MRTF-A-mediated protection against amyloid-ß-induced neuronal injury correlates with restoring autophagy via miR-1273g-3p/mTOR axis in Alzheimer models.

Myocardia-Related Transcription Factors-A (MRTF-A), which is enriched in the hippocampus and cerebral cortex, has been shown to have a protective function against ischemia hypoxia-induced neuronal apoptosis. However, the function of MRTF-A on ß-amyloid peptide (Aß)-induced neurotoxicity and autophagy dysfunction in Alzheimer's disease is still unclear. This study shows that the expression of MRTF-A in the hippocampus of Tg2576 transgenic mice is reduced, and the overexpression of MRTF-A mediated by lentiviral vectors carrying MRTF-A significantly reduces the accumulation of hippocampal ß-amyloid peptide and reduces cognition defect. Overexpression of MRTF-A inhibits neuronal apoptosis, increases the protein levels of microtubule-associated protein 1 light chain 3-II (MAP1LC3/LC3-II) and Beclin1, reduces the accumulation of SQSTM1/p62 protein, and promotes autophagosomes-Lysosomal fusion in vivo and in vitro. Microarray analysis and bioinformatics analysis show that MRTF-A reverses Aß-induced autophagy impairment by up-regulating miR-1273g-3p level leading to negative regulation of the mammalian target of rapamycin (mTOR), which is confirmed in Aß1-42-treated SH-SY5Y cells. Further, overexpression of MRTF-A reduces Aß1-42-induced neuronal apoptosis. And the effect was abolished by miR-1273g-3p inhibitor or MHY1485 (mTOR agonist), indicating that the protection of MRTF-A on neuronal damage is through targeting miR-1273g-3p/mTOR axis. Targeting this signaling may be a promising approach to protect against Aß-induced neuronal injury.

The c-Myc Oncogene Maintains Corneal Epithelial Architecture at Homeostasis, Modulates p63 Expression, and Enhances Proliferation During Tissue Repair.

Purpose: The transcription factor c-Myc (Myc) plays central regulatory roles in both self-renewal and differentiation of progenitors of multiple cell lineages. Here, we address its function in corneal epithelium (CE) maintenance and repair. Methods: Myc ablation in the limbal-corneal epithelium was achieved by crossing a floxed Myc mouse allele (Mycfl/fl) with a mouse line expressing the Cre recombinase gene under the keratin (Krt) 14 promoter. CE stratification and protein localization were assessed by histology of paraffin and plastic sections and by immunohistochemistry of frozen sections, respectively. Protein levels and gene expression were determined by western blot and real-time quantitative PCR, respectively. CE wound closure was tracked by fluorescein staining. Results: At birth, mutant mice appeared indistinguishable from control littermates; however, their rates of postnatal weight gain were 67% lower than those of controls. After weaning, mutants also exhibited spontaneous skin ulcerations, predominantly in the tail and lower lip, and died 45 to 60 days after birth. The mutant CE displayed an increase in stratal thickness, increased levels of Krt12 in superficial cells, and decreased exfoliation rates. Accordingly, the absence of Myc perturbed protein and mRNA levels of genes modulating differentiation and proliferation processes, including ΔNp63ß, Ets1, and two Notch target genes, Hey1 and Maml1. Furthermore, Myc promoted CE wound closure and wound-induced hyperproliferation. Conclusions: Myc regulates the balance among CE stratification, differentiation, and surface exfoliation and promotes the transition to the hyperproliferative state during wound healing. Its effect on this balance may be exerted through the control of multiple regulators of cell fate, including isoforms of tumor protein p63.

Islet transplantation into brown adipose tissue can delay immune rejection.

Type 1 diabetes is an autoimmune disease characterized by insulin-producing ß cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.

UPF1: a potential biomarker in human cancers.

Recently, Up-frameshift protein 1 (UPF1) is reported to be downregulated in various cancers and its low expression is closely correlated with poor prognosis. UPF1 is well known as a master regulator of nonsense-mediated mRNA decay (NMD), which serves as a highly conserved mRNA surveillance process protecting cells from aberrant toxic transcripts. Due to dysfunction of UPF1, NMD fails to proceed, which contributes to tumor initiation and progression. This review shows a brief summary of the aberrant expression, functional roles and molecular mechanisms of UPF1 during tumorigenesis. Increasing evidence has indicated that UPF1 could serve as a potential biomarker for cancer diagnosis and treatment for future clinical applications in cancer.

Targeting hyperactive TGFBR2 for treating MYOCD deficient lung cancer.

Purpose: Clinical success of cancer therapy is severely limited by drug resistance, attributed in large part to the loss of function of tumor suppressor genes (TSGs). Developing effective strategies to treat those tumors is challenging, but urgently needed in clinic. Experimental Design: MYOCD is a clinically relevant TSG in lung cancer patients. Our in vitro and in vivo data confirm its tumor suppressive function. Further analysis reveals that MYOCD potently inhibits stemness of lung cancer stem cells. Mechanistically, MYOCD localizes to TGFBR2 promoter region and thereby recruits PRMT5/MEP50 complex to epigenetically silence its transcription. Conclusions: NSCLC cells deficient of MYOCD are particularly sensitive to TGFBR kinase inhibitor (TGFBRi). TGFBRi and stemness inhibitor synergize with existing drugs to treat MYOCD deficient lung cancers. Our current work shows that loss of function of MYOCD creates Achilles' heels in lung cancer cells, which might be exploited in clinic.

Lost Small Envelope Protein Expression from Naturally Occurring PreS1 Deletion Mutants of Hepatitis B Virus Is Often Accompanied by Increased HBx and Core Protein Expression as Well as Genome Replication.

Hepatitis B virus (HBV) transcribes coterminal mRNAs of 0.7 to 3.5 kb from the 3.2-kb covalently closed circular DNA, with the 2.1-kb RNA being most abundant. The 0.7-kb RNA produces HBx protein, a transcriptional transactivator, while the 3.5-kb pregenomic RNA (pgRNA) drives core and P protein translation as well as genome replication. The large (L) and small (S) envelope proteins are translated from the 2.4-kb and 2.1-kb RNAs, respectively, with the majority of the S protein being secreted as noninfectious subviral particles and detected as hepatitis B surface antigen (HBsAg). pgRNA transcription could inhibit transcription of subgenomic RNAs. The present study characterized naturally occurring in-frame deletions in the 3' preS1 region, which not only codes for L protein but also serves as the promoter for 2.1-kb RNA. The human hepatoma cell line Huh7 was transiently transfected with subgenomic expression constructs for envelope (and HBx) proteins, dimeric constructs, or constructs mimicking covalently closed circular DNA. The results confirmed lost 2.1-kb RNA transcription and HBsAg production from many deletion mutants, accompanied by increases in other (especially 2.4-kb) RNAs, intracellular HBx and core proteins, and replicative DNA but impaired virion and L protein secretion. The highest intracellular L protein levels were achieved by mutants that had residual S protein expression or retained the matrix domain in L protein. Site-directed mutagenesis of a high replicating deletion mutant suggested that increased HBx protein expression and blocked virion secretion both contributed to the high replication phenotype. Our findings could help explain why such deletions are selected at a late stage of chronic HBV infection and how they contribute to viral pathogenesis. IMPORTANCE Expression of hepatitis B e antigen (HBeAg) and overproduction of HBsAg by wild-type HBV are implicated in the induction of immune tolerance to achieve chronic infection. How HBV survives the subsequent immune clearance phase remains incompletely understood. Our previous characterization of core promoter mutations to reduce HBeAg production revealed the ability of the 3.5-kb pgRNA to diminish transcription of coterminal RNAs of 2.4 kb, 2.1 kb, and 0.7 kb. The later stage of chronic HBV infection often selects for in-frame deletions in the preS region. Here, we found that many 3' preS1 deletions prevented transcription of the 2.1-kb RNA for HBsAg production, which was often accompanied by increases in intracellular 3.5-, 0.7-, and especially 2.4-kb RNAs, HBx and core proteins, and replicative DNA but lost virion secretion. These findings established the biological consequences of preS1 deletions, thus shedding light on why they are selected and how they contribute to hepatocarcinogenesis.

Prognostic significance of the expression of metastasis-associated in colon cancer-1 in gynecologic cancers and breast cancer: A protocol for systematic review and meta-analysis.

BACKGROUND: The prognostic role of the expression of metastasis-associated in colon cancer-1 (MACC1) in gynecologic cancers and breast cancer remains unclear. The aim of this systematic review and meta-analysis was to determine the prognostic significance of MACC1 expression in gynecologic cancers and breast cancer. MATERIALS AND METHODS: PubMed, Web of Science and Embase were comprehensively searched up to February 9, 2020. Studies focusing on the relationship between the expression of MACC1 and prognosis in gynecologic cancers and breast cancer were included into the analysis. Pooled hazard ratio (HR) or odd ratio with 95% confidence interval (CI) was used to estimate the prognostic value of the expression of MACC1. RESULTS: A total of 1,811patients with gynecologic cancers or breast cancer were included into the analysis. Patients with high expression of MACC1 tended to suffer a shorter overall survival (HR = 2.76, 95%CI = 2.12-3.59, P < .01) and recurrence-free survival (HR = 2.37, 95%CI = 1.44-3.90, P < .01) compared to those with low expression of MACC1. High expression of MACC1 was significantly associated with worse tumor differentiation (P = .04), more advanced FIGO stage (P < .01) and earlier lymph node metastasis (P < .01) compared to low expression of MACC1. CONCLUSION: Compared to low expression of MACC1, high expression of MACC1 predicts a worse prognosis of gynecologic cancers and breast cancer. The expression of MACC1 can serve as a prognostic indicator of gynecologic cancers and breast cancer.

RSF1 requires CEBP/ß and hSNF2H to promote IL-1ß-mediated angiogenesis: the clinical and therapeutic relevance of RSF1 overexpression and amplification in myxofibrosarcomas.

Myxofibrosarcoma is genetically complex and lacks effective nonsurgical treatment strategies; thus, elucidation of novel molecular drivers is urgently needed. Reanalyzing public myxofibrosarcoma datasets, we identified mRNA upregulation and recurrent gain of RSF1 and characterized this chromatin remodeling gene. Myxofibrosarcoma cell lines were employed to elucidate the oncogenic mechanisms of RSF1 by genetic manipulation and two IL-1ß-neutralizing antibodies (RD24, P2D7KK), highlighting the regulatory basis and targetability of downstream IL-1ß-mediated angiogenesis. Tumor samples were assessed for RSF1, IL-1ß, and microvascular density (MVD) by immunohistochemistry and for RSF1 gene status by FISH. In vivo, RSF1-silenced and P2D7KK-treated xenografts were analyzed for tumor-promoting effects and the IL-1ß-linked therapeutic relevance of RSF1, respectively. In vitro, RSF1 overexpression promoted invasive and angiogenic phenotypes with a stronger proangiogenic effect. RT-PCR profiling identified IL1B as a top-ranking candidate upregulated by RSF1. RSF1 required hSNF2H and CEBP/ß to cotransactivate the IL1B promoter, which increased the IL1B mRNA level, IL-1ß secretion and angiogenic capacity. Angiogenesis induced by RSF1-upregulated IL-1ß was counteracted by IL1B knockdown and both IL-1ß-neutralizing antibodies. Clinically, RSF1 overexpression was highly associated with RSF1 amplification, IL-1ß overexpression, increased MVD and higher grades (all P ≤ 0.01) and independently predicted shorter disease-specific survival (P = 0.019, hazard ratio: 4.556). In vivo, both RSF1 knockdown and anti-IL-1ß P2D7KK (200 µg twice weekly) enabled significant growth inhibition and devascularization in xenografts. In conclusion, RSF1 overexpression, partly attributable to RSF1 amplification, contributes a novel proangiogenic function by partnering with CEBP/ß to cotransactivate IL1B, highlighting its prognostic, pathogenetic, and therapeutic relevance in myxofibrosarcomas.

Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor.

The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human ß-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a ß-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.

lncRNA SNHG1 Knockdown Alleviates Amyloid-ß-Induced Neuronal Injury by Regulating ZNF217 via Sponging miR-361-3p in Alzheimer's Disease.

BACKGROUND: Long noncoding RNAs have been proven to play an important role in the progression of Alzheimer's disease (AD). However, the function of small nucleolar RNA host gene 1 (SNHG1) in AD progression remains to be studied. OBJECTIVE: To explore the role of SNHG1 in AD progression and clarify its potential mechanism. METHODS: Amyloid ß-protein (Aß) was used to construct an AD cell model in vitro. The expression levels of SNHG1 and miR-361-3p were determined by quantitative real-time polymerase chain reaction. Cell viability and apoptosis were measured by cell counting kit 8 assay and flow cytometry. The levels of apoptosis-related proteins and zinc finger gene 217 (ZNF217) protein were evaluated by western blot analysis. Additionally, the contents of inflammatory cytokines and oxidative stress markers were tested by enzyme-linked immunosorbent assay. Furthermore, dual-luciferase reporter and RNA immunoprecipitation assays were used to verify the interaction between miR-361-3p and SNHG1 or ZNF217. RESULTS: Aß could induce cell injury, while resveratrol could reverse this effect. SNHG1 expression was positively regulated by Aß and negatively regulated by resveratrol. SNHG1 knockdown could reverse the promotion effect of Aß on cell injury. Moreover, SNHG1 sponged miR-361-3p, and miR-361-3p targeted ZNF217. Additionally, miR-361-3p overexpression reversed the promotion effect of SNHG1 overexpression on cell injury, and ZNF217 silencing also reversed the promotion effect of miR-361-3p inhibitor on cell injury. CONCLUSION: SNHG1 promoted cell injury by regulating the miR-361-3p/ZNF217 axis, which might provide a theoretical basis for molecular therapy of AD.

Allogeneic stem cell transplantation mitigates the adverse prognostic impact of high diagnostic BAALC and MN1 expression in AML.

For most acute myeloid leukemia (AML) patients, an allogeneic hematopoietic stem cell transplantation (HSCT) offers the highest chance of sustained remissions and long-term survival. At diagnosis, high expression of the AML-associated genes BAALC (brain and acute leukemia, cytoplasmic) and MN1 (meningioma-1) were repeatedly linked to inferior outcomes in patients consolidated with chemotherapy while data for patients receiving HSCT remain limited. Using clinically applicable digital droplet PCR assays, we analyzed the diagnostic BAALC/ABL1 and MN1/ABL1 copy numbers in 302 AML patients. High BAALC/ABL1 and MN1/ABL1 copy numbers associated with common adverse prognostic factors at diagnosis. However, while high diagnostic copy numbers of both genes associated with shorter event free survival (EFS) and overall survival (OS) in patients receiving chemotherapy, there was no prognostic impact in patients undergoing HSCT. Our data suggests that the adverse prognostic impact of high BAALC and MN1 expression are mitigated by allogeneic HSCT. But preHSCT BAALC/ABL1 and MN1/ABL1 assessed in remission prior to HSCT remained prognosticators for EFS and OS independent of the diagnostic expression status. Whether allogeneic HSCT may improve survival for AML patients with high diagnostic BAALC or MN1 expression should be investigated prospectively and may improve informed decisions towards individualized consolidation options in AML.

Tectorigenin enhances PDX1 expression and protects pancreatic ß-cells by activating ERK and reducing ER stress.

Pancreas/duodenum homeobox protein 1 (PDX1) is an important transcription factor that regulates islet ß-cell proliferation, differentiation, and function. Reduced expression of PDX1 is thought to contribute to ß-cell loss and dysfunction in diabetes. Thus, promoting PDX1 expression can be an effective strategy to preserve ß-cell mass and function. Previously, we established a PDX1 promoter-dependent luciferase system to screen agents that can promote PDX1 expression. Natural compound tectorigenin (TG) was identified as a promising candidate that could enhance the activity of the promoter for the PDX1 gene. In this study, we first demonstrated that TG could promote the expression of PDX1 in ß-cells via activating extracellular signal-related kinase (ERK), as indicated by increased phosphorylation of ERK; this effect was observed under either normal or glucotoxic/lipotoxic conditions. We then found that TG could suppress induced apoptosis and improved the viability of ß-cells under glucotoxicity and lipotoxicity by activation of ERK and reduction of reactive oxygen species and endoplasmic reticulum (ER) stress. These effects held true in vivo as well: prophylactic or therapeutic use of TG could obviously inhibit ER stress and decrease islet ß-cell apoptosis in the pancreas of mice given a high-fat/high-sucrose diet (HFHSD), thus dramatically maintaining or restoring ß-cell mass and islet size, respectively. Accordingly, both prophylactic and therapeutic use of TG improved HFHSD-impaired glucose metabolism in mice, as evidenced by ameliorating hyperglycemia and glucose intolerance. Taken together, TG, as an agent promoting PDX1 expression exhibits strong protective effects on islet ß-cells both in vitro and in vivo.

VASN promotes proliferation of prostate cancer through the YAP/TAZ axis.

OBJECTIVE: The purpose of this study was to uncover the role of VASN in regulating proliferative ability of prostate cancer (PCa) cells through the yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ) axis, thus influencing the progression of PCa. PATIENTS AND METHODS: VASN, YAP, and TAZ levels in PCa tissues or in the serum were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The diagnostic value of VASN in PCa was assessed by introducing receiver operating characteristic (ROC) curves. Besides, the regulatory effects of VASN on viability, clonality, and expression levels of YAP/TAZ were evaluated by cell counting kit-8 (CCK-8), colony formation, and Western blot, respectively. Finally, rescue experiments were conducted to uncover the involvement of YAP in VASN-regulated proliferation of PCa. RESULTS: Results manifested that VASN, YA, and TAZ were upregulated in PCa patients, and VASN presented a certain diagnostic value. Knockdown of VASN in LNCaP and C4-2 cells suppressed viability and clonality, and downregulated protein levels of YAP and TAZ. Notably, overexpression of YAP abolished the attenuated viability and clonality in PCa cells with VASN knockdown. CONCLUSIONS: VASN promotes proliferative ability in PCa via regulating the YAP/TAZ axis, thus aggravating the progression of the disease.

In vitro analysis of the effect of Flightless I on murine tenocyte cellular functions.

BACKGROUND: Healing of tendons after injury involves the proliferation of tenocytes and the production of extracellular matrix; however, their capacity to heal is limited by poor cell density and limited growth factor activity. Flightless I (Flii) has previously been identified as an important regulator of cellular proliferation and migration, and the purpose of this study was to evaluate the effect of differential Flii gene expression on tenocyte function in vitro. METHODS: The role of Flii on tenocyte proliferation, migration, and contraction was assessed using established assays. Tenocytes from Flii+/-, wild-type, and Flii overexpressing mice were obtained and the effect of differential Flii expression on migration, proliferation, contraction, and collagen synthesis determined in vitro. Statistical differences were determined using unpaired Student's t test and statistical outliers were identified using the Grubbs' test. RESULTS: Flii overexpressing tenocytes showed significantly improved migration and proliferation as well as increased collagen I secretion. Explanted tendons from Flii overexpressing mice also showed significantly elevated tenocyte outgrowth compared to Flii+/- mice. In contrast to its role in dermal wound repair, Flii positively affects cellular processes in tendons. CONCLUSIONS: These findings suggest that Flii could be a novel target for modulating tenocyte activity and improving tendon repair. This could have significant clinical implications as novel therapeutic targets for improved healing of tendon injuries are urgently needed.

Expression of FAM83H and ZNF16 are associated with shorter survival of patients with gallbladder carcinoma.

BACKGROUND: Recently, FAM83H was reported to have roles in cancer progression in conjunction with oncogenic molecules such as MYC and b-catenin. Moreover, the data from the public database indicates a molecular relationship between FAM83H and zinc finger proteins, especially between FAM83H and ZNF16. However, studies on FAM83H and ZNF16 in gallbladder cancer have been limited. METHODS: This study investigated the expression of FAM83H and ZNF16 in 105 gallbladder carcinomas. RESULTS: In human gallbladder carcinomas, immunohistochemical expression of FAM83H was significantly associated with ZNF16 expression. In univariate analysis, nuclear and cytoplasmic expression of FAM83H or ZNF16 were significantly associated with shorter survival of gallbladder carcinoma patients. Multivariate analysis revealed the nuclear expression of FAM83H as an independent indicator of poor prognosis of overall survival (p = 0.005) and relapse-free survival (p = 0.005) of gallbladder carcinoma patients. Moreover, co-expression patterns of nuclear FAM83H and ZNF16 were also independent indicators of shorter survival of gallbladder carcinoma patients (overall survival; p <  0.001, relapse-free survival; p <  0.001). CONCLUSIONS: This study suggests FAM83H and ZNF16 are associated with the progression of gallbladder carcinoma, and the expressions of FAM83H and ZNF16 might be novel prognostic indicators of gallbladder carcinoma patients.

Aberrant expression of the UPF1 RNA surveillance gene disturbs keratinocyte homeostasis by stabilizing AREG.

The up­frameshift suppressor 1 homolog (UPF1) RNA surveillance gene is a core element in the nonsense­mediated RNA decay (NMD) pathway, which impacts a broad spectrum of biological processes in a cell­specific manner. In the present study, the contribution of the NMD pathway to psoriasis lesions and its moderating effects on the biological processes of keratinocytes was reported. Sanger sequencing for skin scales from two patients with psoriasis identified two mRNA mutations (c.2935_2936insA and c.2030­2081del) in the UPF1 gene. The somatic mutants produced truncated UPF1 proteins and perturbed the NMD pathway in cells, leading to the upregulation of NMD substrates. As the most abundant epidermal growth factor receptor ligand in keratinocytes, it was concluded that amphiregulin (AREG) mRNA is a natural NMD substrate, that is dependent on its 3' untranslated region sequence. Perturbed NMD modulated keratinocyte homeostasis in an AREG­dependent but nonidentical manner, which highlighted the unique characteristics of NMD in keratinocytes. By targeting AREG mRNA post­transcriptionally, the UPF1­NMD pathway contributed to an imbalance between proliferation on the one hand, and apoptosis and abnormal differentiation, migration and inflammatory response on the other, in keratinocytes, which indicated a role of the NMD pathway in the full development of keratinocyte­related morbidity and skin diseases.

CDK7 Inhibition is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition.

CDK7, a transcriptional cyclin-dependent kinase, is emerging as a novel cancer target. Triple-negative breast cancers (TNBC) but not estrogen receptor-positive (ER+) breast cancers have been reported to be uniquely sensitive to the CDK7 inhibitor THZ1 due to the inhibition of a cluster of TNBC-specific genes. However, bioinformatic analysis indicates that CDK7 RNA expression is associated with negative prognosis in all the major subtypes of breast cancer. To further elucidate the effects of CDK7 inhibition in breast cancer, we profiled a panel of cell lines representing different breast cancer subtypes. THZ1 inhibited cell growth in all subtypes (TNBC, HER2+, ER+, and HER2+/ER+) with no apparent subtype selectivity. THZ1 inhibited CDK7 activity and induced G1 arrest and apoptosis in all the tested cell lines, but THZ1 sensitivity did not correlate with CDK7 inhibition or CDK7 expression levels. THZ1 sensitivity across the cell line panel did not correlate with TNBC-specific gene expression but it was found to correlate with the differential inhibition of three genes: CDKN1B, MYC and transcriptional coregulator CITED2. Response to THZ1 also correlated with basal CITED2 protein expression, a potential marker of CDK7 inhibitor sensitivity. Furthermore, all of the THZ1-inhibited genes examined were inducible by EGF but THZ1 prevented this induction. THZ1 had synergistic or additive effects when combined with the EGFR inhibitor erlotinib, with no outward selectivity for a particular subtype of breast cancer. These results suggest a potential broad utility for CDK7 inhibitors in breast cancer therapy and the potential for combining CDK7 and EGFR inhibitors.

TOX3 Promotes Ovarian Estrogen Synthesis: An RNA-Sequencing and Network Study.

Background: Women who undergo chronic exposure to excessive estrogen are at a high risk of developing breast cancer. TOX3 has been reported to be highly expressed in breast tumors and is closely related to estrogen receptors. However, the effect of TOX3 on estrogen synthesis remains poorly understood. Methods: Using lentiviruses as a vector, we stably overexpressed TOX3 in the ovarian granulosa cell line KGN, the cells where estradiol is primarily produced, to investigate its role in estrogen production as well as cell viability and apoptosis. RNA-Sequencing was applied to uncover the global gene expression upon TOX3 overexpression. Results: We observed an increased level of cell viability and a reduced cell apoptosis rate after TOX3 overexpression, and the level of estradiol in the cell culture supernatant also increased significantly. Gene set enrichment analysis of the transcriptome showed that the ovarian steroidogenesis pathway was significantly enriched. Similarly, pathway mapping using the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses also showed that TOX3 overexpression affects the ovarian steroidogenesis pathway. Further experiments showed that upregulated FSHR, CYP19A1, and BMP6 accounted for the enhanced estrogen synthesis. Conclusion: Our study demonstrated that TOX3 quantitatively and qualitatively stimulates estrogen synthesis by enhancing estrogen signaling pathway-related gene expression in ovarian granulosa cells. These findings suggest that TOX3 may play a vital role in the pathogenesis of breast cancer.

Acetylation of alpha-fetoprotein promotes hepatocellular carcinoma progression.

Alpha-fetoprotein (AFP) is a well-established biomarker for hepatocellular carcinoma (HCC). Here, we investigated the acetylation state of AFP in vivo. AFP acetylation was regulated by the acetyltransferase CBP and the deacetylase SIRT1. Acetylation of AFP at lysines 194, 211, and 242 increased the stability of AFP protein by decreasing its ubiquitination and proteasomal degradation. AFP acetylation promoted its oncogenic role by blocking binding to the phosphatase PTEN and the pro-apoptotic protein caspase-3, which increased signaling for proliferation, migration, and invasion and decreased apoptosis. High levels of acetylated AFP in HCC tissues were associated with HBV infection and correlated with poor prognosis and decreased patient survival. In HCC cells, hepatitis B virus X protein (HBx) and palmitic acid (PA) increased the level of acetylated AFP by disrupting SIRT1-mediated deacetylation. AFP acetylation plays an important role in HCC progression and provides a new potential prognostic marker and therapeutic target for HCC.

Moderate alcohol intake promotes pancreatic ductal adenocarcinoma development in mice expressing oncogenic Kras.

Kras mutations are associated with pancreatic ductal adenocarcinoma (PDAC). Although tobacco smoking, pancreatitis, and obesity are known environmental risk factors for PDAC, the contribution of moderate alcohol intake to PDAC remains elusive. In the present study, we tested whether a combination of risk factors or moderate alcohol intake induces PDAC development in mice. Control Pdx1Cre and Pdx1Cre;LSL-KrasG12D mutant mice were fed a Western alcohol diet containing high levels of cholesterol and saturated fat, 3.5% alcohol, and lipopolysaccharide for 5 mo. In addition, mice were treated with cerulein, for induction of pancreatitis, and nicotine every month. Treatment with all of these risk factors promoted development of advanced pancreatic neoplasia and PDAC in the Pdx1Cre;LSL-KrasG12D mice but not in the control Pdx1Cre mice. Moderate alcohol intake or Western diet feeding also significantly promoted advanced neoplasia and PDAC development in Pdx1Cre;LSL-KrasG12D mice compared with mice fed a regular chow. Alcohol, but not Western diet, increased tumor development in the liver in the Pdx1Cre;LSL-KrasG12D mice, but its origin remained elusive due to leakiness of Pdx1Cre in hepatocytes. RNA-seq analysis revealed that alcohol feeding increases expression of markers for tumors (Epcam, Krt19, Prom1, Wt1, and Wwtr1), stroma (Dcn, Fn1, and Tnc), and cytokines (Tgfb1 and Tnf) and decreases expression of Fgf21 and Il6 in the pancreatic tumor tissues. Immunostaining showed heterogeneous expression of nephronectin, S100 calcium-binding protein A6, and vascular cell adhesion molecule 1 in pancreatic tumors surrounded by podoplanin-positive stromal cells. Our data indicate that moderate alcohol drinking is a risk factor for development of PDAC.NEW & NOTEWORTHY Heavy alcohol intake has been suspected to be a risk factor of pancreatic ductal adenocarcinoma (PDAC) in humans. However, the contribution of moderate alcohol intake to PDAC development remains elusive. In the present study, we experimentally show that moderate alcohol feeding significantly induces advanced stages of pancreatic intraepithelial neoplasia development and invasive PDAC in Pdx1Cre;LSL-KrasG12D mutant mice. Our data indicate that moderate alcohol drinking is a risk factor for PDAC.