Further investigation demonstrated that MTDH is overexpressed in more than40%of breast cancers and has been elucidated to play a role in several significant stages of tumor progression, including transformation, initiation of apoptosis, invasion, metastasis, chemoresistance and angiogenesis.MTDH, as a novel oncogene, has been demonstrated to be critically involved in breast cancer progression and metastasis. In this study, we attempted to further investigate the potential role of MTDH in breast cancer development, progression and drug resistance. Based onthis, the thesiswas divided into three parts:Part I Identification of novel variants of Metadherin in breast cancerABSTRACTOncogenes are enssential to normal daily life and therefore are highly conservedin evolution.When the structure or regulatory regions of the oncogenes mutate, gene products increased or its activity enhanced, the cells will be excessive proliferating, thereby forming tumors.Various genetic or environmental factors will cause structural changeof oncogenes, which in turn leads to excessive or persistent cell growth-stimulating signals, causing cell transformation.DNA structural changes of oncogenes include point mutations, chromosomal translocations, insertional mutagenesis, gene amplification and so on. Identification of variants in the host can potentially facilitate the evaluation of the susceptibility of cancer, predict progression of disease or response to treatment. MTDH is a novel oncogene and there are no studies to date assessing variants of the MTDH gene and their potential relationship with breast cancer susceptibility.Here, we investigated the genetic polymorphisms in MTDH by direct full-length gene sequencing in a cohort of108breast cancer cases and100healthycontrols, with the intention of discovering of novel variants and comparing the distribution of variant genotype frequencies of these2affected vs. non-affected cohorts to determine whether a particular variant may influence susceptibility to breast cancer development.In consequence, thirteen variants were detected in the control group compared with eleven variants in the affected breast cancer cohort; of these,9unnamed, novel variants were discovered in sum. The novel and known variants appear to gather at both ends of MTDH gene, with3novel variants displayed in the central part of the gene in case group, but not in the control group.In addition to finding some known variants of MTDH, several novel variants of MTDH were discovered in our study that appear to be correlated to breast cancer susceptibility. However, additional studies need to be conducted in larger sample sizes to validate these findings and to further investigate whether these variants are prognostic in breast cancer patients. PART Ⅱ miR-30a suppresses breast tumor growth and metastasis by targeting Metadherin ABSTRACTIn contrast to well-confined primary breast cancer, metastatic breast cancer is always intractable because of its oftentimes surgically inoperable nature and the resistance of disseminated tumor cells to existing therapeutic agents. Despite much advancement in knowledge from more than a century of research, metastasis remains one of the most enigmatic aspects of the disease and the molecular pathways underlying each step are still obscure. Elucidation of the precise molecular circuitry that governs the metastasis process is critical for the reduction of mortality by breast cancer.Recently, with the latest deciphering of involvement of microRNAs （miRNAs） in numerous biological processes there are new hopes that the above scenario will be rapidly changed. The small （19-25nucleotides）, non-coding RNAs can modulate the expression of their cognate target genes by binding to the3’untranslated region （UTR） of target mRNAs, resulting in either translational inhibition or mRNA cleavage. Accumulating data have pointed to a central regulatory role for miRNAs in the initiation and development of breast cancer. The dysregulation of miRNA expression appears to be a general trait of metastatic breast cancer. Lately, miR-30a has attracted much attention due to its important role in various biological and pathological process, including development, differentiation_ENREF₄6, senescence, apoptosis and autophage. However, whether miR-30a is involved in the progression of breast cancer and the underlying mechanism remains poorly understood.Here, we intend to establish the role of miR-30a in breast cancer development and metastasis. Ectopic expression of miR-30a in breast cancer cell lines resulted in the suppression of cell growth and metastasis in vitro. Consistently, the xenograft mouse model also unveiled the suppressive effects of miR-30a on tumor growth and distal pulmonary metastasis. With dual luciferase reporter assay, we revealed that miR-30a could bind to the3’untranslated region of MTDH gene, thus exerting inhibitory effect on MTDH. Furthermore, we demonstrated that silence of MTDH could recapitulate the effects of miR-30a overexpression, while overexpression of MTDH could partially abrogate miR-30a-mediated suppression. Of significance, expression level of miR-30a was found to be significantly lower in primary breast cancer tissues than in the paired normal tissues. Further evaluation verified that miR-30a was negatively correlated with the extent of lymph node and lung metastasis in patients with breast cancer. In the present study, we showed that miR-30a was associated with breast cancer development and progression by directly targeting metadherin （MTDH）, suggesting a tumor-suppressor role of miR-30a in breast cancer. Our findings suggested that the restoration of miR-30a represents a promising therapeutic strategy for breast cancer treatment. PART IIIMetadherin modulates TRAIL-resistance in breast cancer ABSTRACTMTDH is overexpressed in more than40%of breast cancers. Recent studies have revealed MTDH favors oncogenic courses. In addition, a more general role for MTDH as a possible mediator of broad-spectrum chemoresistance has been indicated recently. NCI60pharmacogenomic data revealed a significant correlation between MTDH overexpression and resistance of cancer cells to a broader spectrum of chemical compounds. Subsequent in vitro and in vivo chemoresistance analyses showed that MTDH knock-down sensitizes several different breast cancer cell lines to paclitaxel, doxorubicin and cisplatin.Tumor necrosis factor-related apoptosis-inducing ligand （TRAIL） is a member of the TNF super family that can initiate apoptosis via activation of the extrinsic apoptosis pathway. It has emerged as a potential therapeutic agent due to its remarkable feature of selectively inducing apoptosis in cancer cells while sparing normal cells. TRAIL can effectively induce apoptosis in various types of cancer cells. Ongoing and completed phase I and phase II clinical trials with TRAIL are showing clinically promising outcomes with no apparent toxicity. However, recent studies have indicated that a variety of cancer cells are resistant to the apoptotic effects of TRAIL, and it is notable that the majority of breast cancer cells are resistant to TRAIL-mediated apoptosis. The mechanisms underlying resistance to TRAIL are not fully understood, and the identification of TRAIL resistance factors could facilitate the development of more effective TRAIL-based cancer therapies.Considering the role of MTDH in drug resistance, we hypothesized that MTDH may be a potential target to overcome TRAIL resistance of breast cancer cells. With a number of breast cancer cell lines and breast tumor samples, we found the relative expression of MTDH correlated with TRAIL sensitivity in breast cancer. In the study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3and PARP processing, increased Bcl-2expression and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD, suggesting that MTDH depletion stimulates activation of caspases. In MCF-7breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2expression contributes to TRAIL resistance. We further confirmed MTDH may control Bcl-2expression partly by suppressing miR-16.Collectively, we demonstrate that MTDH contributes to TRAIL resistance in breast cancer cells both in vitro and in vivo. Mechenicaly, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16mediated Bcl-2up-regulation and the extrinsic apoptosis pathway through caspase-8down-regulation.Our findings suggest that MTDH inhibition may be used to restore TRAIL sensitivity in TRAIL-resistant breast cancers.