Metabolic modulation of CtBP dimeric status impacts the repression of DNA damage repair genes and the platinum sensitivity of ovarian cancer.

Platinum drug-based chemotherapy plays a dominant role in OC (ovarian cancer) treatment. The expression of DNA damage repair (DDR) genes is critical in distinguishing drug-sensitive and drug-refractory patients, as well as in the development of drug resistance in long-term treated patients. CtBP is a highly expressed oncogene in OC and was found to repress DDR genes expression in our previous study. In the present study, the formation of CtBP dimers in live cells was studied, and the functional differences between monomeric and oligomeric CtBP were explored by CHIP-seq and RNA-seq. Besides, the dynamics of CtBP dimer formation in response to the metabolic modulation were investigated by the protein fragment complementation (PCA) assays. We show that dimerized CtBP, but not the dimerization-defective mutant, binds to and represses DDR gene expression in OC cells. Treatment of the mice tumors grown from engrafted OC cells by cisplatin disclosed that high-level CtBP expression promotes the CtBP dimerization and increases the therapeutic effect of cisplatin. Moreover, the CtBP dimerization is responsive to the intracellular metabolic status as represented by the free NADH abundance. Metformin was found to increase the dimerization of CtBP and potentiate the therapeutic effect of cisplatin in a CtBP dimerization-dependent manner. Our data suggest that the CtBP dimerization status is a potential biomarker to predict platinum drug sensitivity in patients with ovarian cancer and a target of metformin to improve the therapeutic effect of platinum drugs in OC treatment.

Glycogen synthesis and beyond, a comprehensive review of GSK3 as a key regulator of metabolic pathways and a therapeutic target for treating metabolic diseases.

Glycogen synthase kinase-3 (GSK3) is a highly evolutionarily conserved serine/threonine protein kinase first identified as an enzyme that regulates glycogen synthase (GS) in response to insulin stimulation, which involves GSK3 regulation of glucose metabolism and energy homeostasis. Both isoforms of GSK3, GSK3α, and GSK3ß, have been implicated in many biological and pathophysiological processes. The various functions of GSK3 are indicated by its widespread distribution in multiple cell types and tissues. The studies of GSK3 activity using animal models and the observed effects of GSK3-specific inhibitors provide more insights into the roles of GSK3 in regulating energy metabolism and homeostasis. The cross-talk between GSK3 and some important energy regulators and sensors and the regulation of GSK3 in mitochondrial activity and component function further highlight the molecular mechanisms in which GSK3 is involved to regulate the metabolic activity, beyond its classical regulatory effect on GS. In this review, we summarize the specific roles of GSK3 in energy metabolism regulation in tissues that are tightly associated with energy metabolism and the functions of GSK3 in the development of metabolic disorders. We also address the impacts of GSK3 on the regulation of mitochondrial function, activity and associated metabolic regulation. The application of GSK3 inhibitors in clinical tests will be highlighted too. Interactions between GSK3 and important energy regulators and GSK3-mediated responses to different stresses that are related to metabolism are described to provide a brief overview of previously less-appreciated biological functions of GSK3 in energy metabolism and associated diseases through its regulation of GS and other functions.

Nuclear HKII-P-p53 (Ser15) Interaction is a Prognostic Biomarker for Chemoresponsiveness and Glycolytic Regulation in Epithelial Ovarian Cancer.

In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

From impossible to possible: the lessons from the control of recent COVID-19 outbreaks in China.

The COVID-19 pandemic has catastrophically impacted the world. Before the success in vaccination, this virus shows no sign of stop spreading. Nearly all the countries have implemented stringent approaches to slow down the transmission of the virus, but the virus still caused over 2 million deaths and the number is increasing. Therefore, preventing the virus spreading is still necessary to protect most people, especially the ones with pre-conditions. Mainland China has successfully eradicated the COVID-19 virus infection in Wuhan in 2020. After that, several small-scale outbreaks occurred in many cities in China, but none of these COVID-19 virus infections caused the widespread. In this review, we would like to give a detailed presentation of the approaches that were implemented by the China government to suppress the virus spreading by considering the unique characteristics of this virus and the paths of the virus transmission. Both the pros and cons of these strategies will also be analyzed. The experiences and lessons learned during the virus-fighting in China, expectedly, will be a useful source of reference for other regions in overcoming the threat caused by the COVID-19 virus.

Applications of the CRISPR-Cas system for infectious disease diagnostics.

INTRODUCTION: Rapid and accurate diagnostic approaches are essential for impeding the spread of infectious diseases. This review aims to summarize current progress of clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems in the applications for diagnostics of infectious diseases including the ongoing COVID-19 epidemic. AREAS COVERED: In this review, we discuss class 2 CRISPR-Cas biosensing systems-based diagnostics in various emerging and reemerging infectious diseases, CRISPR-Cas systems have created a new era for early diagnostics of infectious diseases, especially with the discovery of the collateral cleavage activity of Cas12 and Cas13. We mainly focus on different CRISPR-Cas effectors for the detection of pathogenic microorganisms as well as provide a detailed explanation of the pros and cons of CRISPR-Cas biosensing systems. In addition, we also introduce future research perspectives. EXPERT COMMENTARY: However, further improvement of newly discovered systems and engineering existing ones should be developed to increase the specificity, sensitivity or stability of the diagnostic tools. It may be a long journey to finish the clinical transition from research use. CRISPR-Cas approaches will emerge as more promising and robust tools for infectious disease diagnosis in the future.

Peripheral cytotoxic T lymphocyte predicts first-line progression free survival in HER2-positive advanced breast cancer.

BACKGROUND: The role of peripheral blood lymphocyte (pBL) in breast cancer has long been studied. However, the predictive role of pBL in advanced breast cancer (ABC) is poorly understood. METHODS: A total of 303 patients with ABC were consecutively recruited at our center between January 2015 and September 2019. At baseline, pBL subtypes were detected in all patients with 229 blood samples available for circulating tumor DNA (ctDNA) detection. pBL was analyzed through flow cytometry. ctDNA-based gene mutations were detected using next generation sequencing. The cutoff value of pCTL was estimated by X-tile software. Progression free survival (PFS) was estimated by Kaplan-Meier curve and Cox hazard proportion regression model, with difference detection by log-rank test. RESULTS: Median follow-up time of the study was 21.0 months. The median age of diagnosis was 52.0 years. Among the pBL subtypes, only pCTL level was found predictive for PFS in the HER2+ patients whom received anti-HER2 therapy (13.1 vs. 5.6 months, P = 0.001). However, the predictive role of pCTL was not found in HR-positive (P = 0.716) and TNBC (P = 0.202). pCTL high associated with suppressive immune indictors including lower CD4/CD8 ratio (P = 0.004) and high level of Treg cell (P = 0.004). High occurrence of FGFR1 amplification which has been reported as immune suppressor was also found in HER2+ patients with pCTL high (22.2% vs. 4.3%, P = 0.048). CONCLUSIONS: Higher pCTLs level associated with shorter PFS and FGFR1 mutation in HER2+ ABC patients.

Nuclear localization of Desmoplakin and its involvement in telomere maintenance.

The interaction between genomic DNA and protein fundamentally determines the activity and the function of DNA elements. Capturing the protein complex and identifying the proteins associated with a specific DNA locus is difficult. Herein, we employed CRISPR, the well-known gene-targeting tool in combination with the proximity-dependent labeling tool BioID to capture a specific genome locus associated proteins and to uncover the novel functions of these proteins. By applying this research tool on telomeres, we identified DSP, out of many others, as a convincing telomere binding protein validated by both biochemical and cell-biological approaches. We also provide evidence to demonstrate that the C-terminal domain of DSP is required for its binding to telomere after translocating to the nucleus mediated by NLS sequence of DSP. In addition, we found that the telomere binding of DSP is telomere length dependent as hTERT inhibition or knockdown caused a decrease of telomere length and diminished DSP binding to the telomere. Knockdown of TRF2 also negatively influenced DSP binding to the telomere. Functionally, loss of DSP resulted in the shortened telomere DNA and induced the DNA damage response and cell apoptosis. In conclusion, our studies identified DSP as a novel potential telomere binding protein and highlighted its role in protecting against telomere DNA damage and resultant cell apoptosis.

Non-classical estrogen signaling in ovarian cancer improves chemo-sensitivity and patients outcome.

Deficiency in homologous recombination repair (HRR) is frequently associated with hormone-responsive cancers, especially the epithelial ovarian cancer (EOC) which shows defects of HRR in up to half of cases. However, whether there are molecular connections between estrogen signaling and HRR deficiency in EOC remains unknown. Methods: We analyzed the estrogen receptor α (ERα) binding profile in EOC cell lines and investigated its association with genome instability, HRR deficiency and sensitivity to chemotherapy using extensive public datasets and in vitro/in vivo experiments. Results: We found an inverse correlation between estrogen signaling and HRR activity in EOC, and the genome-wide collaboration between ERα and the co-repressor CtBP. Though the non-classical AP-1-mediated ERα signaling, their targets were highly enriched by HRR genes. We found that depleting ERα in EOC cells up-regulates HRR activity and HRR gene expression. Consequently, estrogen signaling enhances the sensitivity of ovarian cancer cells to chemotherapy agents in vitro and in vivo. Large-scale analyses further indicate that estrogen replacement and ESR1 expression are associated with chemo-sensitivity and the favorable survival of EOC patients. Conclusion: These findings characterize a novel role of ERα in mediating the molecular connection between hormone and HRR in EOC and encourage hormone replacement therapy for EOC patients.

Applications of Protein Fragment Complementation Assays for Analyzing Biomolecular Interactions and Biochemical Networks in Living Cells.

Protein-protein interactions (PPIs) are indispensable for the dynamic assembly of multiprotein complexes that are central players of nearly all of the intracellular biological processes, such as signaling pathways, metabolic pathways, formation of intracellular organelles, establishment of cytoplasmic skeletons, etc. Numerous approaches have been invented to study PPIs both in vivo and in vitro, including the protein-fragment complementation assay (PCA), which is a widely applied technology to study PPIs and biomolecular interactions. PCA is a technology based on the expression of the bait and prey proteins in fusion with two complementary reporter protein fragments, respectively, that will reassemble when in close proximity. The reporter protein can be the enzymes or fluorescent proteins. Recovery of the enzymatic activity or fluorescent signal can be the indicator of PPI between the bait and prey proteins. Significant effort has been invested in developing many derivatives of PCA, along with various applications, in order to address specific questions. Therefore, a prompt review of these applications is important. In this review, we will categorize these applications according to the scenarios that the PCAs were applied and expect to provide a reference guideline for the future selection of PCA methods in solving a specific problem.

PP2ACα deficiency impairs early cortical development through inducing DNA damage in neuroprojenitor cells.

The role of protein phosphatase 2ACα (PP2ACα) in brain development is poorly understood. To understand the function of PP2ACα in neurogenesis, we inactivated Pp2acα gene in the central nervous system (CNS) of mice by Cre/LoxP system and generated the PP2ACα deficient mice (designated as the Pp2acα-/- mice). PP2ACα deletion results in DNA damage in neuroprogenitor cells (NPCs), which impairs memory formation and cortical neurogenesis. We first identify that PP2ACα can directly associate with Ataxia telangiectasia mutant kinase (ATM) and Ataxia telangiectasia/Rad3-related kinase (ATR) in neocortex and NPCs. Importantly, the P53 and hypermethylated in cancer 1 (HIC1) function complex, the newly found down-stream executor of the ATR/ATM cascade, will be translocated into nuclei and interact with homeodomain interacting protein kinase 2 (HIPK2) to respond to DNA damage. Notably, HICI plays a direct transcriptional regulatory role in HIPK2 gene expression. The interplay among P53, HIC1 and HIPK2 maintains DNA stability in neuroprogenitor cells. Taken together, our findings highlight a new role of PP2ACα in regulating early neurogenesis through maintaining DNA stability in neuroprogenitor cells. The P53/HIC/HIPK2 regulation loop, directly targeted by the ATR/ATM cascade, is involved in DNA repair in neuroprogenitor cells.

BioID: A Proximity-Dependent Labeling Approach in Proteomics Study.

Biological activities are mainly executed by proteins and in most of the occasions these activities are accomplished by protein complexes or through protein-protein interactions (PPI). So it is critical to reveal how the protein complexes are organized and demonstrate the PPIs involved in the biological processes. In addition to the traditional biochemical approaches, proximity-dependent labeling (PDL) has recently been proposed to identify the interacting partners of a given protein. PDL requires the fusion expression of the target protein with an enzyme which catalyzes the attachment of a reactive molecule to the interacting partners in a distance-dependent manner. Further analysis of all the proteins that are modified by the reactive molecule discloses the identity of these proteins which are presumed to be interacting partners of the target protein. BioID is one of those representative PDL methods with the most widely applications. The enzyme used in BioID is a biotin ligase BirA which catalyzes the biotinylation of target protein with the presence of biotin. Through streptavidin-mediated pull-down and mass spectrometry analysis, the interacting protein candidates of a given protein can be obtained.

CtBP promotes metastasis of breast cancer through repressing cholesterol and activating TGF-ß signaling.

Metastasis is the process through which the primary cancer cells spread beyond the primary tumor and disseminate to other organs. Most cancer patients die of metastatic disease. EMT is proposed to be the initial event associated with cancer metastasis and how it occurred is still a mystery. CtBP is known as a co-repressor abundantly expressed in many types of cancer and regulates genes involved in cancer initiation, progression, and metastasis. We found that CtBP regulates intracellular cholesterol homeostasis in breast cancer cells by forming a complex with ZEB1 and transcriptionally repressing SREBF2 expression. Importantly, CtBP repression of intracellular cholesterol abundance leads to increased EMT and cell migration. The reason is that cholesterol negatively regulates the stability of TGF-ß receptors on the cell membrane. Interestingly, TGF-ß is also capable of reducing intracellular cholesterol relying on the increased recruitment of ZEB1 and CtBP complex to SREBF2 promoter. Thus, we propose a feedback loop formed by CtBP, cholesterol, and TGF-ß signaling pathway, through which TGF-ß triggers the cascade that mobilizes the cancer cells for metastasis. Consistently, the intravenous injection of breast cancer cells with ectopically CtBP expression show increased lung metastasis depending on the reduction of intracellular cholesterol. Finally, we analyzed the public breast cancer datasets and found that CtBP expression negatively correlates with SREBF2 and HMGCR expressions. High expression of CtBP and low expression of SREBF2 and HMGCR significantly correlates with high EMT of the primary tumors.

Immunogenomic Analyses of Advanced Serous Ovarian Cancer Reveal Immune Score is a Strong Prognostic Factor and an Indicator of Chemosensitivity.

Purpose: Ovarian cancer is one of the first human cancers for which in situ immune response was reported to be important for the clinical outcome. To elucidate the mechanistic relationship between immune repertoire and cancer genotype in ovarian cancer, the development of a well-defined immune score for ovarian cancer is required.Experimental Design: From a collection of 2,203 patient samples of advanced ovarian cancer from public available resources, we evaluated the prognostic values for a compendium of immune marker genes and proposed an immune score. The relationships between immune score, tumor-infiltrating immune cells, cancer genotypes, and their impact on patient outcome were characterized.Results: Loss of chemokine and IFNγ pathway genes is frequent in ovarian cancer and is significantly associated with low immune score and poor outcome. Chemotherapy can increase the immune score of tumors by inducing the expression of IFNγ inducible chemokines. High immune score is significantly associated with BRCA1/2 mutation status and the response to chemotherapy. Multivariate analysis revealed that immune score is a strong predictor of patient survival and the response to immunotherapy.Conclusions: Our results reveal the drivers of the immune repertoire of advanced ovarian cancer and demonstrate the importance of immune score as an independent prognostic signature and a potent indicator of intratumoral immune status. Clin Cancer Res; 24(15); 3560-71. ©2018 AACR.

Molecular link between glucose and glutamine consumption in cancer cells mediated by CtBP and SIRT4.

Glucose and Glutamine are two essential ingredients for cell growth. However, it remains open for investigation whether there is a general mechanism that coordinates the consumption of glucose and glutamine in cancer cells. Glutamine is mainly metabolized through the glutaminolysis pathway and our previous report indicated that CtBP increases GDH activity and promotes glutaminolysis through repressing the expression of SIRT4, a well-known mitochondrion-located factor that inhibits glutaminolysis pathway. CtBP is known to be a sensor of intracellular metabolic status; we thus hypothesized that a consensus CtBP-SIRT4-GDH axis may mediate the crosstalk between glycolysis and glutaminolysis. Herein, supporting this hypothesis, we observed the coordinated consumption of glucose and glutamine across different cell lines. This coordination was found to be related to CtBP repression activity on SIRT4 expression under high level of glucose but not low glucose level. Low level of glucose supply was found to decrease GDH activity via blocking CtBP dimerization. Mechanically, low glucose also abolished CtBP binding to SIRT4 promoter and the repression of SIRT4 expression. Consistently, the CtBP dimerization inhibitor MTOB mimicked low glucose effects on SIRT4 expression, and GDH activity suggest that CtBP requires high glucose supply to act as a suppressor of SIRT4 gene. In conclusion, we propose that a general molecular pathway composed by CtBP-SIRT4-GDH coordinating the metabolism of glucose and glutamine in cancer cells.

Integrated Analysis Reveals Tubal- and Ovarian-Originated Serous Ovarian Cancer and Predicts Differential Therapeutic Responses.

Purpose: The relative importance of fallopian tube (FT) compared with ovarian surface epithelium (OSE) in the genesis of serous type of ovarian cancer (SOC) is still unsettled. Here, we followed an integrated approach to study the tissue origin of SOC, as well as its association with clinical outcome and response to therapeutic drugs.Experimental Design: A collection of transcriptome data of 80 FTs, 89 OSEs, and 2,668 SOCs was systematically analyzed to determine the characteristic of FT-like and OSE-like tumors. A molecular signature was developed for identifying tissue origin of SOC and then was used to reevaluate the prognostic genes and therapeutic biomarkers of SOC of different tissue origins. IHC staining of tissue array and functional experiments on a panel of ovarian cancer cell lines were used to further validate the key findings.Results: The expression patterns of tissue-specific genes, prognostic genes, and molecular markers all support a dualistic tissue origin of SOC, from either FT or OSE. A molecular signature was established to identify the tissue identity of SOCs. Surprisingly, the signature showed a strong association with overall survival (OSE-like vs. FT-like, HR = 4.16; 95% CI, 2.67-6.48; P < 10-9). The pharmacogenomic approach revealed AXL to be a therapeutic target of the aggressive OSE-derived SOC.Conclusions: SOC has two subtypes originated from either FT or OSE, which show different clinical and pathologic features. Clin Cancer Res; 23(23); 7400-11. ©2017 AACR.

Genome-wide methylome and chromatin interactome identify abnormal enhancer to be risk factor of breast cancer.

Enhancer is critical cis regulatory elements in gene expression. To understand whether and how the aberrant enhancer activation may contribute to cancer risk, the differentially methylated enhancers (eDMRs) in normal and malignant breast tissues were identified and analyzed. By incorporating genome-wide chromatin interaction, integrated analysis of eDMRs and target gene expression identified 1,272 enhancer-promoter pairs. Surprisingly, two functionally distinct groups of genes were identified in these pairs, one showing better correlation to enhancer methylation (eRGs) and the other showing better correlation to promoter methylation (pRGs), and the former group is functionally enriched with cancer related genes. Moreover, enhancer methylation based clustering of breast cancer samples is capable of discriminating basal breast cancer from other subtypes. By correlating enhancer methylation status to patient survival, 345 enhancers show the impact on the disease outcome and the majority of their target genes are important regulators of cell survival pathways including known cancer related genes. Together, these results suggest reactivation of enhancers in cancer cells has the add-on effect and contributes to cancer risk in combination.

Epigenetic targeting drugs potentiate chemotherapeutic effects in solid tumor therapy.

Epigenetic therapy is a novel tumor therapeutic method and refers to the targeting of the aberrant epigenetic modifications presumably at cancer-related genes by chemicals which are epigenetic targeting drugs (ETDs). Not like in treating hematopoietic cancer, the clinical trials investigating the potential use of ETDs in the solid tumor is not encouraging. Instead, the curative effects of ETD delivered together with DNA targeting chemo drugs (DTDs) are quite promising according to our meta-analysis. To investigate the synergistic mechanism of ETD and DTD drug combination, the therapeutic effect was studied using both cell lines and mouse engrafted tumors. Mechanically we show that HDAC inhibitors and DNMT inhibitors are capable of increasing the chromatin accessibility to cisplatin (CP) and doxorubicin (Dox) through chromatin decompaction globally. Consequently, the combination of ETD and DTD enhances the DTD induced DNA damage and cell death. Engrafted tumors in SCID mice also show increased sensitivity to irradiation (IR) or CP when the tumors were pretreated by ETDs. Given the limited therapeutic effect of ETD alone, these results strongly suggest that the combination of DTD, including irradiation, and ETD treatment is a very promising choice in clinical solid tumor therapy.

Proximity Labeling of Interacting Proteins: Application of BioID as a Discovery Tool.

Proteins perform biochemical functions by forming complexes, or protein-protein interactions (PPIs). Many different approaches such as phage display, yeast hybridization, etc. were developed to illustrate the PPIs, and disclose the composition and organization of protein complexes. However, none of these approaches are based on the real-time and in vivo PPI analysis. Proximity-dependent labeling (PDL) of interacting proteins has recently been proposed by taking advantage of several enzymes, which are capable of attaching the known reactive groups to the nearby proteins covalently. Among the PDL methods, BioID is the earliest and the most widely used one and has been upgraded from its prototype, making it an extremely convenient research tool. In this review, we describe the BioID technology development, its potential applications according to the nature of the target protein, and some recent efforts to circumvent the technical limitations. Moreover, some comparable PDL methods are introduced, including selective proteomic proximity labeling assay using tyramide, enzyme-mediated activation of radical sources, Proximity Labeling with Ascorbate Peroxidase, in vivo proximal labeling, etc., and we propose that systematic comparison of the working radius of these methods may be helpful to develop a tool box, from which the right method can be selected for a given target protein for PPI research.

Distinct mutation accumulation rates among tissues determine the variation in cancer risk.

Cancer is believed to be a result of accumulated mutations. However, this concept has not been fully confirmed owing to the impossibility of tracking down the ancestral somatic cell. We sought to verify the concept by exploring the correlation between cancer risk and mutation accumulation among different tissues. We hypothesized that the detected mutations through bulk tumor sequencing are commonly shared in majority, if not all, of tumor cells and are therefore largely a reflection of the mutations accumulated in the ancestral cell that gives rise to tumor. We collected a comprehensive list of mutation frequencies revealed by bulk tumor sequencing, and investigated its correlation with cancer risk to mirror the correlation between mutation accumulation and cancer risk. This revealed an approximate 1:1 relationship between mutation frequency and cancer risk in 41 different cancer types based on the sequencing data of 5,542 patients. The correlation strongly suggests that variation in cancer risk among tissues is mainly attributable to distinct mutation accumulation rates. Moreover, the correlation establishes a baseline to evaluate the effect of non-mutagenic carcinogens on cancer risk. Finally, our mathematic modeling provides a reasonable explanation to reinforce that cancer risk is predominantly determined by the first rate-limiting mutation.