Colitis Is Associated with Loss of the Histidine Phosphatase LHPP and Upregulation of Histidine Phosphorylation in Intestinal Epithelial Cells.

Protein histidine phosphorylation (pHis) is a posttranslational modification involved in cell cycle regulation, ion channel activity and phagocytosis. Using novel monoclonal antibodies to detect pHis, we previously reported that the loss of the histidine phosphatase LHPP (phospholysine phosphohistidine inorganic pyrophosphate phosphatase) results in elevated pHis levels in hepatocellular carcinoma. Here, we show that intestinal inflammation correlates with the loss of LHPP in dextran sulfate sodium (DSS)-treated mice and in inflammatory bowel disease (IBD) patients. Increased histidine phosphorylation was observed in intestinal epithelial cells (IECs), as determined by pHis immunofluorescence staining of colon samples from a colitis mouse model. However, the ablation of Lhpp did not cause increased pHis or promote intestinal inflammation under physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but the loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine.

The prognostic significance of CXCR4 and SDF-1 in differentiated thyroid cancer depends on CD8+ density.

BACKGROUND: Tumor infiltration with cytotoxic CD8+ T-cells is associated with a favorable outcome in several neoplasms, including thyroid cancer. The chemokine axis CXCR4/SDF-1 correlates with more aggressive tumors, but little is known concerning the prognostic relevance in relation to the tumor immune microenvironment of differentiated thyroid cancer (DTC). METHODS: A tissue microarray (TMA) of 37 tumor specimens of primary DTC was analyzed by immunohistochemistry (IHC) for the expression of CD8+, CXCR4, phosphorylated CXCR4 and SDF-1. A survival analysis was performed on a larger collective (n = 456) at RNA level using data from The Cancer Genome Atlas (TCGA) papillary thyroid cancer cohort. RESULTS: Among the 37 patients in the TMA-cohort, the density of CD8+ was higher in patients with less advanced primary tumors (median cells/TMA-punch: 12.5 (IQR: 6.5, 12.5) in T1-2 tumors vs. 5 (IQR: 3, 8) in T3-4 tumors, p = 0.05). In the TCGA-cohort, CXCR4 expression was higher in patients with cervical lymph node metastasis compared to N0 or Nx stage (CXCR4high/low 116/78 vs. 97/116 vs. 14/35, respectively, p = 0.001). Spearman's correlation analysis of the TMA-cohort demonstrated that SDF-1 was significantly correlated with CXCR4 (r = 0.4, p = 0.01) and pCXCR4 (r = 0.5, p = 0.002). In the TCGA-cohort, density of CD8+ correlated with CXCR4 and SDF-1 expression (r = 0.58, p < 0.001; r = 0.4, p < 0.001). The combined marker analysis of the TCGA cohort demonstrated that high expression of both, CXCR4 and SDF-1 was associated with reduced overall survival in the CD8 negative TCGA cohort (p = 0.004). CONCLUSION: These findings suggest that the prognostic significance of CXCR4 and SDF-1 in differentiated thyroid cancer depends on the density of CD8 positive T-lymphocytes. Further studies with larger sample sizes are needed to support our findings and inform future investigations of new treatment and diagnostic options for a more personalized approach for patients with differentiated thyroid cancer.

GATA3 and MDM2 are synthetic lethal in estrogen receptor-positive breast cancers.

Synthetic lethal interactions, where the simultaneous but not individual inactivation of two genes is lethal to the cell, have been successfully exploited to treat cancer. GATA3 is frequently mutated in estrogen receptor (ER)-positive breast cancers and its deficiency defines a subset of patients with poor response to hormonal therapy and poor prognosis. However, GATA3 is not yet targetable. Here we show that GATA3 and MDM2 are synthetically lethal in ER-positive breast cancer. Depletion and pharmacological inhibition of MDM2 significantly impaired tumor growth in GATA3-deficient models in vitro, in vivo and in patient-derived organoids/xenograft (PDOs/PDX) harboring GATA3 somatic mutations. The synthetic lethality requires p53 and acts via the PI3K/Akt/mTOR pathway. Our results present MDM2 as a therapeutic target in the substantial cohort of ER-positive, GATA3-mutant breast cancer patients. With MDM2 inhibitors widely available, our findings can be rapidly translated into clinical trials to evaluate in-patient efficacy.

ZNRF3 and RNF43 cooperate to safeguard metabolic liver zonation and hepatocyte proliferation.

AXIN2 and LGR5 mark intestinal stem cells (ISCs) that require WNT/ß-Catenin signaling for constant homeostatic proliferation. In contrast, AXIN2/LGR5+ pericentral hepatocytes show low proliferation rates despite a WNT/ß-Catenin activity gradient required for metabolic liver zonation. The mechanisms restricting proliferation in AXIN2+ hepatocytes and metabolic gene expression in AXIN2+ ISCs remained elusive. We now show that restricted chromatin accessibility in ISCs prevents the expression of ß-Catenin-regulated metabolic enzymes, whereas fine-tuning of WNT/ß-Catenin activity by ZNRF3 and RNF43 restricts proliferation in chromatin-permissive AXIN2+ hepatocytes, while preserving metabolic function. ZNRF3 deletion promotes hepatocyte proliferation, which in turn becomes limited by RNF43 upregulation. Concomitant deletion of RNF43 in ZNRF3 mutant mice results in metabolic reprogramming of periportal hepatocytes and induces clonal expansion in a subset of hepatocytes, ultimately promoting liver tumors. Together, ZNRF3 and RNF43 cooperate to safeguard liver homeostasis by spatially and temporally restricting WNT/ß-Catenin activity, balancing metabolic function and hepatocyte proliferation.

Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo-Independent Regulation of Heat Shock Protein 70 Family Members.

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up-regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo. Additionally, RNA sequencing analysis of TEAD4-overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up-regulation of genes involved in epithelial-to-mesenchymal transition, proliferation, and protein-folding pathways. Among the most up-regulated genes following TEAD4 overexpression were the 70-kDa heat shock protein (HSP70) family members HSPA6 and HSPA1A. Chromatin immunoprecipitation-quantitative real-time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4-overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes-associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ)-knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo-independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members.

Nestin and CD34 expression in colorectal cancer predicts improved overall survival.

BACKGROUND: Nestin, a class VI intermediate filament protein of the cytoskeleton, and CD34, a transmembrane phosphoglycoprotein, are markers of progenitor cells. This study aimed to evaluate their expression and clinical significance in colorectal cancer. METHODS: A clinically annotated tissue microarray, including 599 patients with colorectal cancer, was analyzed by immunohistochemistry. Furthermore, nestin and CD34 correlations with HIF-1a and a panel of cytokines and chemokines were assessed using quantitative reverse transcription PCR and The Cancer Genome Atlas dataset. RESULTS: Expression of nestin and CD34 was observed only in the tumor stroma. Patients displaying high expression of nestin and CD34 demonstrated higher rates of T1 and T2 tumors (p = .020), lower vascular invasion (p < .001) and improved 5-year overall survival (65%; 95% CI = 55-73 vs 45%; 95% CI = 37-53) after adjusting for clinicopathological characteristics (HR: 0.67; 95% CI = 0.46-0.96). A moderate to strong correlation (r = 0.37-0.78, p < .03) of nestin and CD34 was demonstrated for the following markers; HIF-1α, CD4, CD8, FOXP3, IRF1, GATA3, CCL2, CCL3, CXCL12 and CCL21. CONCLUSIONS: Combined expression of nestin and CD34 expression is associated with better overall survival possibly by modulating a favorable immune response.

Adenylosuccinate lyase is oncogenic in colorectal cancer by causing mitochondrial dysfunction and independent activation of NRF2 and mTOR-MYC-axis.

Rationale: Adenylosuccinate lyase (ADSL) is an essential enzyme for de novo purine biosynthesis. Here we sought to investigate the putative role of ADSL in colorectal carcinoma (CRC) carcinogenesis and response to antimetabolites. Methods: ADSL expression levels were assessed by immunohistochemistry or retrieved from The Cancer Genome Atlas (TCGA) dataset. The effects of ADSL silencing or overexpression were evaluated on CRC cell proliferation, cell migration and cell-cycle. In vivo tumor growth was assessed by the chicken chorioallantoic membrane (CAM). Transfected cell lines or patient-derived organoids (PDO) were treated with 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP) and drug response was correlated with ADSL expression levels. Metabolomic and transcriptomic profiling were performed to identify dysregulated pathways and ADSL downstream effectors. Mitochondrial respiration and glycolytic capacity were measured using Seahorse; mitochondrial membrane potential and the accumulation of ROS were measured by FACS using MitoTracker Red and MitoSOX staining, respectively. Activation of canonical pathways was assessed by immunohistochemistry and immunoblotting. Results: ADSL expression is significantly increased in CRC tumors compared to non-tumor tissue. ADSL-high CRCs show upregulation of genes involved in DNA synthesis, DNA repair and cell cycle. Accordingly, ADSL overexpression accelerated progression through the cell cycle and significantly increased proliferation and migration in CRC cell lines. Additionally, ADSL expression increased tumor growth in vivo and sensitized CRCs to 6-MP in vitro, ex vivo (PDOs) and in vivo (CAM model). ADSL exerts its oncogenic function by affecting mitochondrial function via alteration of the TCA cycle and impairment of mitochondrial respiration. The KEAP1-NRF2 and mTORC1-cMyc axis are independently activated upon ADSL overexpression and may favor the survival and proliferation of ROS-accumulating cells, favoring DNA damage and tumorigenesis. Conclusions: Our results suggest that ADSL is a novel oncogene in CRC, modulating mitochondrial function, metabolism and oxidative stress, thus promoting cell cycle progression, proliferation and migration. Our results also suggest that ADSL is a predictive biomarker of response to 6-mercaptopurine in the pre-clinical setting.

Prognostic significance of CD8+ T-cells density in stage III colorectal cancer depends on SDF-1 expression.

Since colorectal cancer (CRC) remains one of the most common malignancies, a tremendous amount of studies keep taking place in this field. Over the past 25 years, a notable part of the scientific community has focused on the association between the immune system and colorectal cancer. A variety of studies have shown that high densities of infiltrating CD8+ T-cells are associated with improved disease-free and overall survival in CRC. Stromal cell-derived factor-1 (SDF-1) is a protein that regulates leukocyte trafficking and is variably expressed in several healthy and malignant tissues. There is strong evidence that SDF-1 has a negative prognostic impact on a variety of solid tumors. However, the existing data do not provide sufficient evidence that the expression of SDF-1 has an influence on CRC. Knowing nowadays, that the microenvironment plays a crucial role in the development of cancer, we hypothesized that the expression of SDF-1 in CRC could influence the prognostic significance of CD8+ T-cells, as an indicator of the essential role of the immune microenvironment in cancer development. Therefore, we explored the combined prognostic significance of CD8+ T-cell density and SDF-1 expression in a large CRC collective. We analyzed a tissue microarray of 613 patient specimens of primary CRCs by immunohistochemistry (IHC) for the CD8 + T-cells density and the expression of SDF-1 by tumor cells and tumor-infiltrating immune cells. Besides, we analyzed the expression of SDF-1 at the RNA level in The Cancer Genome Atlas cohort. We found that the combined high CD8+ T-cell infiltration and expression of SDF-1 shows a favorable 5-year overall survival rate (66%; 95% CI 48-79%) compared to tumors showing a high expression of CD8+ T-cell only (55%; 95% CI 45-64%; p = 0.0004). After stratifying the patients in nodal negative and positive groups, we found that the prognostic significance of CD8+ T-cell density in nodal positive colorectal cancer depends on SDF-1 expression. Univariate and multivariate Hazard Cox regression survival analysis considering the combination of both markers revealed that the combined high expression of SDF-1 and CD8+ T-cell density was an independent, favorable, prognostic marker for overall survival (HR = 0.34, 95% CI 0.17-0.66; p = 0.002 and HR = 0.45, 95% CI 0.23-0.89; p = 0.021, respectively). In our cohort there was a very weak correlation between SDF-1 and CD8+ T-cells (rs = 0.13, p = 0.002) and in the trascriptomic expression of these two immune markers display a weak correlation (rs = 0.28, p < 0.001) which was significantly more pronounced in stage III cancers (rs = 0.40, p < 0.001). The combination of high CD8+ T-cell density and expression of SDF-1 represents an independent, favorable, prognostic condition in CRC, mostly in patients with stage III disease.

Infiltration by IL22-Producing T Cells Promotes Neutrophil Recruitment and Predicts Favorable Clinical Outcome in Human Colorectal Cancer.

Immune cell infiltration in colorectal cancer effectively predicts clinical outcome. IL22, produced by immune cells, plays an important role in inflammatory bowel disease, but its relevance in colorectal cancer remains unclear. Here, we addressed the prognostic significance of IL22+ cell infiltration in colorectal cancer and its effects on the composition of tumor microenvironment. Tissue microarrays (TMA) were stained with an IL22-specific mAb, and positive immune cells were counted by expert pathologists. Results were correlated with clinicopathologic data and overall survival (OS). Phenotypes of IL22-producing cells were assessed by flow cytometry on cell suspensions from digested specimens. Chemokine production was evaluated in vitro upon colorectal cancer cell exposure to IL22, and culture supernatants were used to assess neutrophil migration in vitro Evaluation of a testing (n = 425) and a validation TMA (n = 89) revealed that high numbers of IL22 tumor-infiltrating immune cells were associated with improved OS in colorectal cancer. Ex vivo analysis indicated that IL22 was produced by CD4+ and CD8+ polyfunctional T cells, which also produced IL17 and IFNγ. Exposure of colorectal cancer cells to IL22 promoted the release of the neutrophil-recruiting chemokines CXCL1, CXCL2, and CXCL3 and enhanced neutrophil migration in vitro Combined survival analysis revealed that the favorable prognostic significance of IL22 in colorectal cancer relied on the presence of neutrophils and was enhanced by T-cell infiltration. Altogether, colorectal cancer-infiltrating IL22-producing T cells promoted a favorable clinical outcome by recruiting beneficial neutrophils capable of enhancing T-cell responses.

High Expression of FAP in Colorectal Cancer Is Associated With Angiogenesis and Immunoregulation Processes.

Fibroblast activation protein α (FAP) plays an important role in tissue remodeling and helps tumor cells invade surrounding tissue. We sought to investigate FAP as a prognostic molecular marker in colorectal cancer (CRC) using immunohistochemical and transcriptomic data. FAP expression and clinicopathological information were obtained from The Cancer Genome Atlas data set. The association of FAP expression and tissue cellular heterogeneity landscape was explored using the xCell method. We evaluated FAP protein expression in a cohort of 92 CRCs and 19 non-tumoral tissues. We observed that FAP was upregulated in tumors both at the mRNA and protein levels, and its expression was associated with advanced stages, poor survival, and consensus molecular subtype 4. FAP expression was also associated with angiogenesis and collagen degradation. We observed an enrichment in immune-cell process-related genes associated with FAP overexpression. Colorectal cancers with high FAP expression display an inflamed phenotype enriched for macrophages and monocytes. Those tumors showed enrichment for regulatory T cell populations and depletion of TH1 and natural killer T cells, pointing to an immunosuppressive environment. Colorectal cancers with high levels of stromal FAP are associated with aggressive disease progression and survival. Our results suggest that FAP plays additional roles in tumor progression such as modulation of angiogenesis and immunoregulation in the tumor microenvironment.

A Pygopus 2-Histone Interaction Is Critical for Cancer Cell Dedifferentiation and Progression in Malignant Breast Cancer.

Pygopus 2 (Pygo2) is a coactivator of Wnt/ß-catenin signaling that can bind bi- or trimethylated lysine 4 of histone-3 (H3K4me2/3) and participate in chromatin reading and writing. It remains unknown whether the Pygo2-H3K4me2/3 association has a functional relevance in breast cancer progression in vivo. To investigate the functional relevance of histone-binding activity of Pygo2 in malignant progression of breast cancer, we generated a knock-in mouse model where binding of Pygo2 to H3K4me2/3 was rendered ineffective. Loss of Pygo2-histone interaction resulted in smaller, differentiated, and less metastatic tumors, due, in part, to decreased canonical Wnt/ß-catenin signaling. RNA- and ATAC-sequencing analyses of tumor-derived cell lines revealed downregulation of TGFß signaling and upregulation of differentiation pathways such as PDGFR signaling. Increased differentiation correlated with a luminal cell fate that could be reversed by inhibition of PDGFR activity. Mechanistically, the Pygo2-histone interaction potentiated Wnt/ß-catenin signaling, in part, by repressing the expression of Wnt signaling antagonists. Furthermore, Pygo2 and ß-catenin regulated the expression of miR-29 family members, which, in turn, repressed PDGFR expression to promote dedifferentiation of wild-type Pygo2 mammary epithelial tumor cells. Collectively, these results demonstrate that the histone binding function of Pygo2 is important for driving dedifferentiation and malignancy of breast tumors, and loss of this binding activates various differentiation pathways that attenuate primary tumor growth and metastasis formation. Interfering with the Pygo2-H3K4me2/3 interaction may therefore serve as an attractive therapeutic target for metastatic breast cancer. SIGNIFICANCE: Pygo2 represents a potential therapeutic target in metastatic breast cancer, as its histone-binding capability promotes ß-catenin-mediated Wnt signaling and transcriptional control in breast cancer cell dedifferentiation, EMT, and metastasis.

Hepatocellular Carcinoma Xenografts Established From Needle Biopsies Preserve the Characteristics of the Originating Tumors.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Treatment options for patients with advanced-stage disease are limited. A major obstacle in drug development is the lack of an in vivo model that accurately reflects the broad spectrum of human HCC. Patient-derived xenograft (PDX) tumor mouse models could overcome the limitations of cancer cell lines. PDX tumors maintain the genetic and histologic heterogeneity of the originating tumors and are used for preclinical drug development in various cancers. Controversy exists about their genetic and molecular stability through serial passaging in mice. We aimed to establish PDX models from human HCC biopsies and to characterize their histologic and molecular stability during serial passaging. A total of 54 human HCC needle biopsies that were derived from patients with various underlying liver diseases and tumor stages were transplanted subcutaneously into immunodeficient, nonobese, diabetic/severe combined immunodeficiency gamma-c mice; 11 successfully engrafted. All successfully transplanted HCCs were Edmondson grade III or IV. HCC PDX tumors retained the histopathologic, transcriptomic, and genomic characteristics of the original HCC biopsies over 6 generations of retransplantation. These characteristics included Edmondson grade, expression of tumor markers, tumor gene signature, tumor-associated mutations, and copy number alterations. Conclusion: PDX mouse models can be established from undifferentiated HCCs, with an overall success rate of approximately 20%. The transplanted tumors represent the entire spectrum of the molecular landscape of HCCs and preserve the characteristics of the originating tumors through serial passaging. HCC PDX models are a promising tool for preclinical personalized drug development.

Genomic Analysis Revealed New Oncogenic Signatures in TP53-Mutant Hepatocellular Carcinoma.

The TP53 gene is the most commonly mutated gene in human cancers and mutations in TP53 have been shown to have either gain-of-function or loss-of-function effects. Using the data generated by The Cancer Genome Atlas, we sought to define the spectrum of TP53 mutations in hepatocellular carcinomas (HCCs) and their association with clinicopathologic features, and to determine the oncogenic and mutational signatures in TP53-mutant HCCs. Compared to other cancer types, HCCs harbored distinctive mutation hotspots at V157 and R249, whereas common mutation hotspots in other cancer types, R175 and R273, were extremely rare in HCCs. In terms of clinicopathologic features, in addition to the associations with chronic viral infection and high Edmondson grade, we found that TP53 somatic mutations were less frequent in HCCs with cholestasis or tumor infiltrating lymphocytes, but were more frequent in HCCs displaying necrotic areas. An analysis of the oncogenic signatures based on the genetic alterations found in genes recurrently altered in HCCs identified four distinct TP53-mutant subsets, three of which were defined by CTNNB1 mutations, 1q amplifications or 8q24 amplifications, respectively, that co-occurred with TP53 mutations. We also found that mutational signature 12, a liver cancer-specific signature characterized by T>C substitutions, was prevalent in HCCs with wild-type TP53 or with missense TP53 mutations, but not in HCCs with deleterious TP53 mutations. Finally, whereas patients with HCCs harboring deleterious TP53 mutations had worse overall and disease-free survival than patients with TP53-wild-type HCCs, patients with HCCs harboring missense TP53 mutations did not have worse prognosis. In conclusion, our results highlight the importance to consider the genetic heterogeneity among TP53-mutant HCCs in studies of biomarkers and molecular characterization of HCCs.