Hepatic Tmem30a Deficiency Causes Intrahepatic Cholestasis by Impairing Expression and Localization of Bile Salt Transporters.

Mutations in ATP8B1 or ATP11C (members of P4-type ATPases) cause progressive familial intrahepatic cholestasis type 1 in human or intrahepatic cholestasis in mice. Transmembrane protein 30A (TMEM30A), a ß-subunit, is essential for the function of ATP8B1 and ATP11C. However, its role in the etiology of cholestasis remains poorly understood. To investigate the function of TMEM30A in bile salt (BS) homeostasis, we developed Tmem30a liver-specific knockout (LKO) mice. Tmem30a LKO mice experienced hyperbilirubinemia, hypercholanemia, inflammatory infiltration, ductular proliferation, and liver fibrosis. The expression and membrane localization of ATP8B1 and ATP11C were significantly reduced in Tmem30a LKO mice, which correlated with the impaired expression and localization of BS transporters, such as OATP1A4, OATP1B2, NTCP, BSEP, and MRP2. The proteasome inhibitor bortezomib partially restored total protein levels of BS transporters but not the localization of BS transporters in the membrane. Furthermore, the expression of nuclear receptors, including FXRα, RXRα, HNF4α, LRH-1, and SHP, was also down-regulated. A cholic acid-supplemented diet exacerbated the liver damage in Tmem30a LKO mice. TMEM30A deficiency led to intrahepatic cholestasis in mice by impairing the expression and localization of BS transporters and the expression of related nuclear receptors. Therefore, TMEM30A may be a novel genetic determinant of intrahepatic cholestasis.

MicroRNAs in hepatocellular carcinoma: regulation, function, and clinical implications.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the third cause of cancer-related death. Poor understanding of the mechanisms underlying the pathogenesis of HCC makes it difficult to be diagnosed and treated at early stage. MicroRNAs (miRNAs), a class of noncoding single-stranded RNAs of ~22 nucleotides in length, posttranscriptionally regulate gene expression by base pairing with the 3' untranslated regions (3'UTRs) of target messenger RNAs (mRNAs). Aberrant expression of miRNAs is found in many if not all cancers, and many deregulated miRNAs have been proved to play crucial roles in the initiation and progression of cancers by regulating the expression of various oncogenes or tumor suppressor genes. In this Paper, we will summarize the regulations and functions of miRNAs aberrantly expressed in HCC and discuss the potential application of miRNAs as diagnostic and prognostic biomarkers of HCC and their potential roles in the intervention of HCC.

Targeting Trop-2 in cancer: Recent research progress and clinical application.

The development of new antitumor drugs depends mainly upon targeting tumor cells precisely. Trophoblast surface antigen 2 (Trop-2) is a type I transmembrane glycoprotein involved in Ca2+ signaling in tumor cells. It is highly expressed in various tumor tissues than in normal tissues and represents a novel and promising molecular target for caner targeted therapy. Up to now, the mechanisms and functions associated with Trop-2 have been extensively studied in a variety of solid tumors. According to these findings, Trop-2 plays an important role in cell proliferation, apoptosis, cell adhesion, epithelial-mesenchymal transition, as well as tumorigenesis and tumor progression. In addition, Trop-2 related drugs are also being developed widely. There are a number of Trop-2 related ADC drugs that have demonstrated potent antitumor activity and are currently been studied, such as Sacituzumab Govitecan (SG) and Datopotamab Deruxtecan (Dato-Dxd). In this study, we reviewed the progress of Trop-2 research in solid tumors. We also sorted out the composition and rationale of Trop-2 related drugs and summarized the related clinical trials. Finally, we discussed the current status of Trop-2 research and expanded our perspectives on its future research directions. Importantly, we found that Trop-2 targeted ADCs have great potential for combination with other antitumor therapies. Trop-2 targeted ADCs can reprogramme tumor microenvironment through multiple signaling pathways, ultimately activating antitumor immunity.

Krüppel-like factors in breast cancer: Function, regulation and clinical relevance.

Breast cancer has accounted for the leading cause of cancer-related mortality among women worldwide. Although the progress in its diagnosis and treatment has come at a remarkable pace during the past several decades, there are still a wide array of problems regarding its progression, metastasis and treatment resistance that have not yet been fully clarified. Recently, an increasing number of studies have revealed that some members of Krüppel-like factors(KLFs) are significantly associated with cell proliferation, apoptosis, metastasis, cancer stem cell regulation and prognostic and predictive value for patients in breast cancer, indicating their promising prognostic and predictive potential for breast cancer survival and outcome. In this review, we will summarize our current knowledge of the functions, regulations and clinical relevance of KLFs in breast cancer.

CXXC4 activates apoptosis through up-regulating GDF15 in gastric cancer.

Worldwide, gastric cancer is one of the most fatal cancers. Epigenetic alterations in gastric cancer play important roles in silencing of tumor suppressor genes. We previously found that CXXC finger protein 4 (CXXC4) was a novel tumor suppressor in gastric cancer. In this report, we demonstrated that CXXC4 inhibited growth of gastric cancer cells as a pro-apoptotic factor. This inhibition could be reversed by the pan-caspase inhibitor called Z-VAD-FMK. However, CXXC4 with mutations in its DNA binding domain failed to induce apoptosis. Growth differentiation factor 15 (GDF15) was identified as one of potential targets responsible for CXXC4-induced apoptosis. CXXC4 activated GDF15 transcription through enhancing the interaction of transcription factor Sp1 with GDF15 promoter. In summary, the nuclear protein CXXC4 activated apoptosis in gastric cancer through up-regulating its novel potential downstream target GDF15. GDF15 might be a promising target for clinical treatment of gastric cancer with CXXC4 deficiency.

Regulation and role of post-translational modifications of enhancer of zeste homologue 2 in cancer development.

Post-translational modifications (PTMs) are critical molecular events which alter protein conformation after their synthesis and diversity protein properties by modulating their stability, localization, interacting partners or the activity of their substrates, consequently exerting pivotal roles in regulating the functions of many important eukaryotic proteins. It has been well acknowledged that PTMs are of great importance in a broad range of biological processes such as gene regulation, cell proliferation, differentiation and apoptosis, tissue development, diseases, tumor progression and drug resistance. As the core and contributing catalytic subunit of Polycomb repressive complex 2(PRC2), Enhancer of zeste homolog 2 (EZH2) is a master epigenetic regulator, often serving as a highly conserved histone methyltransferase (HMTase) to induce histone H3 lysine 27 trimethylation (H3K27me3) and repress gene transcription and expression. Dysregulated EZH2 expression is frequently associated with cancer development and poor prognosis in a wide variety of cancers. Considered its essential role in carcinogenesis, EZH2 is a potential candidate for cancer targeted therapy. Remarkably, mounting evidence highlights that EZH2 expression, activity and stability can be regulated by PTMs including phosphorylation, acetylation, ubiquitination, sumoylation and GlcNAcylation aside from its well-validated modifications in transcriptional and post-transcriptional levels. However, the precise regulatory mechanisms underlying EZH2 PTMs and whether other types of PTMs orchestrate in EZH2 remain largely unclear. In this review, we summarize current advances in the understanding of EZH2 regulation by PTMs and their associated biological functions during tumorigenesis.

MicroRNAs as potential biomarkers in cancer: opportunities and challenges.

MicroRNAs (miRNAs) are a group of small noncoding RNAs (ncRNAs) that posttranscriptionally regulate gene expression by targeting their corresponding messenger RNAs (mRNAs). Dysregulated miRNAs have been considered as a new type of ''oncomiRs" or ''tumor suppressors," playing essential roles in cancer initiation and progression. Using genome-wide detection methods, ubiquitously aberrant expression profiles of miRNAs have been identified in a broad array of human cancers, showing great potential as novel diagnostic and prognostic biomarkers of cancer with high specificity and sensitivity. The detectable miRNAs in tissue, blood, and other body fluids with high stability provide an abundant source for miRNA-based biomarkers in human cancers. Despite the fact that an increasing number of potential miRNA biomarkers have been reported, the transition of miRNAs-based biomarkers from bench to bedside still necessitates addressing several challenges. In this review, we will summarize our current understanding of miRNAs as potential biomarkers in human cancers.

Oncogenic Ras suppresses ING4-TDG-Fas axis to promote apoptosis resistance.

Ras is aberrantly activated in many cancers and active DNA demethylation plays a fundamental role to establish DNA methylation pattern which is of importance to cancer development. However, it was unknown whether and how Ras regulate DNA demethylation during carcinogenesis. Here we found that Ras downregulated thymine-DNA glycosylase (TDG), a DNA demethylation enzyme, by inhibiting the interaction of transcription activator ING4 with TDG promoter. TDG recruited histone lysine demethylase JMJD3 to the Fas promoter and activated its expression, thus restoring sensitivity to apoptosis. TDG suppressed in vivo tumorigenicity of xenograft pancreatic cancer. Thus, we speculate that reversing Ras-mediated ING4 inhibition to activate Fas expression is a potential therapeutic approach for Ras-driven cancers.

New tumor suppressor CXXC finger protein 4 inactivates mitogen activated protein kinase signaling.

As a well-characterized master player in epigenetic regulatory network, EZH2 is widely implicated in the development of many malignancies. We previously found that EZH2 promoted Wnt/ß-catenin activation through downregulation of CXXC4 expression. In this report, we demonstrated that CXXC4 inhibited MAPK signaling through binding to ERK-1/2 and abrogating the interaction of ERK 1/2 with MEK1/2. L183, the critical residue in CXXC4 ERK D domain, was found to be essential for CXXC4-ERK 1/2 interaction and the growth inhibitory effect of CXXC4 in human cancer cells. In summary, CXXC4 directly disrupted MEK1/2-ERK 1/2 interaction to inactivate MAPK signaling. L183 site is indispensable for the binding of CXXC4 to ERK1/2 and growth inhibitory effect of CXXC4. Therefore, EZH2 can activate MAPK signaling by inhibiting CXXC4 expression.

Downregulation of histone deacetylase 1 by microRNA-520h contributes to the chemotherapeutic effect of doxorubicin.

Doxorubicin induces DNA damage to exert its anti-cancer function. Histone deacetylase 1 (HDAC1) can protect the genome from DNA damage. We found that doxorubicin specifically downregulates HDAC1 protein expression and identified HDAC1 as a target of miR-520h, which was upregulated by doxorubicin. Doxorubicin-induced cell death was impaired by exogenous HDAC1 or by miR-520h inhibitor. Moreover, HDAC1 reduced the level of γH2AX by preventing the interaction of doxorubicin with DNA. In summary, doxorubicin downregulates HDAC1 protein expression, by inducing the expression of HDAC1-targeting miR-520h, to exacerbate DNA-doxorubicin interaction. The upregulation of HDAC1 protein may contribute to drug resistance of human cancer cells and targeting HDAC1 is a promising strategy to increase the clinical efficacy of DNA damage-inducing chemotherapeutic drugs.

YY1-MIR372-SQSTM1 regulatory axis in autophagy.

Autophagy is a self-proteolytic process that degrades intracellular material to enable cellular survival under unfavorable conditions. However, how autophagy is activated in human carcinogenesis remains largely unknown. Herein we report an epigenetic regulation of autophagy in human cancer cells. YY1 (YY1 transcription factor) is a well-known epigenetic regulator and is upregulated in many cancers. We found that YY1 knockdown inhibited cell viability and autophagy flux through downregulating SQSTM1 (sequestosome 1). YY1 regulated SQSTM1 expression through the epigenetic modulation of the transcription of MIR372 (microRNA 372) which was found to target SQSTM1 directly. During nutrient starvation, YY1 was stimulated to promote SQSTM1 expression and subsequent autophagy activation by suppressing MIR372 expression. Similar to YY1 depletion, MIR372 overexpression blocked autophagy activation and inhibited in vivo tumor growth. SQSTM1 upregulation and competent autophagy flux thus contributed to the oncogenic function of YY1. YY1-promoted SQSTM1 upregulation might be a useful histological marker for cancer detection and a potential target for drug development.

Regulation and function of mitophagy in development and cancer.

Beyond its role in recycling intracellular components nonselectively to sustain survival in response to metabolic stresses, autophagy can also selectively degrade specific cargoes such as damaged or dysfunctional organelles to maintain cellular homeostasis. Mitochondria, known as the power plant of cells, are the critical and dynamic organelles playing a fundamental role in cellular metabolism. Mitophagy, the selective autophagic elimination of mitochondria, has been identified both in yeast and in mammalian cells. Moreover, defects in mitophagy may contribute to a variety of human disorders such as neurodegeneration and myopathies. However, the role of mitophagy in development and cancer remains largely unclear. In this review, we summarize our current knowledge of the regulation and function of mitophagy in development and cancer.

Annexin A6 is down-regulated through promoter methylation in gastric cancer.

BACKGROUND: The aberrant activation of oncogenic signaling such as Ras/MAPK signaling is a frequent event in human cancers. In addition to genetic changes, epigenetic silencing of inhibitors in Ras/MAPK signaling contributes to the activation of Ras/MAPK signaling. Recently, ANXA6 has been shown to interact with Ras-GAP1 and inhibit Ras activation in human breast cancer. However, whether and how it is involved in human cancers remain unknown. METHODS: Real-time PCR was used to determine ANXA6 expression in gastric cancer cells and primary gastric carcinomas. Next, we explored the methylation of ANXA6 promoter in cell lines and tumor tissues with methylation-specific PCR and bisulfite genomic sequencing. We also investigated the function of ANXA6 in gastric cancer cells with colony formation assay and western blotting analysis. RESULTS: ANXA6 was down-regulated in gastric cancer cells and primary gastric carcinomas. Ectopic ANXA6 expression inhibited the growth of gastric cancer cells and the activity of Ras/MAPK signaling. Its expression was restored after pharmaceutical demethylation. ANXA6 promoter was methylated in gastric cancer cell lines (6/6) and primary gastric carcinoma tissues (29/156). Interestingly, the knockdown of oncoprotein Yin Yang 1 (YY1) also restored ANXA6 expression and promoted the demethylation of ANXA6 promoter. However, ANXA6 methylation was not associated with clinical parameters such as differentiation, and TNM staging. Neither Kaplan-Meier Curve nor Cox regression analysis revealed a significant role of ANXA methylation to predict the survival of gastric cancer patients. CONCLUSIONS: We firstly reported that ANXA6 is epigenetically silenced through promoter methylation in human cancers and YY1 is important to initiate or maintain ANXA6 promoter methylation in gastric cancer cells. ANXA6 functions as a tumor suppressor in gastric cancer cells through the inhibition of Ras/MAPK signaling. ANXA6 methylation is not a prognostic factor for gastric cancer patients.

Histone deacetylase 3 inhibits expression of PUMA in gastric cancer cells.

During cancer development, tumor suppressor genes were silenced by promoter methylation or histone deacetylation. Histone deacetylases (HDACs) are important to maintain histone deacetylation. HDAC inhibitors (HDACis) were thus proposed as a new therapeutic approach to cancer. The current study aims to understand the effect and molecular mechanisms of HDACis on gastric cancer cells. Trichostatin A (TSA) significantly inhibited the growth of gastric cancer cells by inducing apoptosis. Gene profiling results showed PUMA (p53 upregulated modulator of apoptosis) as one of 122 genes upregulated in TSA-treated gastric cancer cells. PUMA was downregulated in gastric cancer cell lines and primary gastric carcinoma tissues. Patients with low PUMA expression had significant decreases in overall survival (HR, 2.04; p = 0.047). Ectopic PUMA expression inhibited the growth of gastric cancer cells while PUMA depletion promoted cellular growth. The knockdown of HDAC3 but not other HDACs upregulated PUMA expression. HDAC3 could bind to PUMA promoter, which was abrogated after TSA treatment. In contrast to TSA and SB, HDAC3 siRNA failed to upregulate p53 expression but promoted the interaction of p53 with PUMA promoter. In summary, proapoptotic PUMA was downregulated in gastric cancer and its mRNA expression level is a valuable prognosis factor for gastric cancer. HDAC3 is important to downregulate PUMA expression in gastric cancer and HDACis, like TSA, promoted PUMA expression through stabilizing p53 in addition to HDAC3 inhibition. In combination with chemotherapy, targeting HDAC3 might be a promising strategy to induce apoptosis of gastric cancer cells.

Yin Yang-1 suppresses differentiation of hepatocellular carcinoma cells through the downregulation of CCAAT/enhancer-binding protein alpha.

As a member of the GLI-Kruppel family of transcriptional factors, Yin Yang-1 (YY1) functions as an oncogene in various types of cancers. However, the role of YY1 in hepatocellular carcinogenesis remains unknown. In this report, we investigated the relevance of YY1 to hepatocellular carcinoma (HCC) development. We found that YY1 was upregulated in HCC cell lines. Ectopic YY1 expression promoted the growth of non-tumor liver cells that expressed low level of YY1. In contrast, YY1 depletion inhibited the growth of HCC cells which was accompanied with distinct morphological changes. Moreover, the phenotypic changes induced by YY1 depletion were attributed to cellular differentiation rather than cellular senescence. CCAAT/enhancer-binding protein alpha (CEBPA) which was important to regulate differentiation of hepatocytes was found as the direct target downregulated by YY1. Restoration of CEBPA in YY1-expressing HCC cells induced cellular differentiation and growth inhibition while knockdown of CEBPA expression in non-tumor liver cells promoted cell growth. In summary, our study demonstrated that YY1 could promote hepatocellular carcinogenesis and inhibit cellular differentiation through the downregulation of CEBPA expression.

Human mammaglobin: a superior marker for reverse-transcriptase PCR in detecting circulating tumor cells in breast cancer patients.

Breast cancer is the most frequent cancer in women in the USA and the second most common cause of death in females who develop cancer. Recently, the detection of circulating tumor cells has emerged as a promising tool for monitoring the progression of clinically occult micrometastases in breast cancer patients. Sensitive molecular techniques, primarily based upon the reverse-transcriptase PCR, using various molecules as markers, have been developed to detect circulating tumor cells. Among those molecules, human mammaglobin mRNA has been found to be the most specific marker for the hematogenous spread of breast cancer cells. In this article, we review the current knowledge regarding the use of reverse-transcriptase PCR for detecting human mammaglobin mRNA as a biomarker for circulating tumor cells in breast cancer patients, and evaluate the clinical implications of human mammaglobin since it was first isolated in 1996.

Klotho is silenced through promoter hypermethylation in gastric cancer.

As one of major epigenetic changes to inactivate tumor suppressor genes in human carcinogenesis, promoter hypermethylation was proposed as a marker to define novel tumor suppressor genes and predict the prognosis of cancer patients. In the present study, we found KL (klotho) as a novel tumor suppressor gene silenced through promoter hypermethylation in gastric cancer, the second leading cause of cancer death worldwide. KL expression was downregulated in primary gastric carcinoma tissues (n=22, p<0.05) and all of gastric cancer cells lines examined. Ectopic expression of KL inhibited the growth of gastric cancer cells partially through the induction of apoptosis, demonstrating a tumor suppressive role of KL in gastric cancer. Demethylation with 5-aza-2'-deoxycytidine (Aza) increased KL expression and KL promoter was hypermethylated in gastric cancer cell lines as well as some of primary gastric carcinoma tissues (47/99) but none of normal gastric tissues. Importantly, promoter methylation of KL was significantly associated with the poor outcome of gastric cancer patients (p=0.025, Log-rank test), highlighting the relevance of epigenetic inactivation of KL in gastric carcinogenesis. As a summary, we found that KL is a novel tumor suppressor gene epigenetically inactivated in gastric cancer and promoter methylation of KL could be used to predict the prognosis of gastric cancer patients.