Post-translational Modification of PD-1: Potential Targets for Cancer Immunotherapy.

Activation of effector T cells leads to upregulation of PD-1, which can inhibit T-cell activity following engagement with its ligand PD-L1. Post-translational modifications (PTM), including glycosylation, phosphorylation, ubiquitination, and palmitoylation, play a significant role in regulating PD-1 protein stability, localization, and interprotein interactions. Targeting PTM of PD-1 in T cells has emerged as a potential strategy to overcome PD-1-mediated immunosuppression in cancer and enhances antitumor immunity. The regulatory signaling pathways that induce PTM of PD-1 can be suppressed with small-molecule inhibitors, and mAbs can directly target PD-1 PTMs. Preliminary outcomes from exploratory studies suggest that focusing on the PTM of PD-1 has strong therapeutic potential and can enhance the response to anti-PD-1.

GMI, a Ganoderma microsporum protein, abolishes focal adhesion network to reduce cell migration and metastasis of lung cancer.

BACKGROUND: Cancer metastasis is a major cause of cancer-related deaths, emphasizing the urgent need for effective therapies. Although it has been shown that GMI, a fungal protein from Ganoderma microsporum, could suppress primary tumor growth in a wide spectrum of cancer types, it is still unclear whether GMI exhibits anti-metastasis properties, particularly in lung cancers. Further investigation is needed. AIMS AND OBJECTIVES: The objective of this study is to investigate the potential inhibitory effects of GMI on lung cancer metastasis in vivo. Utilizing systematic and comprehensive approaches, our research aims to elucidate the underlying molecular mechanisms responsible for the anti-metastatic effects. MATERIALS AND METHODS: In vitro migration and cell adhesion assays addressed the epithelial-to-mesenchymal transition (EMT)-related phenotype. Proteomic and bioinformatic analyses identified the GMI-regulated proteins and cellular responses. GMI-treated LLC1-bearing mice were analyzed using IVIS Spectrum to assess the anti-metastatic effect. KEY FINDINGS: GMI inhibits EMT as well as cell migration. GMI disrupts cell adhesion and downregulates integrin, resulting in inhibition of phosphorylated FAK. GMI induces macropinocytosis and lysosome-mediated degradation of integrin αv, α5, α6 and ß1. GMI downregulates Slug via inhibition of FAK activity, which in turn enhances expressions of epithelial-related markers and decreases cell mobility. Mechanistically, GMI-induced FAK inhibition engenders MDM2 expression and enhances MDM2/p21/Slug complex formation, leading to Slug degradation. GMI treatment reduces the metastatic pulmonary lesion and prolongs the survival of LLC1-bearing mice. SIGNIFICANCE: Our findings highlight GMI as a promising therapeutic candidate for metastatic lung cancers, offering potential avenues for further research and drug development.

DoSurvive: A webtool for investigating the prognostic power of a single or combined cancer biomarker.

We present DoSurvive, a user-friendly survival analysis web tool and a cancer prognostic biomarker centered database. DoSurvive is the first database that allows users to perform multivariant survival analysis for cancers with customized gene/patient list. DoSurvive offers three survival analysis methods, Log rank test, Cox regression and accelerated failure time model (AFT), for users to analyze five types of quantitative features (mRNA, miRNA, lncRNA, protein and methylation of CpG islands) with four survival types, i.e. overall survival, disease-specific survival, disease-free interval, and progression-free interval, in 33 cancer types. Notably, the implemented AFT model provides an alternative method for genes/features which failed the proportional hazard assumption in Cox regression. With the unprecedented number of survival models implemented and high flexibility in analysis, DoSurvive is a unique platform for the identification of clinically relevant targets for cancer researcher and practitioners. DoSurvive is freely available at http://dosurvive.lab.nycu.edu.tw/.

DDX3 suppresses hepatocellular carcinoma progression through modulating the secretion and composition of exosome.

Due to the lack of predictive biomarkers and the lack of conspicuous symptoms at the early stage, hepatocellular carcinoma (HCC) remains difficult to diagnose and treat effectively. During cancer development, exosomes secreted from tumor cells carry functional molecules to surrounding recipient cells, thereby participating in the regulation of cancer progression. DDX3, a DEAD-box RNA helicase, performs many important functions in several cellular processes and is therefore implicated as a tumor suppressor in HCC. However, whether DDX3 affects the secretion and cargo sorting of HCC exosomes remains obscure. In this study, our results revealed that reduced DDX3 expression in HCC cells promoted the release of exosomes and enhanced the expression of several exosome biogenesis-associated proteins, such as exosome markers (e.g., TSG101, Alix, and CD63) and Rab proteins (e.g., Rab5, Rab11, and Rab35). By double knockdown of the expression of DDX3 and these exosome biogenesis-related factors, we confirmed that DDX3 participated in the regulation of exosome secretion by modulating the expression of these cellular factors in HCC cells. In addition, exosomes derived from DDX3-knockdown HCC cells enhanced cancer stem cell properties, including self-renewal capability, migration, and drug resistance, in recipient HCC cells. Moreover, up-regulation of the exosome markers TSG101, Alix, and CD63 as well as down-regulation of tumor-suppressive miR-200b and miR-200c were observed in exosomes derived from DDX3-knockdown HCC cells, which may account for the enhanced hepatic cancer stemness of the recipient cells treated with DDX3-knockdown HCC cell-derived exosomes. Taken together, our findings provide a new molecular mechanism supporting the tumor-suppressor role of DDX3 in HCC, which may contribute to the development of new therapeutic strategies against HCC.

Deglycosylation of SLAMF7 in breast cancers enhances phagocytosis.

N-linked glycosylation of proteins is one of the post-translational modifications (PTMs) that shield tumor antigens from immune attack. Signaling lymphocytic activation molecule family 7 (SLAMF7) suppresses cancer cell phagocytosis and is an ideal target under clinical development. PTM of SLAMF7, however, remains less understood. In this study, we investigated the role of N-glycans on SLAMF7 in breast cancer progression. We identified seven N-linked glycosylation motifs on SLAMF7, which are majorly occupied by complex structures. Evolutionally conserved N98 residue is enriched with high mannose and sialylated glycans. Hyperglycosylated SLAMF7 was associated with STT3A expression in breast cancer cells. Inhibition of STT3A by a small molecule inhibitor, N-linked glycosylation inhibitor-1 (NGI-1), reduced glycosylation of SLAMF7, resulting in enhancing antibody affinity and phagocytosis. To provide an on-target effect, we developed an antibody-drug conjugate (ADC) by coupling the anti-SLAMF7 antibody with NGI-1. Deglycosylation of SLAMF7 increases antibody recognition and promotes macrophage engulfment of breast cancer cells. Our work suggests deglycosylation by ADC is a potential strategy to enhance the response of immunotherapeutic agents.

Mutant p53-microRNA-200c-ZEB2-Axis-Induced CPT1C Elevation Contributes to Metabolic Reprogramming and Tumor Progression in Basal-Like Breast Cancers.

TP53 is mutated in more than 80% of basal-like breast cancers (BLBCs). BLBCs with TP53 mutation are usually high-grade and have worse responses to chemotherapy, leading to poor clinical outcomes. Wild-type p53 (WTp53) is well-accepted to promote fatty acid oxidation (FAO); however, in this study, we demonstrate that mutant p53 (Mutp53) enhances FAO activity through constitutively upregulating CPT1C via dysregulating the miR-200c-ZEB2 axis. Sustained CPT1C expression contributes to the metabolic preference of FAO, epithelial-mesenchymal transition (EMT) phenotypes, migration, invasion, and cancer stemness in BLBC, which is mediated by modulating the redox status. Furthermore, interference of CPT1C expression impairs tumor growth and pulmonary colonization of BLBC cells in vivo, and even postpones the occurrence of spontaneous metastasis, resulting in a prolonged disease-specific survival (DSS). Consistently, clinical validation reveals that high CPT1C is observed in breast cancer patients with metastasis and is correlated with poor overall, disease-free, progression-free, and disease-specific survival in BLBC patients. Together, unlike WTp53 which transiently transactivates CPT1C, Mutp53 provides long-term benefits through sustaining CPT1C expression by disturbing the miR-200c-ZEB2 axis, which potentiates FAO and facilitates tumor progression in BLBC, suggesting that targeting Mutp53-CPT1C-driven metabolic reprogramming is promising to serve as novel therapeutic strategies for BLBC in the future.

p53-Mediated Indirect Regulation on Cellular Metabolism: From the Mechanism of Pathogenesis to the Development of Cancer Therapeutics.

The transcription factor p53 is the most well-characterized tumor suppressor involved in multiple cellular processes, which has expanded to the regulation of metabolism in recent decades. Accumulating evidence reinforces the link between the disturbance of p53-relevant metabolic activities and tumor development. However, a full-fledged understanding of the metabolic roles of p53 and the underlying detailed molecular mechanisms in human normal and cancer cells remain elusive, and persistent endeavor is required to foster the entry of drugs targeting p53 into clinical use. This mini-review summarizes the indirect regulation of cellular metabolism by wild-type p53 as well as mutant p53, in which mechanisms are categorized into three major groups: through modulating downstream transcriptional targets, protein-protein interaction with other transcription factors, and affecting signaling pathways. Indirect mechanisms expand the p53 regulatory networks of cellular metabolism, making p53 a master regulator of metabolism and a key metabolic sensor. Moreover, we provide a brief overview of recent achievements and potential developments in the therapeutic strategies targeting mutant p53, emphasizing synthetic lethal methods targeting mutant p53 with metabolism. Then, we delineate synthetic lethality targeting mutant p53 with its indirect regulation on metabolism, which expands the synthetic lethal networks of mutant p53 and broadens the horizon of developing novel therapeutic strategies for p53 mutated cancers, providing more opportunities for cancer patients with mutant p53. Finally, the limitations and current research gaps in studies of metabolic networks controlled by p53 and challenges of research on p53-mediated indirect regulation on metabolism are further discussed.

Sumoylation participates in the regulation of YB-1-mediated mismatch repair deficiency and alkylator tolerance.

Numerous reports indicate that enhanced expression of Y-box binding protein-1 (YB-1) in tumor cells is strongly associated with tumorigenesis, aggressiveness, drug resistance, as well as poor prognosis in several types of cancers, and YB-1 is considered to be an oncogene. The molecular mechanism contributing to the regulation of the biological activities of YB-1 remains obscure. Sumoylation, a post-translational modification involving the covalent conjugation of small ubiquitin-like modifier (SUMO) proteins to a target protein, plays key roles in the modulation of protein functions. In this study, our results revealed that YB-1 is sumoylated and that Lys26 is a critical residue for YB-1 sumoylation. Moreover, YB-1 was found to directly interact with SUMO proteins, and disruption of the SUMO-interacting motif (SIM) of YB-1 not only interfered with this interaction but also diminished YB-1 sumoylation. The subcellular localization, protein stability, and transcriptional regulatory activity of YB-1 were not significantly affected by sumoylation. However, decreased sumoylation disrupted the interaction between YB-1 and PCNA as well as YB-1-mediated inhibition of the MutSα/PCNA interaction and MutSα mismatch binding activity, indicating a functional role of YB-1 sumoylation in inducing DNA mismatch repair (MMR) deficiency and spontaneous mutations. The MMR machinery also recognizes alkylator-modified DNA adducts to signal for cell death. We further demonstrated that YB-1 sumoylation is crucial for the inhibition of SN1-type alkylator MNNG-induced cytotoxicity, G2/M-phase arrest, apoptosis, and the MMR-dependent DNA damage response. Collectively, these results provide molecular explanations for the impact of YB-1 sumoylation on MMR deficiency and alkylator tolerance, which may provide insight for designing therapeutic strategies for malignancies and alkylator-resistant cancers associated with YB-1 overexpression.

Mutant p53 Attenuates Oxidative Phosphorylation and Facilitates Cancer Stemness through Downregulating miR-200c-PCK2 Axis in Basal-Like Breast Cancer.

miR-200c is a tumor suppressor miRNA that plays a critical role in regulating epithelial phenotype and cancer stemness. p53 deficiency downregulates the expression of miR-200c and leads to epithelial-mesenchymal transition (EMT) and stemness phenotype, which contributes to the progression of breast cancers. In this study, we demonstrated that CRISPR-mediated knockout (KO) of miR-200c induces metabolic features similar to the metabolic rewiring caused by p53 hot-spot mutations, and that impairing this metabolic reprogramming interferes with miR-200c deficiency-induced stemness and transformation. Moreover, restoring miR-200c expression compromised EMT, stem-cell properties, and the Warburg effect caused by p53 mutations, suggesting that mutant p53 (MTp53) induces EMT-associated phenotypes and metabolic reprogramming by downregulating miR-200c. Mechanistically, decreased expression of PCK2 was observed in miR-200c- and p53-deficient mammary epithelial cells, and forced expression of miR-200c restored PCK2 in p53 mutant-expressing cells. Reduced PCK2 expression not only led to attenuated oxidative phosphorylation (OXPHOS) and increased stemness in normal mammary epithelial cells but also compromised the enhanced OXPHOS and suppression of cancer stemness exerted by miR-200c in p53 mutation-bearing basal-like breast cancer (BLBC) cells. Clinically, PCK2 expression is negatively associated with EMT markers and is downregulated in basal-like subtype and cases with low miR-200c expression or p53 mutation. Notably, low expression of PCK2 is associated with poor overall survival (OS) in patients with breast cancer. IMPLICATIONS: Together, our results suggest that p53 and miR-200c regulate OXPHOS and stem/cancer stemness through PCK2, and loss of the p53-miR-200c-PCK2 axis might provide metabolic advantages that facilitate cancer stemness, leading to the progression of BLBCs.

Epithelial-mesenchymal transition induced by SARS-CoV-2 required transcriptional upregulation of Snail.

The engagement of human angiotensin-converting enzyme 2 (hACE2) and SARS-CoV-2 spike protein facilitate virus spread. Thus far, ACE2 and TMPRSS2 expression is correlated with the epithelial-mesenchymal transition (EMT) gene signature in lung cancer. However, the mechanism for SARS-CoV-2-induced EMT has not been thoroughly explored. Here, we showed that SARS-CoV-2 induces EMT phenotypic change and stemness in breast cancer cell model and subsequently identified Snail as a modulator for this regulation. The in-depth analysis identifies the spike protein (S), but not envelope (E), nucleocapsid (N), or membrane protein (M), of SARS-CoV-2 induces EMT marker changes. Suppression of Snail expression in these cells abrogates S protein-induced invasion, migration, stemness, and lung metastasis, suggesting that Snail is required for SARS-CoV-2-mediated aggressive phenotype in cancer. This study reveals an important oncogenic role of SARS-CoV-2 in triggering breast cancer metastasis through Snail upregulation.

Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1.

The Ca2+ entry mechanism that sustains the Ca2+ oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca2+ stores were depleted using cyclopiazonic acid (CPA); others were inseminated. Changes in the oocytes' cytosolic free Ca2+ concentration were monitored, while interaction between the expressed fusion proteins was investigated using fluorescence resonance energy transfer (FRET). Store depletion led to an increase of the FRET signal in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that Ca2+ release was followed by an interaction between these proteins. A similar FRET increase in response to CPA was also detected in oocytes co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming punctae after store depletion. ML-9, an inhibitor that can interfere with STIM1 puncta formation, blocked store-operated Ca2+ entry (SOCE) induced by Ca2+ add-back after a CPA treatment; it also disrupted the Ca2+ oscillations in fertilized oocytes. In addition, oocytes overexpressing mVenus-STIM1 showed high-frequency Ca2+ oscillations when fertilized, arguing for an active role of the protein. High-frequency Ca2+ oscillations were also detected in fertilized oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, and both of these high-frequency Ca2+ oscillations could be stopped by inhibitors of SOCE. Importantly, in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, we were also able to detect cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in pig oocytes, SOCE is involved in the maintenance of the repetitive Ca2+ transients at fertilization.

RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP.

Multifunctional RNA helicase DDX3 participates in HCV infection, one of the major causes of hepatic steatosis. Here, we investigated the role of DDX3 in hepatic lipid metabolism. We found that HCV infection severely reduced DDX3 expression. Analysis of intracellular triglyceride and secreted ApoB indicated that lipid accumulations were increased while ApoB secretion were decreased in DDX3 knockdown HuH7 and HepG2 cell lines. Down-regulation of DDX3 significantly decreased protein and transcript expression of microsomal triglyceride transfer protein (MTP), a key regulator of liver lipid homeostasis. Moreover, DDX3 interacted with hepatocyte nuclear factor 4 (HNF4) and small heterodimer partner (SHP), and synergistically up-regulated HNF4-mediated transactivation of MTP promoter via its ATPase activity. Further investigation revealed that DDX3 interacted with CBP/p300 and increased the promoter binding affinity of HNF4 by enhancing HNF4 acetylation. Additionally, DDX3 partially relieved the SHP-mediated suppression on MTP promoter by competing with SHP for HNF4 binding which disrupted the inactive HNF4/SHP heterodimer while promoted the formation of the active HNF4 homodimer. Collectively, these results imply that DDX3 regulates MTP gene expression and lipid homeostasis through interplay with HNF4 and SHP, which may also reveal a novel mechanism of HCV-induced steatosis.

Y-Box Binding Protein 1 Stabilizes Hepatitis C Virus NS5A via Phosphorylation-Mediated Interaction with NS5A To Regulate Viral Propagation.

UNLABELLED: Replication of hepatitis C virus (HCV) is dependent on virus-encoded proteins and numerous cellular factors. DDX3 is a well-known host cofactor of HCV replication. In this study, we investigated the role of a DDX3-interacting protein, Y-box binding protein 1 (YB-1), in the HCV life cycle. Both YB-1 and DDX3 interacted with the viral nonstructural protein NS5A. During HCV infection, YB-1 partially colocalized with NS5A and the HCV replication intermediate double-stranded RNA (dsRNA) in HCV-infected Huh-7.5.1 cells. Despite sharing the same interacting partners, YB-1 participated in HCV RNA replication but was dispensable in steady-state HCV RNA replication, different from the action of DDX3. Moreover, knockdown of YB-1 in HCV-infected cells prevented infectious virus production and reduced the ratio of hyperphosphorylated (p58) to hypophosphorylated (p56) forms of NS5A, whereas DDX3 silencing did not affect the ratio of the p58 and p56 phosphoforms of NS5A. Interestingly, silencing of YB-1 severely reduced NS5A protein stability in NS5A-ectopically expressing, replicon-containing, and HCV-infected cells. Furthermore, mutations of serine 102 of YB-1 affected both YB-1-NS5A interaction and NS5A-stabilizing activity of YB-1, indicating that this Akt phosphorylation site of YB-1 plays an important role in stabilizing NS5A. Collectively, our results support a model in which the event of YB-1 phosphorylation-mediated interaction with NS5A results in stabilizing NS5A to sustain HCV RNA replication and infectious HCV production. Overall, our study may reveal a new aspect for the development of novel anti-HCV drugs. IMPORTANCE: Chronic hepatitis C virus (HCV) infection induces liver cirrhosis and hepatocellular carcinoma. The viral nonstructural protein NS5A co-opting various cellular signaling pathways and cofactors to support viral genome replication and virion assembly is a new strategy for anti-HCV drug development. NS5A phosphorylation is believed to modulate switches between different stages of the HCV life cycle. In this study, we identified the cellular protein YB-1 as a novel NS5A-interacting protein. YB-1 is a multifunctional protein participating in oncogenesis and is an oncomarker of hepatocellular carcinoma (HCC). We found that YB-1 protects NS5A from degradation and likely regulates NS5A phosphorylation through its phosphorylation-dependent interaction with NS5A, which might be controlled by HCV-induced signaling pathways. Our observations suggest a model in which HCV modulates NS5A level and the ratio of the p58 and p56 phosphoforms for efficient viral propagation via regulation of cellular signaling inducing YB-1 phosphorylation. Our finding may provide new aspects for developing novel anti-HCV drugs.

MicroRNA-205 signaling regulates mammary stem cell fate and tumorigenesis.

Dysregulation of epigenetic controls is associated with tumorigenesis in response to microenvironmental stimuli; however, the regulatory pathways involved in epigenetic dysfunction are largely unclear. We have determined that a critical epigenetic regulator, microRNA-205 (miR-205), is repressed by the ligand jagged1, which is secreted from the tumor stroma to promote a cancer-associated stem cell phenotype. Knockdown of miR-205 in mammary epithelial cells promoted epithelial-mesenchymal transition (EMT), disrupted epithelial cell polarity, and enhanced symmetric division to expand the stem cell population. Furthermore, miR-205-deficient mice spontaneously developed mammary lesions, while activation of miR-205 markedly diminished breast cancer stemness. These data provide evidence that links tumor microenvironment and microRNA-dependent regulation to disruption of epithelial polarity and aberrant mammary stem cell division, which in turn leads to an expansion of stem cell population and tumorigenesis. This study elucidates an important role for miR-205 in the regulation of mammary stem cell fate, suggesting a potential therapeutic target for limiting breast cancer genesis.

Epithelial-mesenchymal transition induced by TNF-α requires NF-κB-mediated transcriptional upregulation of Twist1.

Proinflammatory cytokines produced in the tumor microenvironment facilitate tumor development and metastatic progression. In particular, TNF-α promotes cancer invasion and angiogenesis associated with epithelial-mesenchymal transition (EMT); however, the mechanisms underlying its induction of EMT in cancer cells remain unclear. Here we show that EMT and cancer stemness properties induced by chronic treatment with TNF-α are mediated by the upregulation of the transcriptional repressor Twist1. Exposure to TNF-α rapidly induced Twist1 mRNA and protein expression in normal breast epithelial and breast cancer cells. Both IKK-ß and NF-κB p65 were required for TNF-α-induced expression of Twist1, suggesting the involvement of canonical NF-κB signaling. In support of this likelihood, we defined a functional NF-κB-binding site in the Twist1 promoter, and overexpression of p65 was sufficient to induce transcriptional upregulation of Twist1 along with EMT in mammary epithelial cells. Conversely, suppressing Twist1 expression abrogated p65-induced cell migration, invasion, EMT, and stemness properties, establishing that Twist1 is required for NF-κB to induce these aggressive phenotypes in breast cancer cells. Taken together, our results establish a signaling axis through which the tumor microenvironment elicits Twist1 expression to promote cancer metastasis. We suggest that targeting NF-κB-mediated Twist1 upregulation may offer an effective a therapeutic strategy for breast cancer treatment.

p53 regulates epithelial-mesenchymal transition and stem cell properties through modulating miRNAs.

The epithelial-mesenchymal transition (EMT) has recently been linked to stem cell phenotype. However, the molecular mechanism underlying EMT and regulation of stemness remains elusive. Here, using genomic approaches, we show that tumour suppressor p53 has a role in regulating both EMT and EMT-associated stem cell properties through transcriptional activation of the microRNA miR-200c. p53 transactivates miR-200c through direct binding to the miR-200c promoter. Loss of p53 in mammary epithelial cells leads to decreased expression of miR-200c and activates the EMT programme, accompanied by an increased mammary stem cell population. Re-expressing miR-200c suppresses genes that mediate EMT and stemness properties and thereby reverts the mesenchymal and stem-cell-like phenotype caused by loss of p53 to a differentiated epithelial cell phenotype. Furthermore, loss of p53 correlates with a decrease in the level of miR-200c, but an increase in the expression of EMT and stemness markers, and development of a high tumour grade in a cohort of breast tumours. This study elucidates a role for p53 in regulating EMT-MET (mesenchymal-epithelial transition) and stemness or differentiation plasticity, and reveals a potential therapeutic implication to suppress EMT-associated cancer stem cells through activation of the p53-miR-200c pathway.

EZH2 promotes expansion of breast tumor initiating cells through activation of RAF1-ß-catenin signaling.

It has been proposed that an aggressive secondary cancer stem cell population arises from a primary cancer stem cell population through acquisition of additional genetic mutations and drives cancer progression. Overexpression of Polycomb protein EZH2, essential in stem cell self-renewal, has been linked to breast cancer progression. However, critical mechanism linking increased EZH2 expression to BTIC (breast tumor initiating cell) regulation and cancer progression remains unclear. Here, we identify a mechanism in which EZH2 expression-mediated downregulation of DNA damage repair leads to accumulation of recurrent RAF1 gene amplification in BTICs, which activates p-ERK-ß-catenin signaling to promote BTIC expansion. We further reveal that AZD6244, a clinical trial drug that inhibits RAF1-ERK signaling, could prevent breast cancer progression by eliminating BTICs.

The activated Notch1 receptor cooperates with alpha-enolase and MBP-1 in modulating c-myc activity.

The Notch signal pathway plays multifaceted roles to promote or suppress tumorigenesis. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, activates the c-myc proto-oncogene. The complex of N1IC and transcription factor YY1 binds to the human c-myc promoter to enhance c-myc expression in a CBF1-independent manner. Here we demonstrated that N1IC interacted with the c-Myc-regulating proteins alpha-enolase and c-myc promoter binding protein 1 (MBP-1). Both alpha-enolase and MBP-1 suppressed the N1IC-enhanced activity of the c-myc promoter in a CBF1-independent manner. The YY1 response element in front of the P2 c-myc promoter was essential and sufficient for the modulation of c-myc by N1IC and alpha-enolase or MBP-1. Furthermore, N1IC, YY1, and alpha-enolase or MBP-1 but not CBF1 bound to the c-myc promoter through associating with the YY1 response element. Hemin-induced erythroid differentiation was suppressed by N1IC in K562 cells. This suppression was relieved by the expression of alpha-enolase and MBP-1. In addition, both alpha-enolase and MBP-1 suppressed the N1IC-enhanced colony-forming ability through c-myc. These results indicate that the activated Notch1 receptor and alpha-enolase or MBP-1 cooperate in controlling c-myc expression through binding the YY1 response element of the c-myc promoter to regulate tumorigenesis.

DDX3, a DEAD box RNA helicase with tumor growth-suppressive property and transcriptional regulation activity of the p21waf1/cip1 promoter, is a candidate tumor suppressor.

DDX3 is a DEAD box RNA helicase with diverse biological functions. Using colony formation assay, our results revealed that DDX3 inhibited the colony formation ability of various tumor cells, and this inhibition might be due to a reduced growth rate caused by DDX3. Additionally, we identified p21(waf1/cip1), a cyclin-dependent kinase inhibitor, as a target gene of DDX3, and the up-regulation of p21(waf1/cip1) expression accounted for the colony-suppressing activity of DDX3. Moreover, DDX3 exerted its transactivation function on p21(waf1/cip1) promoter through an ATPase-dependent but helicase-independent mechanism, and the four Sp1 sites located within the -123 to -63 region, relative to the transcription start site of p21(waf1/cip1) promoter, were essential for the response to DDX3. Furthermore, DDX3 interacted and cooperated with Sp1 to up-regulate the promoter activity of p21(waf1/cip1). To determine the relevance of DDX3 in clinical cancers, the expression profile of DDX3 in various tumors was also examined. A declined expression of DDX3 mRNA and protein was found in approximately 58% to 73% of hepatoma specimens, which led to the reduction of p21(waf1/cip1) expression in a manner independent of p53 status. Additionally, an alteration of subcellular localization from nuclei to cytoplasm was also observed in >70% of cutaneous squamous cell carcinoma samples. Because DDX3 exhibits tumor suppressor functions, such as a growth-suppressive property and transcriptional activation of the p21(waf1/cip1) promoter, and is inactivated through down-regulation of gene expression or alteration of subcellular localization in tumor cells, all these features together suggest that DDX3 might be a candidate tumor suppressor.

Hepatitis C viral proteins interact with Smad3 and differentially regulate TGF-beta/Smad3-mediated transcriptional activation.

Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine implicated as a pathogenic mediator in various liver diseases. Enhanced TGF-beta production and lack of TGF-beta responses are often observed during hepatitis C virus (HCV) infection. In this study, we demonstrate that TGF-beta-mediated transactivation is decreased in cells exogenously expressing the intact HCV polyprotein. Among 10 viral products of HCV, only core and nonstructural protein 3 (NS3) physically interact with the MH1 (Mad homology 1) region of the Smad3 and block TGF-beta/Smad3-mediated transcriptional activation through interference with the DNA-binding ability of Smad3, not the nuclear translocation. However, the interactive domain of NS3 extends to the MH2 (Mad homology 2) region of Smad3 and a distinction is found between effects mediated, respectively, by these two viral proteins. HCV core, in the presence or absence of TGF-beta, has a stronger suppressive effect on the DNA-binding and transactivation ability of Smad3 than NS3. Although HCV core, NS3, and the HCV subgenomic replicon all attenuate TGF-beta/Smad3-mediated apoptosis, only HCV core represses TGF-beta-induced G1 phase arrest through downregulation of the TGF-beta-induced p21 promoter activation. Along with this, HCV core, rather than NS3, exhibits a significant inhibitory effect on the binding of Smad3/Sp1 complex to the proximal p21 promoter in response to TGF-beta. In conclusion, HCV viral proteins interact with the TGF-beta signaling mediator Smad3 and differentially impair TGF-beta/Smad3-mediated transactivation and growth inhibition. This functional counteraction of TGF-beta responses provides insights into possible mechanisms, whereby the HCV oncogenic proteins antagonize the host defenses during hepatocarcinogenesis.