The YAP-TEAD4 complex promotes tumor lymphangiogenesis by transcriptionally upregulating CCBE1 in colorectal cancer.

The secreted protein collagen and calcium-binding EGF domain 1 (CCBE1) is critical for embryonic lymphatic development through its role in the proteolytic activation of mature vascular endothelial growth factor C (VEGFC). We previously reported that CCBE1 is overexpressed in colorectal cancer (CRC) and that its transcription is negatively regulated by the TGFß-SMAD pathway, but the transcriptional activation mechanism of CCBE1 in CRC remains unknown. Recent studies have revealed the vital role of the hippo effectors YAP/TAZ in lymphatic development; however, the role of YAP/TAZ in tumor lymphangiogenesis has not been clarified. In this study, we found that high nuclear expression of transcription factor TEAD4 is associated with lymph node metastasis and high lymphatic vessel density in patients with CRC. YAP/TAZ-TEAD4 complexes transcriptionally upregulated the expression of CCBE1 by directly binding to the enhancer region of CCBE1 in both CRC cells and cancer-associated fibroblasts, which resulted in enhanced VEGFC proteolysis and induced tube formation and migration of human lymphatic endothelial cells in vitro and lymphangiogenesis in a CRC cell-derived xenograft model in vivo. In addition, the bromodomain and extraterminal domain (BET) inhibitor JQ1 significantly inhibited the transcription of CCBE1, suppressed VEGFC proteolysis, and inhibited tumor lymphangiogenesis in vitro and in vivo. Collectively, our study reveals a new positive transcriptional regulatory mechanism of CCBE1 via YAP/TAZ-TEAD4-BRD4 complexes in CRC, which exposes the protumor lymphangiogenic role of YAP/TAZ and the potential inhibitory effect of BET inhibitors on tumor lymphangiogenesis.

Clostridium difficile toxin A and toxin B inhibit YAP in the colonic epithelial cells.

Toxin A (TcdA) and toxin B (TcdB), the two exotoxins of Clostridium difficile, are main causal agents for the colonic epithelium damage in Clostridium difficile infection (CDI). The Hippo pathway is crucial for the control of tissue homeostasis and regeneration of intestines. However, the dysregulation of Hippo pathway in CDI is unclear. Here we show that YAP and TAZ, the transcriptional coactivators downstream of the Hippo pathway, are sequestered in the cytoplasm, degraded, and inactivated by treatment with TcdA and TcdB in colonic epithelial cells. The overexpression of YAP restores the messenger RNA expressions of YAP target genes, attenuates the disruption of cytoskeleton and cell rounding, and rescues the cell proliferation of colonic epithelial cells under exposure of the two toxins. Our results demonstrate that inhibition of YAP and TAZ is involved in the pathogenesis of CDI, implicating that increasing YAP activity could be a potential therapeutic strategy for the CDI treatment.

Increased IGF2BP3 expression promotes the aggressive phenotypes of colorectal cancer cells in vitro and vivo.

Previous literatures reported insulin-like growth factor-2 messenger RNA-binding protein 3 (IGF2BP3) is a poor prognostic marker for colorectal cancer (CRC) patients. However, basic research on the effect and biological role of IGF2BP3 in CRC was still scare. Real-time quantitative polymerase chain reaction and western blot analysis were used to examine IGF2BP3 expression level in tumors and paired normal tissues from CRC patients. Tissue microarrays with 192 CRC patients were subjected to immunohistochemical staining to analyze the prognostic value of IGF2BP3. Proliferation assays, migration assays, and xenograft tumor formation in nude mice were performed to assess the biological role of IGF2BP3 in CRC cells. IGF2BP3 expression was significantly upregulated in tumor tissues compared with the matched normal tissues both in messenger RNA and protein level and was associated with worse prognosis. IGF2BP3 knockdown made cell cycle arrest to impair the proliferation ability of CRC cells and further inhibited the xenograft tumor growth in nude mice, also inhibited the migration ability of CRC cells via inducing epithelial-mesenchymal transition. Therefore, the research demonstrated that increased IGF2BP3 expression promoted the aggressive phenotypes of CRC cells. Targeted IGF2BP3 could be a novel and effective gene therapy for CRC patients to make a better prognosis.

Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein.

The Yes-associated protein (YAP) is a transcription coactivator that plays a crucial role in organ size control by promoting cell proliferation and inhibiting apoptosis. The Hippo tumor suppressor pathway inhibits YAP through phosphorylation-induced cytoplasmic retention and degradation. Here we report a novel mechanism of YAP regulation by angiomotin (AMOT) family proteins via a direct interaction. Knockdown of AMOT family protein AMOTL2 in polarized Madin-Darby canine kidney (MDCK) cells leads to YAP activation, as indicated by decreased YAP tight junction localization, attenuated YAP phosphorylation, accumulation of nuclear YAP, and induction of YAP target gene expression. Transcriptional coactivator with PDZ-binding motif (TAZ), the YAP paralog, is also regulated by AMOT in a similar fashion. Furthermore, AMOTL2 knockdown results in loss of cell contact inhibition in a manner dependent on the functions of YAP and TAZ. Our results indicate a potential tumor-suppressing role of AMOT family proteins as components of the Hippo pathway, and demonstrate a novel mechanism of YAP and TAZ inhibition by AMOT-mediated tight junction localization. These observations provide a potential link between the Hippo pathway and cell contact inhibition.

ETS (E26 transformation-specific) up-regulation of the transcriptional co-activator TAZ promotes cell migration and metastasis in prostate cancer.

Prostate cancer is a very common malignant disease and a leading cause of death for men in the Western world. Tumorigenesis and progression of prostate cancer involves multiple signaling pathways, including the Hippo pathway. Yes-associated protein (YAP) is the downstream transcriptional co-activator of the Hippo pathway, is overexpressed in prostate cancer, and plays a vital role in the tumorigenesis and progression of prostate cancer. However, the role of the YAP paralog and another downstream effector of the Hippo pathway, transcriptional co-activator with PDZ-binding motif (TAZ), in prostate cancer has not been fully elucidated. Here, we show that TAZ is a basal cell marker for the prostate epithelium. We found that overexpression of TAZ promotes the epithelial-mesenchymal transition (EMT), cell migration, and anchorage-independent growth in the RWPE1 prostate epithelial cells. Of note, knock down of TAZ in the DU145 prostate cancer cells inhibited cell migration and metastasis. We also found that SH3 domain binding protein 1 (SH3BP1), a RhoGAP protein that drives cell motility, is a direct target gene of TAZ in the prostate cancer cells, mediating TAZ function in enhancing cell migration. Moreover, the prostate cancer-related oncogenic E26 transformation-specific (ETS) transcription factors, ETV1, ETV4, and ETV5, were required for TAZ gene transcription in PC3 prostate cancer cells. MAPK inhibitor U0126 treatment decreased TAZ expression in RWPE1 cells, and ETV4 overexpression rescued TAZ expression in RWPE1 cells with U0126 treatment. Our results show a regulatory mechanism of TAZ transcription and suggest a significant role for TAZ in the progression of prostate cancer.

PARD3 induces TAZ activation and cell growth by promoting LATS1 and PP1 interaction.

The Hippo pathway plays a major role in organ size control, and its dysregulation contributes to tumorigenesis. The major downstream effectors of the Hippo pathway are the YAP/TAZ transcription co-activators, which are phosphorylated and inhibited by the Hippo pathway kinase LATS1/2. Here, we report a novel mechanism of TAZ regulation by the tight junction protein PARD3. PARD3 promotes the interaction between PP1A and LATS1 to induce LATS1 dephosphorylation and inactivation, therefore leading to dephosphorylation and activation of TAZ. The cytoplasmic, but not the tight junction complex associated, PARD3 is responsible for TAZ regulation. Our study indicates a potential molecular basis for cell growth-promoting function of PARD3 by modulating the Hippo pathway signaling in response to cell contact and cell polarity signals.

Interleukin 22 protects colorectal cancer cells from chemotherapy by activating the STAT3 pathway and inducing autocrine expression of interleukin 8.

Resistance to chemotherapy is the major cause of colorectal cancer (CRC) treatment failure. The cytokine IL-22, which is produced by T cells and NK cells, is associated with tumorigenesis and tumor progression in cancers. However, the role of IL-22 in chemoresistance has not been investigated. We found that IL-22 levels in tumor tissues and peripheral blood were associated with chemoresistance and indicate poor prognosis for patients who received FOLFOX chemotherapy. In CRC cells, IL-22 was able to attenuate the cytotoxic and apoptosis-inducing effects of 5-FU and OXA by activating the STAT3 pathway and subsequently increasing the expression of anti-apoptotic genes. In addition, IL-22 conferred resistance to 5-FU and OXA by inducing IL-8 autocrine expression through STAT3 activation. Our findings identify IL-22 as a novel chemoresistance cytokine and may be a useful prognostic biomarker for CRC patients receiving FOLFOX chemotherapy.

YAP/TAZ enhances P-body formation to promote tumorigenesis.

The role of processing bodies (P-bodies) in tumorigenesis and tumor progression is not well understood. Here, we showed that the oncogenes YAP/TAZ promote P-body formation in a series of cancer cell lines. Mechanistically, both transcriptional activation of the P-body-related genes SAMD4A, AJUBA, and WTIP and transcriptional suppression of the tumor suppressor gene PNRC1 are involved in enhancing the effects of YAP/TAZ on P-body formation in colorectal cancer (CRC) cells. By reexpression of PNRC1 or knockdown of P-body core genes (DDX6, DCP1A, and LSM14A), we determined that disruption of P-bodies attenuates cell proliferation, cell migration, and tumor growth induced by overexpression of YAP5SA in CRC. Analysis of a pancancer CRISPR screen database (DepMap) revealed co-dependencies between YAP/TEAD and the P-body core genes and correlations between the mRNA levels of SAMD4A, AJUBA, WTIP, PNRC1, and YAP target genes. Our study suggests that the P-body is a new downstream effector of YAP/TAZ, which implies that reexpression of PNRC1 or disruption of P-bodies is a potential therapeutic strategy for tumors with active YAP.

Computer-aided molecular design and optimization of potent inhibitors disrupting APC‒Asef interaction.

Colorectal cancer (CRC) is the second leading cause of cancer mortality worldwide. At initial diagnosis, approximately 20% of patients are diagnosed with metastatic CRC (mCRC). Although the APC‒Asef interaction is a well-established target for mCRC therapy, the discovery and development of effective and safe drugs for mCRC patients remains an urgent and challenging endeavor. In this study, we identified a novel structural scaffold based on MAI inhibitors, the first-in-class APC‒Asef inhibitors we reported previously. ONIOM model-driven optimizations of the N-terminal cap and experimental evaluations of inhibitory activity were performed, and 24-fold greater potency was obtained with the best inhibitor compared to the parental compound. In addition, the cocrystal structure validated that the two-layer π‒π stacking interactions were essential for inhibitor stabilization in the bound state. Furthermore, in vitro and in vivo studies have demonstrated that novel inhibitors suppressed lung metastasis in CRC by disrupting the APC‒Asef interaction. These results provide an intrinsic structural basis to further explore drug-like molecules for APC‒Asef-mediated CRC therapy.

AMBRA1 promotes intestinal inflammation by antagonizing PP4R1/PP4c mediated IKK dephosphorylation in an autophagy-independent manner.

IκB kinase (IKK) complex is central regulators of the NF-κB pathway, and dysregulation of IKK phosphorylation leads to hyperactivation of proinflammatory response in various chronic inflammatory diseases, including inflammatory bowel disease (IBD). However, the dynamic modulation of IKK phosphorylation and dephosphorylation in intestinal inflammation remains uncharacterized. Here, we found that autophagy/beclin-1 regulator 1 (AMBRA1) was highly expressed in inflamed colons in a colitis mouse model and in clinical IBD samples. Importantly, AMBRA1 deletion significantly decreased proinflammatory cytokine expression and enhanced the therapeutic effect of infliximab on intestinal inflammation. Mechanistically, the N-term F1 domain of AMBRA1 was required for AMBRA1 to competitively interact with protein phosphatase 4 regulatory subunit 1 (PP4R1) and catalytic protein phosphatase 4 (PP4c) to suppress their interactions with IKK, promote the dissociation of the PP4R1/PP4c complex, and antagonize the dephosphorylation activity of this complex towards the IKK complex. In response to TNF-α stimulation, IKKα phosphorylates AMBRA1 at S1043 to stabilize AMBRA1 expression by impairing its binding to Cullin4A (CUL4A) to decrease its CUL4A-mediated K48-linked ubiquitination. Overall, our study identifies an autophagy-independent function of AMBRA1 as a positive modulator of IKK phosphorylation to promote intestinal inflammation, thus providing a new targeted therapeutic strategy for patients with refractory IBD.

Comprehensive Proteogenomic Profiling Reveals the Molecular Characteristics of Colorectal Cancer at Distinct Stages of Progression.

Colorectal cancer (CRC) is the second most common malignant tumor world-wide. Analysis of the changes that occur during CRC progression could provide insights into the molecular mechanisms driving CRC development and identify improved treatment strategies. Here, we performed an integrated multi-omics analysis of 435 trace-tumor-samples from 148 colorectal cancer (CRC) patients, covering non-tumor (NT), intraepithelial neoplasia (IEN), infiltration (IFT), and advanced-stage CRC (A-CRC) phases. Proteogenomics analyses demonstrated that KRAS and BRAF mutations were mutually exclusive and elevated oxidation phosphorylation in the IEN phase. Chr17q loss and chr20q gain were also mutually exclusive, occurred predominantly in the IEN and IFT phases, respectively, and impacted the cell cycle. Mutation of TP53 was frequent in the A-CRC phase and associated with tumor microenvironment, including increased extracellular matrix rigidity and stromal infiltration. Analysis of the profiles of CRC based on CMS and CRIS classifications revealed the progression paths of each subtype and indicated that microsatellite instability was associated with specific subtype classifications. Additional comparison of molecular characteristics of CRC based on location showed that ANKRD22 amplification by chr10q23.31 gain enhanced glycolysis in the right-sided CRC. The AOM/DSS-induced CRC carcinogenesis mouse model in mice indicated that DDX5 deletion due to chr17q loss promoted CRC development, consistent with the findings from the patient samples. Collectively, this study provides an informative resource for understanding the driving events of different stages of CRC and identifying the potential therapeutic targets.

Aspirin-Mediated Acetylation of SIRT1 Maintains Intestinal Immune Homeostasis.

Aspirin, also named acetylsalicylate, can directly acetylate the side-chain of lysine in protein, which leads to the possibility of unexplained drug effects. Here, the study used isotopic-labeling aspirin-d3 with mass spectrometry analysis to discover that aspirin directly acetylates 10 HDACs proteins, including SIRT1, the most studied NAD+-dependent deacetylase. SIRT1 is also acetylated by aspirin in vitro. It is also identified that aspirin directly acetylates lysine 408 of SIRT1, which abolishes SIRT1 deacetylation activity by impairing the substrates binding affinity. Interestingly, the lysine 408 of SIRT1 can be acetylated by CBP acetyltransferase in cells without aspirin supplement. Aspirin can inhibit SIRT1 to increase the levels of acetylated p53 and promote p53-dependent apoptosis. Moreover, the knock-in mice of the acetylation-mimic mutant of SIRT1 show the decreased production of pro-inflammatory cytokines and maintain intestinal immune homeostasis. The study indicates the importance of the acetylated internal functional site of SIRT1 in maintaining intestinal immune homeostasis.

ELK4 Promotes Colorectal Cancer Progression by Activating the Neoangiogenic Factor LRG1 in a Noncanonical SP1/3-Dependent Manner.

Although the MAPK/MEK/ERK pathway is prevalently activated in colorectal cancer (CRC), MEK/ERK inhibitors show limited efficiency in clinic. As a downstream target of MAPK, ELK4 is thought to work primarily by forming a complex with SRF. Whether ELK4 can serve as a potential therapeutic target is unclear and the transcriptional regulatory mechanism has not been systemically analyzed. Here, it is shown that ELK4 promotes CRC tumorigenesis. Integrated genomics- and proteomics-based approaches identified SP1 and SP3, instead of SRF, as cooperative functional partners of ELK4 at genome-wide level in CRC. Serum-induced phosphorylation of ELK4 by MAPKs facilitated its interaction with SP1/SP3. The pathological neoangiogenic factor LRG1 is identified as a direct target of the ELK4-SP1/SP3 complex. Furthermore, targeting the ELK4-SP1/SP3 complex by combination treatment with MEK/ERK inhibitor and the relatively specific SP1 inhibitor mithramycin A (MMA) elicited a synergistic antitumor effect on CRC. Clinically, ELK4 is a marker of poor prognosis in CRC. A 9-gene prognostic model based on the ELK4-SP1/3 complex-regulated gene set showed robust prognostic accuracy. The results demonstrate that ELK4 cooperates with SP1 and SP3 to transcriptionally regulate LRG1 to promote CRC tumorigenesis in an SRF-independent manner, identifying the ELK4-SP1/SP3 complex as a potential target for rational combination therapy.

Innovative molecular subtypes of multiple signaling pathways in colon cancer and validation of FMOD as a prognostic-related marker.

PURPOSE: Colon cancer is highly heterogeneous in terms of the immune and stromal microenvironment, genomic integrity, and oncogenic properties; therefore, molecular subtypes of the four characteristic dimensions are expected to provide novel clues for immunotherapy of colon cancer. METHODS: According to the enrichment of four dimensions, we performed consensus cluster analysis and identified three robust molecular subtypes for colon cancer, namely immune enriched, immune deficiency, and stroma enriched. We characterized and validated the immune infiltration, gene mutations, copy number variants, methylation, protein expression, and clinical features in different datasets. Finally, we developed an 8-gene risk prognostic model and proposed the innovative RiskScore. In addition, a nomogram model was constructed combining clinical characteristics and RiskScore to validate its excellent clinical predictive power. RESULTS: Combining clinical patient tissue samples and histochemical microarray data, we found that high FMOD expression in tumor epithelial cells was associated with poorer patient prognosis, but FMOD expression in the mesenchyme was not associated with prognosis. In pan-cancer, RiskScore, a prognostic model constructed based on characteristic pathway scores, was a poor prognostic factor for malignancy and was negatively associated with immunotherapy response. CONCLUSION: The identification of molecular subtypes could provide innovative ideas for immunotherapy of colon cancer.

ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

The proliferative potential of mast cells after activation for 3-4h was found to be decreased, which suggests that mast cell degranulation and cell proliferation are differentially regulated. ELK4, a member of the ternary complex factor (TCF) subfamily of Ets transcription factors, is one of the downstream effectors of MAPK signaling that is critical for cell proliferation. And Elk4 has been identified to be vital for macrophage activation in response to zymosan and the transcriptional response to 12-O-tetrade canoyl phorbol-13-acetate (TPA) stimulation in fibroblast. However, the effect of ELK4 on the mast cell transcriptional response to FcϵRI and GPCR mediated activation and its potential functional significance in mast cells remain unclear. Here, we showed that ELK4 expression is downregulated in activated mast cells. Elk4 knockout suppresses cell proliferation and impedes the cell cycle in bone marrow-derived mast cells (BMMCs), which is associated with decreased transcription of cell cycle genes. Additionally, the transcriptional activation of cytokines and chemokines is diminished while mast cell degranulation is enhanced in Elk4 knockout BMMCs. Mechanistically, ELK4 might positively modulate Hdc, Ccl3 and Ccl4 transcription by interacting with MITF and negatively regulate the transcription of degranulation-related genes by complexing with SIRT6. Overall, our study identifies a new physiological role of the transcription factor ELK4 in mast cell proliferation and activation.

Effects of tumor-infiltrating lymphocytes on nonresponse rate of neoadjuvant chemotherapy in patients with invasive breast cancer.

High level of tumor-infiltrating lymphocytes (TILs) can predict the rate of total pathological complete remission (tpCR) of breast cancer patients who receive neoadjuvant chemotherapy (NACT). This study focused on evaluating the data of patients whose primary tumor and/or lymph node metastasis show nonresponse (NR) to NACT, trying to provide a basis for the clinical decision which patients will develop NACT resistance. The study included breast cancers from 991 patients who received NACT. ROC curve analysis confirmed that TILs showed significant predictive value for NR of hormone receptor (HR)+HER2- and triple-negative breast cancer (TNBC). Among HR+HER2- breast cancer, TILs ≥ 10% was an independent predictor for low NR rate. Furthermore, positive correlation of TILs with Ki67 index and Miller-Payne grade, and negative correlation with ER and PR H-scores were only identified in this subgroup. In TNBC, TILs ≥ 17.5% was an independent predictor for low NR rate. The predictive value of low TILs on NR may facilitate to screen patients with HR+HER2- or TNBC who may not benefit from NACT. HR+HER2- breast cancer with low levels of TILs should be carefully treated with neoadjuvant chemotherapy, and other alternatives such as neoadjuvant endocrine therapy can be considered.

PP1 cooperates with ASPP2 to dephosphorylate and activate TAZ.

The Hippo pathway regulates organ size by controlling both cell proliferation and apoptosis. TAZ functions as a transcriptional co-activator downstream of the Hippo pathway and has been implicated in human cancer development. A key step in the Hippo-TAZ pathway is phosphorylation of TAZ by LATS kinase, which leads to TAZ inhibition by both cytoplasmic retention and degradation. However, the mechanism of TAZ dephosphorylation and the responsible phosphatase are unknown. Here, we identified PP1 as a bona fide TAZ phosphatase. PP1A dephosphorylates TAZ at Ser-89 and Ser-311, promotes TAZ nuclear translocation, and stabilizes TAZ by disrupting the binding to the SCF E3 ubiquitin ligase. Furthermore, ASPP2 facilitates the interaction between TAZ and PP1 to promote TAZ dephosphorylation. As a result, PP1 and ASPP2 increase TAZ-dependent gene expression. This study demonstrates that PP1A and ASPP2 play a critical role in promoting TAZ function by antagonizing the LATS kinase through TAZ dephosphorylation.

CK2-induced cooperation of HHEX with the YAP-TEAD4 complex promotes colorectal tumorigenesis.

Dysregulation of Hippo pathway leads to hyperactivation of YAP-TEAD transcriptional complex in various cancers, including colorectal cancer (CRC). In this study, we observed that HHEX (Hematopoietically expressed homeobox) may enhance transcription activity of the YAP-TEAD complex. HHEX associates with and stabilizes the YAP-TEAD complex on the regulatory genomic loci to coregulate the expression of a group of YAP/TEAD target genes. Also, HHEX may indirectly regulate these target genes by controlling YAP/TAZ expression. Importantly, HHEX is required for the pro-tumorigenic effects of YAP during CRC progression. In response to serum stimulation, CK2 (Casein Kinase 2) phosphorylates HHEX and enhances its interaction with TEAD4. A CK2 inhibitor CX-4945 diminishes the interaction between HHEX and TEAD4, leading to decreased expression of YAP/TEAD target genes. CX-4945 synergizes the antitumor activity of YAP-TEAD inhibitors verteporfin and Super-TDU. Elevated expression of HHEX is correlated with hyperactivation of YAP/TEAD and associated with poor prognosis of CRC patients. Overall, our study identifies HHEX as a positive modulator of YAP/TEAD to promote colorectal tumorigenesis, providing a new therapeutic strategy for targeting YAP/TEAD in CRC.

Rational design of a sensitivity-enhanced tracer for discovering efficient APC-Asef inhibitors.

The adenomatous polyposis coli (APC)-Rho guanine nucleotide exchange factor 4 (Asef) protein-protein interaction (PPI) is essential for colorectal cancer metastasis, making it a promising drug target. Herein, we obtain a sensitivity-enhanced tracer (tracer 7) with a high binding affinity (Kd = 0.078 µM) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescence-polarization assays for APC-Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC50 of 0.041 ± 0.004 µM and a conjugated transcriptional transactivating sequence for generating cell-permeable MAIT-516. MAIT-516 inhibits CRC cell migration by specifically hindering the APC-Asef PPI. Furthermore, MAIT-516 exhibits no cytotoxic effects on normal intestinal epithelial cell and colorectal cancer cell growth. Overall, we develop a sensitivity-enhanced tracer for fluorescence polarization assays, which is used for the precise quantification of high-activity APC-Asef inhibitors, thereby providing insight into PPI drug development.