Combined use of alcohol in conventional chemical-induced mouse liver cancer model improves the simulation of clinical characteristics of human hepatocellular carcinoma.

Liver cancer is the one of most common types of cancer and the 2nd cause of cancer-associated mortalities worldwide. Establishing appropriate animal models of liver cancer is essential for basic and translational studies. The present study evaluated the effects of the combined use of alcohol with a conventional chemical-induced mouse liver cancer model. The treatment of alcohol/diethylnitrosamine (DEN)/carbon tetrachloride (CCl4) in the mice of experimental groups resulted in a series of pathological changes in the liver. Liver inflammation, fibrosis, cirrhosis and hepatocellular carcinoma were identified, and this method used less time (1-5 months) for inducement compared with the conventional chemical-induced method alone. In addition, murine α-fetoprotein (mAFP) was expressed throughout and ultrastructural features met the criteria for liver cancer. Fatty degeneration of pancreas, reduced blood glucose levels, and increased spleen weight were observed. These results indicated that an AFP-secreting hepatocellular carcinoma model of BALB/c mouse was successfully developed. The disease process and morphological changes met the criterion of the liver cancer process. Therefore the model developed in the present study may be an ideal animal model for studying the occurrence and development of liver cancer.

Metastasis-Associated Protein 1 Deficiency Results in Compromised Pulmonary Alveolar Capillary Angiogenesis in Mice.

BACKGROUND The aim of this study was to investigate the effects of metastasis-associated protein 1 (MTA1) deficiency during angiogenesis of pulmonary alveolar capillaries in mice and to determine the molecular mechanisms involved. MATERIAL AND METHODS The expressions of MTA1, CD34, vascular endothelial growth factor (VEGF), alpha smooth muscle actin (α-SMA), and HIF-1α were analyzed in the lungs of MTA1-knockout (KO) and wild-type mice at embryonic day 18.5 and 2 months by quantitative PCR, immunoblotting, and immunohistochemistry. The morphological changes were investigated during pulmonary alveolar capillary formation. The heart weight/body weight (HW/BW) ratio and the size of the right ventricular wall cardiomyocytes were also measured. Regulation of MTA1 on HIF-1α was determined in vitro. RESULTS MTA1 deficiency reduced the number of pulmonary alveolar capillaries compared to the wild-type mice. MTA1-KO mice exhibited a decreased expression of HIF-1α and VEGF in the lungs. The retarded growth of the MTA1-KO mice was also noticed during the first week after birth. Accordingly, MTA1 deficiency resulted in increased infant mortality. In surviving adult mice, MTA1 deficiency induced myocardial hypertrophy, highlighted by an increased heart weight/body weight ratio and larger cardiomyocytes. In cultured cells, HIF-1α and VEGF levels were significantly upregulated upon MTA1 overexpression, suggesting a close relationship between all 3 molecules. CONCLUSIONS MTA1 participates in the formation of pulmonary capillaries via stabilization of HIF-1α. This finding sheds new light on the function of MTA1 in lung development, opening new avenues for the diagnosis/treatment of related pulmonary diseases.

Epithelial-mesenchymal transition as strategic microenvironment mimicry for cancer cell survival and immune escape?

Epithelial-mesenchymal transition (EMT) is the phenotypic transition of epithelial cells to mesenchymal cells characterized by loss of epithelial markers, loss of intercellular adherence and acquirement of mesenchymal cell markers and increased locomotive ability. EMT is widely considered to be a gene regulated process necessary for cancer metastasis. Yet it is a highly controversial issue. We here propose that EMT is an environmentally induced cell behavior. It is the mimicry of their living environment. It is a survival strategy, a way of immune escape. We also propose here that the epithelial cell markers may functionally act as tumor antigens since in the mesenchymal surroundings there are no other structures bearing the same antigens as epithelial cells.

Differential distribution of immune cells in breast invasive carcinoma vs. breast carcinoma in situ and its significance in interpretation of immune surveillance.

Immune surveillance is a highly controversial subject in both the field of immunology and cancer biology. On one hand, in spite of extensive studies, there is no cancer specific antigens identified. Yet, the organisms do exert immune response to tumors. On the other hand, it is believed that immune surveillance suppresses tumorigenesis by eradicating mutated cells. However, it is also widely known that tumorigenesis is promoted by inflammation, which is in nature immune reaction. In the present study, we tried to find immune cells in early tumor lesions for the supportive or negative evidence of immune surveillance. We used immunohistochemistry to observe the localization and distribution of immune cells in the in situ carcinoma lesions and in the invasive cancer of breast. Interestingly, we did not see immune cells in either ductal carcinoma in situ (DCIS) or lobular carcinoma in situ (LCIS) of breast, the two basic supposed early cancer forms. In contrast, we observed extensive infiltration of immune cells in the invasive breast cancer, and close contact between immune cells and tumor cells. Based on these findings, we propose that the tumor antigens of breast cancer are not derived from the gene mutation or amplification such as HER2, but rather from misplacement of epithelial cells in the mesenchymal tissue. To avoid being targeted by the immune system, the carcinoma cells exert epithelial-mesenchymal transition (EMT). Therefore, immunosurveillance could be regarded as preventing the intrusion of epithelial cells to mesenchymal tissues, and EMT is a form of immune escape by the strategy of mimicry.

Structure, expression and functions of MTA genes.

Metastatic associated proteins (MTA) are integrators of upstream regulatory signals with the ability to act as master coregulators for modifying gene transcriptional activity. The MTA family includes three genes and multiple alternatively spliced variants. The MTA proteins neither have their own enzymatic activity nor have been shown to directly interact with DNA. However, MTA proteins interact with a variety of chromatin remodeling factors and complexes with enzymatic activities for modulating the plasticity of nucleosomes, leading to the repression or derepression of target genes or other extra-nuclear and nucleosome remodeling and histone deacetylase (NuRD)-complex independent activities. The functions of MTA family members are driven by the steady state levels and subcellular localization of MTA proteins, the dynamic nature of modifying signals and enzymes, the structural features and post-translational modification of protein domains, interactions with binding proteins, and the nature of the engaged and resulting features of nucleosomes in the proximity of target genes. In general, MTA1 and MTA2 are the most upregulated genes in human cancer and correlate well with aggressive phenotypes, therapeutic resistance, poor prognosis and ultimately, unfavorable survival of cancer patients. Here we will discuss the structure, expression and functions of the MTA family of genes in the context of cancer cells.

Reasons for cancer metastasis: A holistic perspective.

Over several years, scientists investigating cancer have focused their efforts on elucidating the mechanisms underlying cancer metastasis, with the aim of finding a way to inhibit this process. These mechanisms, however, only explain the process of cancer metastasis, but do not explain why cancer would metastasize in the first place. Cancer metastasizes due to several factors, namely attack by the immune system, lack of oxygen and necessary nutrients, large amounts of lactic acid produced by glycolysis and increased cell death. Therefore, the majority of the presently available treatments for cancer also bear the potential to induce metastasis. Thus, it is crucial in medical practice to minimize the risk of cancer metastasis during a time when there are no effective means to inhibit this process.

MTA1--a stress response protein: a master regulator of gene expression and cancer cell behavior.

Gene mutation's role in initiating carcinogenesis has been controversial, but it is consensually accepted that both carcinogenesis and cancer metastasis are gene-regulated processes. MTA1, a metastasis-associated protein, has been extensively researched, especially regarding its role in cancer metastasis. In this review, I try to elucidate MTA1's role in both carcinogenesis and metastasis from a different angle. I propose that MTA1 is a stress response protein that is upregulated in various stress-related situations such as heat shock, hypoxia, and ironic radiation. Cancer cells are mostly living in a stressful environment of hypoxia, lack of nutrition, and immune reaction attacks. To cope with all these stresses, MTA1 expression is upregulated, plays a role of master regulator of gene expression, and helps cancer cells to survive and migrate out of their original dwelling.

Resistance to apoptosis should not be taken as a hallmark of cancer.

In the research community, resistance to apoptosis is often considered a hallmark of cancer. However, pathologists who diagnose cancer via microscope often see the opposite. Indeed, increased apoptosis and mitosis are usually observed simultaneously in cancerous lesions. Studies have shown that increased apoptosis is associated with cancer aggressiveness and poor clinical outcome. Furthermore, overexpression of Bcl-2, an antiapoptotic protein, is linked with better survival of cancer patients. Conversely, Bax, CD95, Caspase-3, and other apoptosis-inducing proteins have been found to promote carcinogenesis. This notion of the role of apoptosis in cancer is not new; cancer cells were found to be short-lived 88 years ago. Given these observations, resistance to apoptosis should not be considered a hallmark of cancer.

c-Myc-mediated epigenetic silencing of MicroRNA-101 contributes to dysregulation of multiple pathways in hepatocellular carcinoma.

UNLABELLED: The MYC oncogene is overexpressed in hepatocellular carcinoma (HCC) and has been associated with widespread microRNA (miRNA) repression; however, the underlying mechanisms are largely unknown. Here, we report that the c-Myc oncogenic transcription factor physically interacts with enhancer of zeste homolog 2 (EZH2), a core enzymatic unit of polycomb repressive complex 2 (PRC2). Furthermore, miR-101, an important tumor-suppressive miRNA in human hepatocarcinomas, is epigenetically repressed by PRC2 complex in a c-Myc-mediated manner. miR-101, in turn, inhibits the expression of two subunits of PRC2 (EZH2 and EED), thus creating a double-negative feedback loop that regulates the process of hepatocarcinogenesis. Restoration of miR-101 expression suppresses multiple malignant phenotypes of HCC cells by coordinate repression of a cohort of oncogenes, including STMN1, JUNB, and CXCR7, and further increases expression of endogenous miR-101 by inhibition of PRC2 activation. In addition, co-overexpression of c-Myc and EZH2 in HCC samples was closely associated with lower expression of miR-101 (P < 0.0001) and poorer prognosis of HCC patients (P < 0.01). CONCLUSIONS: c-Myc collaborates with EZH2-containing PRC2 complex in silencing tumor-suppressive miRNAs during hepatocarcinogenesis and provides promising therapeutic candidates for human HCC.

Invasive cancers are not necessarily from preformed in situ tumours - an alternative way of carcinogenesis from misplaced stem cells.

Cancers are thought to be the result of accumulated gene mutations in cells. Carcinomas, which are cancers arising from epithelial tissues usually go through several stages of development: atypical hyperplasia, carcinoma in situ and then invasive carcinoma, which might further metastasize. However, we think that the present pathological data are enough to prove that there might be an alternative way of carcinogenesis. We propose that majority of invasive cancers arise in the connective tissue stroma de novo, from the misplaced epithelial stem cells which come to the wrong land of connective tissue stroma by accident. The in situ carcinomas, which are mostly curable, should not be considered genuine cancer, but rather as quasi-cancer. We design this new theory of carcinogenesis as the stem cell misplacement theory (SCMT). Our SCMT theory chains together other carcinogenesis theories such as the inflammation-cancer chain, the stem cell theory and the tissue organization field theory. However, we deny the pathway of somatic mutation theory as the major pathway of carcinogenesis.

MTA1 promotes STAT3 transcription and pulmonary metastasis in breast cancer.

Overexpression of the prometastatic chromatin modifier protein metastasis tumor antigen 1 (MTA1) in human cancer contributes to tumor aggressiveness, but the role of endogenous MTA1 in cancer has not been explored. Here, we report the effects of selective genetic depletion of MTA1 in a physiologically relevant spontaneous mouse model of breast cancer pulmonary metastasis. We found that MTA1 acts as a mandatory modifier of breast-to-lung metastasis without effects on primary tumor formation. The underlying mechanism involved MTA1-dependent stimulation of STAT3 transcription through action on the MTA1/STAT3/Pol II coactivator complex, and, in turn, on the expression and functions of STAT3 target genes including Twist1. Accordingly, we documented a positive correlation between levels of MTA1 and STAT3 in publicly available breast cancer data sets. Together, our findings reveal an essential modifying role of the physiologic level of MTA1 in supporting pulmonary metastasis of breast cancer.

Apoptosis drives cancer cells proliferate and metastasize.

Cancer has been considered to be the result of accumulated gene mutations, which result in uncontrolled cell proliferations for a long time. Cancers are also regarded to be capable of immune evasion. Furthermore, resistance to apoptosis was recognized as an important trait of cancer in the last score of years. However, there are numerous paradoxical issues in this whole set of theory. For example, there is no known set of genes of which mutations are responsible for human cancers. As for the trait of 'resistance to apoptosis', the fact is that cancer has increased frequency of apoptosis. The more malignant the tumour is, the more apoptosis shows. In this study, we propose a new theory that apoptosis plays a key role in the malignant progression and metastasis of cancer. The growth of tumour is the difference between tumour cell proliferation and attrition plus the hyperplastic growth of stroma. Increased and unpreventable death caused by innate or environmental factors such as ischaemia and inflammation drives the tumour cells to proliferate relentlessly, move to new lands to establish colonies. In short, increased cell death is the origin of malignancy.

TBX2 expression is regulated by PAX3 in the melanocyte lineage.

The paired box homeotic gene 3 (PAX3) is a crucial regulator for the maintenance of melanocytic progenitor cells and has a poorly defined role in melanoma. To understand how PAX3 affects melanocyte and melanoma proliferation, we identified potential PAX3 downstream targets through gene expression profiling. Here, we identify T-box 2 (TBX2), a key developmental regulator of cell identity and an antisenescence factor in melanoma, as a directly regulated PAX3 target. We also found that TBX2 is involved in the survival of melanoma cells and is overexpressed in some melanoma specimens. The identification of TBX2 as a target for PAX3 provides a key insight into how PAX3 may contribute to melanoma evolution and may provide opportunities for prosenescence therapeutic intervention aimed at disrupting the ability of PAX3 to regulate TBX2.

[Expression and potential role of metastasis-associated protein 1 in the induced carcinogenesis of mouse liver].

AIM: To establish a hepatocellular carcinoma model of BALB/c mouse and to study the expression and potential role of metastasis-associated protein 1 (MTA1) in the carcinogenesis process. METHODS: Normal adult male BALB/c mice were induced by the combined dimethylnitrosamine (DEN)/carbon tetrachloride (CCl(4);)/alcohol for 150 d. The morphological changes in liver cells and the expression of MTA1 in the liver lesions were observed by HE and immunohistochemical stainings, respectively. RESULTS: The pathological changes of the survivals' livers in experimental group were liver inflammation, fibrosis and cancer in sequence. The level of MTA1 increased in the carcinogenesis process, and MTA1 was mainly expressed in the cytoplasm of the cells suffering cirrhosis. CONCLUSION: The changes of the expression sites and quantity of MTA1 in the DEN-induced carcinogenesis of mouse liver indicate that MTA1 may play an important role in the whole process of liver carcinogenesis.

HER2 interacts with CD44 to up-regulate CXCR4 via epigenetic silencing of microRNA-139 in gastric cancer cells.

BACKGROUND & AIMS: Human epidermal growth factor receptor 2 (HER2) (neu/ERBB2) is overexpressed on many types of cancer cells, including gastric cancer cells; HER2 overexpression has been associated with metastasis and poor prognosis. We investigated the mechanisms by which HER2 regulates cell migration and invasion. METHODS: HER2 expression or activity was reduced in gastric cancer cell lines using small interfering RNAs or the monoclonal antibody, trastuzumab. We identified proteins that interact with HER2 or microRNAs (miRNAs) involved in HER2 signaling. We used various software programs to identify miRNAs that regulate factors in the HER2 signaling pathway. We analyzed expression patterns of these miRNAs in gastric cancer cell lines and tumor samples from patients. RESULTS: We found that CD44 binds directly to HER2, which up-regulates the expression of metastasis-associated protein-1, induces deacetylation of histone H3 lysine 9, and suppresses transcription of microRNA139 (miR-139) to inhibit expression of its target gene, C-X-C chemokine receptor type 4 (CXCR4). Knockdown of HER2 and CD44 reduced invasive activity of cultured gastric cancer cells and suppressed tumor growth in nude mice. Lymph node metastasis was associated with high levels of HER2, CD44, and CXCR4, and reduced levels of miR-139 in human metastatic gastric tumors. Cultures of different types of metastatic cancer cells with histone deacetylase inhibitors and/or DNA methyltransferase resulted in up-regulation of miR-139. CONCLUSIONS: HER2 interaction with CD44 up-regulates CXCR4 by inhibiting expression of miR-139, at the epigenetic level, in gastric cancer cells. These findings indicate how HER2 signaling might promote gastric tumor progression and metastasis.

Notch1 expression, which is related to p65 Status, is an independent predictor of prognosis in colorectal cancer.

PURPOSE: Notch1 has been proven to be aberrantly expressed in colorectal cancer and related to tumor differentiation status. However, few previous studies concentrated on the predictive role of Notch1 expression on the overall survival of patients with colorectal cancer. This study explored expression of Notch1 and its relationship with p65 and prognosis in colorectal cancer. EXPERIMENTAL DESIGN: Two independent study cohorts were involved in the present study. Clinical specimens from 941 eligible patients were constructed into tissue microarrays. The expression of Notch1 and p65 protein was investigated by immunohistochemistry. RESULTS: Statistically significant positive correlations were found between protein expression of Notch1 and p65 in both retrospective and prospective study cohorts. Patients with higher Notch1 expression showed a trend of having shorter survival time, whereas patients with lower Notch1 expression had better survival in both study cohorts. In multivariate analysis, Notch1 expression was proven to be an independent predictor of prognosis. Moreover, the prognostic value of Notch1 might differ according to p65 status. CONCLUSIONS: Notch1 is an independent predictor of prognosis for patients with colorectal cancer. In addition, the predictive role of Notch1 on clinical outcome might be modified by p65 status, suggesting that targeting Notch1 and nuclear factor κB (NF-κB) might be a promising strategy for colorectal cancer treatment.

Rapamycin inhibits re-endothelialization after percutaneous coronary intervention by impeding the proliferation and migration of endothelial cells and inducing apoptosis of endothelial progenitor cells.

Endothelial-cell function is important in the healing of damaged endothelium after percutaneous coronary artery damage. In 3 different animal models, we sought to determine whether rapamycin (sirolimus) affects the proliferation and migration of endothelial cells and endothelial progenitor cells. First, after we implanted stents in dogs, we found that re-endothelialization was impeded more by drug-eluting stents than by bare-metal stents, 30 days after percutaneous coronary intervention. Second, in vitro in rats, we found that 1-100 ng/mL of rapamycin time- and dose-dependently inhibited proliferation over 72 hr (with effects evident as early as 24 hr) and also dose-dependently induced endothelial progenitor-cell apoptosis. Finally, in vivo in rats, we observed that vascular endothelial growth factor expression was decreased after 5 days of rapamycin treatment. We conclude that rapamycin impedes re-endothelialization after drug-eluting stent implantation by inhibiting the proliferation and migration of coronary endothelial cells, inducing endothelial progenitor-cell apoptosis, and decreasing vascular endothelial growth factor expression in the circulation.

Metastasis tumor antigen 1 is involved in the resistance to heat stress-induced testicular apoptosis.

Our previous study documented the expression of Mta1 during spermatogenesis. Here, we present evidence for a possible involvement of Mta1 in the regulation of testicular function, possibly by interacting with p53. A notable decrease of Mta1 expression was revealed at postsurgical day 6, consistent with the previously reported upregulation of p53 in mouse cryptorchidism. Furthermore, in vitro over-expression of Mta1 could remarkably elevate the resistance capability of spermatogenic tumor cells against heat-induced apoptosis with a marked impairment of p53 expression. These findings indicate that Mta1 may operate as a negative modifier of apoptosis by interacting with p53 during gametogenesis.

Repression of Six3 by a corepressor regulates rhodopsin expression.

Here, we provide gain-of-function, loss-of function, and molecular evidence supporting genetic interactions between metastasis associated protein 1 (MTA1) and Six3 and between Six3 and rhodopsin. We discovered that MTA1 physically interacts with the Six3 chromatin in a histone deacetylase-dependent manner, leading to transcriptional suppression of the Six3 gene. MTA1 is also a Six3-interacting corepressor that contributes to a self-negative regulation of Six3 transcription by Six3. In contrast, deletion of the MTA1 alleles in murine embryonic fibroblasts or its knockdown in rat retinal ganglion cells stimulates Six3 expression. MTA1 inactivation in the MTA1-null mice results in an elevated Six3 level and proliferation of the retina cells with no obvious abnormities in eye formation. However, unexpectedly, we discovered an enhanced recruitment of Six3 to the rhodopsin chromatin in retina from the MTA1-null mice; Six3's homeodomain interacts with specific DNA elements in the rhodopsin promoter to stimulate its transcription, resulting in increased rhodopsin expression. Further, in holoprosencephaly patients, Six3 protein with a naturally occurring deletion mutation in the helix 3 of the homeodomain does not bind to rhodopsin DNA or stimulate rhodopsin transcription, implying a potential defective rhodopsin pathway in the affected holoprosencephaly patients. Further Six3 cooperates with Crx or NRL in stimulating transcription from the rhodopsin-luc. These findings reveal a previously unrecognized role for the MTA1 as an upstream modifier of Six3 and indicate that Six3 is a direct stimulator of rhodopsin expression, thus revealing a putative role for the MTA1/Six3/rhodopsin pathway in vertebrate eye.

P21-activated kinase 1 regulation of estrogen receptor-alpha activation involves serine 305 activation linked with serine 118 phosphorylation.

Here, we investigated the role of P21-activated kinase 1 (Pak1) signaling in the function of estrogen receptor-alpha (ER-alpha) as assessed by serine 305 (S305) activation and transactivation activity of ER. We found that Pak1 overexpression interfered with the antiestrogenic action of tamoxifen upon the ER transactivation function in hormone-sensitive cells. In addition, tamoxifen stimulation led to up-regulation of ER target genes in breast cancer cells with increased Pak1 expression. Tamoxifen also increased Pak1-ER interaction in tamoxifen-resistant but not in tamoxifen-sensitive cells. Results from the mutational studies discovered a role of ER-S305 phosphorylation in triggering a subsequent phosphorylation of serine 118 (S118), and these effects were further potentiated by tamoxifen treatment. We found that S305 activation-linked ER transactivation function requires a functional S118, and active Pak1 signaling is required for a sustaining S118 phosphorylation of the endogenous ER. All of these events were positively influenced by tamoxifen and thus may contribute toward the loss of antiestrogenic effect of tamoxifen. These findings suggest that Pak1 signaling-dependent activation of ER-S305 leads to an enhanced S118 phosphorylation presumably due to a conformational change, and such structural modifications may participate in the development of tamoxifen resistance.