MTDH/AEG-1-based DNA vaccine suppresses lung metastasis and enhances chemosensitivity to doxorubicin in breast cancer

The gene MTDH/AEG-1 is overexpressed in more than 40% of breast cancer patients, and it is associated with poor clinical outcomes. Previous studies have indicated that MTDH/AEG-1 could promote metastatic lung-seeding and enhance chemoresistance. Therefore, MTDH/AEG-1 could be a candidate target against breast cancer lung metastasis. We demonstrated that MTDH/AEG-1-based DNA vaccine, delivered by attenuated Salmonella typhimurium, could evoke strong CD8⁺ cytotoxic-T-cell mediated immune responses against breast cancer. This vaccine showed anti-tumor growth and metastasis efficacy in a prophylactic setting. Importantly, in a therapeutic model, MTDH/AEG-1 vaccine was proved to increase chemosensitivity to doxorubicin and inhibit breast cancer lung metastasis. This vaccine could also prolong the life span of tumor-bearing mice without significant side effects in vivo. These results suggested that this novel DNA vaccine was effective in the inhibition of breast cancer growth and metastasis, and this vaccine in combination with chemotherapies offered new strategies for the clinical therapeutics of breast cancer metastasis.     

Knockdown of MTDH sensitizes endometrial cancer cells to cell death induction by death receptor ligand TRAIL and HDAC inhibitor LBH589 co-treatment

Understanding the molecular underpinnings of chemoresistance is vital to design therapies to restore chemosensitivity. In particular, metadherin (MTDH) has been demonstrated to have a critical role in chemoresistance. Over-expression of MTDH correlates with poor clinical outcome in breast cancer, neuroblastoma, hepatocellular carcinoma and prostate cancer. MTDH is also highly expressed in advanced endometrial cancers, a disease for which new therapies are urgently needed. In this present study, we focused on the therapeutic benefit of MTDH depletion in endometrial cancer cells to restore sensitivity to cell death. Cells were treated with a combination of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), which promotes death of malignant cells of the human reproductive tract, and histone deacetylase (HDAC) inhibitors, which have been shown to increase the sensitivity of cancer cells to TRAIL-induced apoptosis. Our data indicate that depletion of MTDH in endometrial cancer cells resulted in sensitization of cells that were previously resistant in response to combinatorial treatment with TRAIL and the HDAC inhibitor LBH589. MTDH knockdown reduced the proportion of cells in S and increased cell arrest in G2/M in cells treated with LBH589 alone or LBH589 in combination with TRAIL, suggesting that MTDH functions at the cell cycle checkpoint to accomplish resistance. Using microarray technology, we identified 57 downstream target genes of MTDH, including calbindin 1 and galectin-1, which may contribute to MTDH-mediated therapeutic resistance. On the other hand, in MTDH depleted cells, inhibition of PDK1 and AKT phosphorylation along with increased Bim expression and XIAP degradation correlated with enhanced sensitivity to cell death in response to TRAIL and LBH589. These findings indicate that targeting or depleting MTDH is a potentially novel avenue for reversing therapeutic resistance in patients with endometrial cancer.     

Prognostic impact of Metadherin–SND1 interaction in colon cancer

The interaction between Metadherin (MTDH) and Staphylococcal nuclease homology domain containing 1 (SND1) is involved in tumorigenesis and tumor progression of several human malignancies. However, its roles in colon cancer are still unclear. To investigate the clinical value of MTDH and SND1 expression in colon cancer. Immunohistochemical staining was performed to detect the expression of MTDH and SND1 using human colon cancer and their corresponding non-cancerous colon tissues from 196 patients’ biopsies. Positive expression of MTDH and SND1 were both increased in colon cancer tissues compared to paired non-cancerous colon tissues. There was a positive correlation between MTDH and SND1 expression in colon cancer tissues (r?=?0.86, p?<?0.001). In addition, their positive expression were both significantly associated with nodal status (both p?=?0.02), pathological stage (p?=?0.006 and 0.008, respectively) and differentiation (both p?=?0.03). Moreover, the overall survival in colon cancer patients with positive expression of MTDH and SND1 were significantly shorter than those without their expression (both p?=?0.01). Furthermore, multivariate Cox regression analysis suggested that positive expression of MTDH and SND1 was an independent poor prognostic predictor in colon cancer. Our data suggest that the increased expression of MTDH and/or SND1 is closely related to carcinogenesis, progression, and prognosis of colon cancer. The co-expression of MTDH/SND1 may be a novel distinctive marker to benefit us in prediction of the prognosis in colon cancer.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

Metadherin mediates lipopolysaccharide-induced migration and invasion of breast cancer cells

Background

Breast cancer is the most prevalent cancer in women worldwide and metastatic breast cancer has very poor prognosis. Inflammation has been implicated in migration and metastasis of breast cancer, although the exact molecular mechanism remains elusive.

Principal Findings

We show that the pro-inflammatory endotoxin Lipopolysaccharide (LPS) upregulates the expression of Metadherin (MTDH), a recently identified oncogene, in a number of breast cancer lines. Stable knockdown of MTDH by shRNA in human breast MDA-MB-231 cells abolishes LPS-induced cell migration and invasion as determined by several in vitro assays. In addition, knockdown of MTDH diminishes Nuclear Factor-kappa B (NF-κB) activation by LPS and inhibited LPS-induced IL-8 and MMP-9 production.

Conclusions

These results strongly suggest that MTDH is a pivotal molecule in inflammation-mediated tumor metastasis. Since NF-κB, IL-8 and MMP-9 play roles in LPS-induced invasion or metastasis, the mechanism of MTDH-promoted invasion and metastasis may be through the activation of NF-κB, IL-8 and MMP-9, also suggesting a role of MTDH in regulating both inflammatory responses and inflammation-associated tumor invasion. These findings indicate that MTDH is involved in inflammation-induced tumor progression, and support that MTDH targeting therapy may hold promising prospects in treating breast cancer.     

Nicotine induces cell survival and chemoresistance by stimulating Mcl-1 phosphorylation and its interaction with Bak in lung cancer

Nicotine is a major carcinogen in cigarettes, which can enhance cell proliferation and metastasis and increase the chemoresistance of cancer cells. Our previous data found that nicotine promotes cell survival in lung cancer by affecting the expression of antiapoptotic protein Mcl-1, suggesting that the Mcl-1 may be a therapeutic target for patients with lung cancer. In this study, we found that the effects of drug resistance on nicotine-induced lung cancer cell lines were shown to influence the phosphorylation of Mcl-1. Moreover, nicotine induces Mcl-1 phosphorylation exclusively at the T163 site, which results in enhancement of the antiapoptotic activity of Mcl-1 and increased cell survival. Meanwhile, nicotine can reduce the sensitivity of H1299 cells to CDDP via enhancement of the binding of Mcl-1 to Bak, which inhibits the proapoptotic effect of Bak and ultimately leads to increased survival and drug resistance of lung cancer cells. Thus, nicotine-induced cell survival and chemoresistance may occur in a mechanism by stimulating Mcl-1 phosphorylation and its interaction with Bak, which may contribute to improving the efficacy of chemotherapy in the treatment of human lung cancer.     

shRNA下调MTDH基因抑制人乳腺癌MCF-7细胞HIF-1α及VEGF表达

目的：探讨转移粘附基因（metadherin,MTDH）的表达对人乳腺癌细胞中肿瘤血管生成相关分子标志物缺氧诱导因子-1α（HIF-1α）及血管内皮生长因子（VEGF）表达的影响。方法：将针对MTDH基因的干扰质粒MTDH-shRNA转染乳腺癌MCF-7细胞,RT-PCR及Western blot验证其对MTDH基因的沉默效果;应用Western blot检测转染前后MCF-7细胞中缺氧诱导因子-1α（HIF-1α）及血管内皮生长因子（VEGF）在蛋白水平上的表达变化;MTT实验检测下调MTDH对MCF-7细胞增殖情况的影响。结果：MCF-7细胞转染48小时后,MTDH-shRNA转染组和MTDH-shRNA-neg转染组转染效率约70%。MTDH-shRNA转染组中MTDH在mRNA及蛋白水平上表达明显下调,此外HIF-1α及VEGF蛋白表达明显降低,与对照组比较差异有统计学意义（P〈0.05）。MTDH-shRNA转染组MCF-7细胞增殖明显受到抑制,与对照组比较差异有统计学意义（P〈0.05）。结论：在乳腺癌MCF-7细胞中下调MTDH基因可以抑制HIF-1α、VEGF表达及细胞增殖,提示MTDH基因可能对乳腺癌肿瘤血管生成有促进作用。     

Molecular Modification of Metadherin/MTDH Impacts the Sensitivity of Breast Cancer to Doxorubicin

BackgroundBreast cancer is a leading cause of death in women and with an increasing worldwide incidence. Doxorubicin, as a first-line anthracycline-based drug is conventional used on breast cancer clinical chemotherapy. However, the drug resistances limited the curative effect of the doxorubicin therapy in breast cancer patients, but the molecular mechanism determinants of breast cancer resistance to doxorubicin chemotherapy are not fully understood. In order to explore the association between metadherin (MTDH) and doxorubicin sensitivity, the differential expressions of MTDH in breast cancer cell lines and the sensitivity to doxorubicin of breast cancer cell lines were investigated.MethodsThe mRNA and protein expression of MTDH were determined by real-time PCR and Western blot in breast cancer cells such as MDA-MB-231, MCF-7, MDA-MB-435S, MCF-7/ADR cells. Once MTDH gene was knocked down by siRNA in MCF-7/ADR cells and overexpressed by MTDH plasmid transfection in MDA-MB-231 cells, the cell growth and therapeutic sensitivity of doxorubicin were evaluated using MTT and the Cell cycle assay and apoptosis rate was determined by flow cytometry.ResultsMCF-7/ADR cells revealed highly expressed MTDH and MDA-MB-231 cells had the lowest expression of MTDH. After MTDH gene was knocked down, the cell proliferation was inhibited, and the inhibitory rate of cell growth and apoptosis rate were enhanced, and the cell cycle arrest during the G0/G1 phase in the presence of doxorubicin treatment. On the other hand, the opposite results were observed in MDA-MB-231 cells with overexpressed MTDH gene.ConclusionMTDH gene plays a promoting role in the proliferation of breast cancer cells and its high expression may be associated with doxorubicin sensitivity of breast cancer.     

Extracellular ATP promotes breast cancer chemoresistance via HIF-1α signaling

We have previously demonstrated that extracellular adenosine 5''-triphosphate (ATP) promotes breast cancer cell chemoresistance. However, the underlying mechanism remains unclear. Using a cDNA microarray, we demonstrated that extracellular ATP can stimulate hypoxia-inducible factor (HIF) signaling. In this study, we report that hypoxia-inducible factor 1α (HIF-1α) was upregulated after ATP treatment and mediated the ATP-driven chemoresistance process. We aimed to investigate the mechanisms and identify potential clinically relevant targets that are involved. Using mass spectrometry, we found that aldolase A (ALDOA) interacts with HIF-1α and increases HIF-1α expression. We then demonstrated that STAT3-ALDOA mediates ATP-HIF-1α signaling and upregulates the HIF-1 target genes adrenomedullin (ADM) and phosphoinositide-dependent kinase-1 (PDK1). Moreover, we show that PI3K/AKT acts upstream of HIF-1α in ATP signaling and contributes to chemoresistance in breast cancer cells. In addition, HIF-1α-knockdown or treatment with direct HIF inhibitors combined with the ATP hydrolase apyrase in MDA-MB-231 cells induced enhanced drug sensitivity in nude BALB/c mice. We then used in vitro spheroid formation assays to demonstrate the significance of ATP-HIF-1α in mediating chemoresistance. Furthermore, considering that indirect HIF inhibitors are effective in clinical cancer therapy, we treated tumor-bearing BALB/c mice with STAT3 and PI3K/AKT inhibitors and found that the dual-targeting strategy sensitized breast cancer to cisplatin. Finally, using breast cancer tissue microarrays, we found that ATP-HIF-1α signaling is associated with cancer progression, poor prognosis, and resistance to chemotherapy. Taken together, we suggest that HIF-1α signaling is vital in ATP-driven chemoresistance and may serve as a potential target for breast cancer therapies.Subject terms: Breast cancer, Cell signalling     

A novel long non-coding RNA AC073352.1 promotes metastasis and angiogenesis via interacting with YBX1 in breast cancer

Breast cancer is the major cause of cancer death worldwide in women. Patients with metastasis have poor prognosis and the mechanisms of breast cancer metastasis are not completely understood. Long non-coding RNAs (lncRNAs) have been shown to have crucial roles in breast cancer development and progression. However, the underlying mechanisms by which lncRNA-driven breast cancer metastasis are unknown. The main objective of this paper is to explore a functional lncRNA and its mechanisms in breast cancer. Here we identified a novel lncRNA AC073352.1 that was significantly upregulated in breast cancer tissues and was associated with advanced TNM stages and poor prognosis in breast cancer patients. In addition, AC073352.1 was found to promote the migration and invasion of breast cancer cells in vitro and enhance breast cancer metastasis in vivo. Mechanistically, we elucidated that AC073352.1 interacted with YBX1 and stabilized its protein expression. Knock down of YBX1 reduced breast cancer cell migration and invasion and could partially reverse the stimulative effects of AC073352.1 overexpressed on breast cancer metastasis. Moreover, AC073352.1 might be packaged into exosomes by binding to YBX1 in breast cancer cells resulting in angiogenesis. Collectively, our results demonstrated that AC073352.1 promoted breast cancer metastasis and angiogenesis via binding YBX1, and it could serve as a promising, novel biomarker for prognosis and a therapeutic target in breast cancer.Subject terms: Breast cancer, Cell invasion, Long non-coding RNAs     

乳腺癌中Abi1和c-Abl及WAVE 2的表达及其相互关系

目的研究乳腺癌组织中Abi1、c-Abl和WAVE2蛋白的表达及其相互关系。方法应用免疫组化SP法检测66例乳腺癌组织和24例正常乳腺组织中Abi1、c-Abl和WAVE2蛋白的表达情况。结果 1.正常乳腺组织与乳腺癌组织相比,Abi1和WAVE2蛋白表达有显著性差异(P0.05),而c-Abl蛋白表达无显著性差异(P0.05),但有定位的改变。2.Abi1强阳性率与乳腺癌的肿瘤大小、组织学分级、淋巴结转移及临床分期呈负相关性(P0.05);与患者年龄无关(P0.05)。c-Abl阳性率及WAVE2强阳性率均与乳腺癌的组织学分级、淋巴结转移及临床分期呈负相关性(P0.05);与患者年龄及肿瘤大小无关(P0.05)。3.Abi1蛋白表达与c-Abl和WAVE2蛋白表达呈正相关(P0.05)。结论乳腺癌中Abi1低表达与预后不良有关。乳腺癌中Abi1蛋白表达的变化可影响c-Abl蛋白的定位和WAVE2蛋白的表达。推测Abi1在Abl/Abi1/WAVE2通路中具有至关重要的地位。     

YTHDF1 promotes breast cancer cell growth,DNA damage repair and chemoresistance

Chemoresistance represents a major obstacle to the treatment of human cancers. Increased DNA repair capacity is one of the important mechanisms underlying chemoresistance. In silico analysis indicated that YTHDF1, an m6A binding protein, is a putative tumor promoter in breast cancer. Loss of function studies further showed that YTHDF1 promotes breast cancer cell growth in vitro and in vivo. YTHDF1 facilitates S-phase entry, DNA replication and DNA damage repair, and accordingly YTHDF1 knockdown sensitizes breast cancer cells to Adriamycin and Cisplatin as well as Olaparib, a PARP inhibitor. E2F8 is a target molecule by YTHDF1 which modulates E2F8 mRNA stability and DNA damage repair in a METTL14-dependent manner. These data demonstrate that YTHDF1 has a tumor-promoting role in breast cancer, and is a novel target to overcome chemoresistance.Subject terms: Breast cancer, Breast cancer     

Genome-wide 5-Hydroxymethylcytosine Profiling Analysis Identifies MAP7D1 as A Novel Regulator of Lymph Node Metastasis in Breast Cancer

Although DNA 5-hydroxymethylcytosine(5 hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5 hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5 hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5 hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genomewide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated5 hmC signatures, and it is found that increased levels of DNA 5 hmC and gene expression of MAP7 D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7 D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic5 hmC profiles during lymph node metastasis suggest a link between DNA 5 hmC and lymph node metastasis. Meanwhile, the role of MAP7 D1 in breast cancer progression suggests that the metastasis-associated 5 hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.     

Identification of novel variants of metadherin in breast cancer

Metadherin (MTDH, also known as AEG-1, and Lyric) has been demonstrated to play a potential role in several significant aspects of tumor progression. It has been reported that overexpression of MTDH is associated with progression of disease and poorer prognosis in breast cancer. However, there are no studies to date assessing variants of the MTDH gene and their potential relationship with breast cancer susceptibility. Thus, we investigated all variants of the MTDH gene and explored the association of the variants with breast cancer development. Our cohort consisted of full-length gene sequencing of 108 breast cancer cases and 100 healthy controls; variants were detected in 11 breast cancer cases and 13 controls. Among the variants detected, 9 novel variants were discovered and 2 were found to be associated with the susceptibility of breast cancer. 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.     

Nuclear import of PTPN18 inhibits breast cancer metastasis mediated by MVP and importin β2

Distant metastasis is the primary cause of breast cancer-associated death. The existing information, such as the precise molecular mechanisms and effective therapeutic strategies targeting metastasis, is insufficient to combat breast cancer. This study demonstrates that the protein tyrosine phosphatase PTPN18 is downregulated in metastatic breast cancer tissues and is associated with better metastasis-free survival. Ectopic expression of PTPN18 inhibits breast cancer cell metastasis. PTPN18 is translocated from the cytoplasm to the nucleus by MVP and importin β2 in breast cancer. Then, nuclear PTPN18 dephosphorylates ETS1 and promotes its degradation. Moreover, nuclear PTPN18 but not cytoplasmic PTPN18 suppresses transforming growth factor-β signaling and epithelial-to-mesenchymal transition by targeting ETS1. Our data highlight PTPN18 as a suppressor of breast cancer metastasis and provide an effective antimetastatic therapeutic strategy.Subject terms: Breast cancer, Epithelial-mesenchymal transition     

GTSE1 Is a Microtubule Plus-End Tracking Protein That Regulates EB1-Dependent Cell Migration

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.