Tofacitinib restores psoriatic arthritis fibroblast-like synoviocytes function via autophagy and mitochondrial quality control modulation.

OBJECTIVES: To evaluate the in vitro effect of tofacitinib on autophagy activity of psoriatic arthritis (PsA) fibroblast-like synoviocytes (FLS), and to confirm its activity on inflammatory and invasive properties of FLS and synovial cells, deepening the impact on mitochondrial function. METHODS: FLS, peripheral blood mononuclear cells (PBMCs), and synovial cells from active PsA patients were cultured with tofacitinib 1 µM or vehicle control for 24 h. Autophagy was measured by Western blot and by fluorescence microscopy. Chemokines/cytokines released into culture supernatants were quantified by ELISA, while invasive properties of FLS by migration assays. Specific mitochondrial probes were adopted to measure intracellular reactive oxygen species (ROS), mitochondrial potential, morphology, turnover and mitophagy. Oxygen consumption rate (OCR), reflecting oxidative phosphorylation, was quantified using the Seahorse technology. Differences were determined by adopting the non-parametric Wilcoxon signed rank test. RESULTS: 18 patients with moderately-to-severely active PsA were enrolled. Tofacitinib significantly increased the levels of the autophagy markers LC3-II and ATG7 in PsA FLS compared to vehicle control, suggesting an increase in spontaneous autophagy activity; no effect was highlighted in PBMCs and synovial cells cultures. Tofacitinib reduced migration properties of PsA FLS, and reduced MCP-1 and IL-6 release into FLS and synovial cells cultures supernatants. Furthermore, tofacitinib decreased intracellular ROS production, increased basal OCR, ATP production and maximal respiratory capacity, and enhanced mitophagy and mitochondrial turnover. CONCLUSIONS: The JAK inhibitor tofacitinib reduces the pro-invasive and pro-inflammatory properties of PsA FLS. Autophagy induction and mitochondrial quality control modulation by tofacitinib might contribute to FLS function restoration.

Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

The Day-To-Day Practice of MMR and MSI Assessment in Colorectal Adenocarcinoma: What We Know and What We Still Need to Explore.

BACKGROUND: The DNA mismatch repair (MMR) system is a highly preserved protein complex recognizing short insertions, short deletions, and single base mismatches during DNA replication and recombination. MMR protein status is identified using immunohistochemistry. Deficit in one or more MMR proteins, configuring deficient MMR status (dMMR), leads to frameshift mutations particularly clustered in microsatellite repeats. Thus, microsatellite instability (MSI) is the epiphenomenon of dMMR. In colorectal cancer (CRC), MMR/MSI status is a biomarker with prognostic and predictive value of resistance to 5-fluorouracil and response to immune checkpoint inhibitor therapy. SUMMARY: In this Review, we describe the challenges the practicing pathologist may face in relation to the assessment of MMR/MSI status and any open issues which still need to be addressed, focusing on pre-analytic issues, pitfalls in the interpretation, and technical aspects of the different assays. KEY MESSAGES: The current methods of detecting dMMR/MSI status have been optimized for CRCs, and whether these techniques can be applied to all tumor and specimen types is still not fully understood. Following the Food and Drug Administration (FDA), tissue/site agnostic drug approval of pembrolizumab for advanced/metastatic MSI tumors, MMR/MSI status in gastrointestinal tract is a common request from the oncologist. In this setting, several issues still need to be addressed, including criteria for sample adequacy.

COVID-19 induced aorto duodenal fistula following evar in the so called "negative" patient.

OBJECTIVES: Since October 2019, SARS-CoV-2 pandemic represents a challenge for the international healthcare system and for the treatment and survival of patients. We normally focus on symptomatic patients, and symptoms can range from the respiratory to the gastrointestinal system. In addition, we consider patients without fever and respiratory symptoms, with both a negative RT nasopharyngeal swab and lung CT, as a "Covid-19 negative patient." In this article, we present a so called Covid-19 "negative" patient, with an unsuspected vascular clinical onset of the viral infection. METHODS: An 80 y.o. man, who previously underwent endovascular aortic repair for an infrarenal abdominal aortic aneurysm, presented to our department with an atypical presentation of an aorto-enteric fistula during the pandemic. While in hospital, weekly nasopharyngeal swab tests were always negative for SARS-CoV-2. However, the absence of aortic endograft complications, the gross anatomy of duodenal ischemic injury, and the recent history of the patient who lived the last months in Bergamo, the Italian city with the highest number of COVID-19 deaths, lead the senior Author to suspect an occult SARS-CoV-2 infection. The patient underwent to resection of the fourth portion of the duodenum and the first jejunal loop, with subsequent duodenum-jejunal latero-lateral anastomosis and the direct suture of the aortic wall. The intestinal specimen was investigated as suspected SARS-CoV-2 bowel infection by the means of immune-histochemistry (IHC). An ileum sample obtained in the pre-COVID-19 era was used as a control tissue. RESULTS: The histological analysis of the bowel revealed sustained wall ischemia and liponecrosis of the duodenal wall, with intramural blood vessels thrombosis. Blood vessel endotheliitis and neo-angiogenesis were also observed. Finally, the IHC was strongly positive for SARS-CoV-2 RNA and for HLA-G presence, with a particular concentration both in blood vessels and in the intestinal villi. The control tissue sample was not positive for both SARS-CoV-2 and HLA-G. CONCLUSIONS: Coronavirus pandemic continues to be an international challenge and more studies and trials must be done to learn its pathogenesis and its complications. As for thromboembolic events caused by SARS-COV-2, vascular surgeons are involved in treatment and prevention of the complications of this syndrome and must be ready with general surgeons to investigate atypical and particular cases such as the one discussed in this article.

Evaluation of the Synovial Effects of Biological and Targeted Synthetic DMARDs in Patients with Psoriatic Arthritis: A Systematic Literature Review and Meta-Analysis.

The aims of this systematic literature review (SLR) were to identify the effects of approved biological and targeted synthetic disease modifying antirheumatic drugs (b/tsDMARDs) on synovial membrane of psoriatic arthritis (PsA) patients, and to determine the existence of histological/molecular biomarkers of response to therapy. A search was conducted on MEDLINE, Embase, Scopus, and Cochrane Library (PROSPERO:CRD42022304986) to retrieve data on longitudinal change of biomarkers in paired synovial biopsies and in vitro studies. A meta-analysis was conducted by adopting the standardized mean difference (SMD) as a measure of the effect. Twenty-two studies were included (19 longitudinal, 3 in vitro). In longitudinal studies, TNF inhibitors were the most used drugs, while, for in vitro studies, JAK inhibitors or adalimumab/secukinumab were assessed. The main technique used was immunohistochemistry (longitudinal studies). The meta-analysis showed a significant reduction in both CD3+ lymphocytes (SMD -0.85 [95% CI -1.23; -0.47]) and CD68+ macrophages (sublining, sl) (SMD -0.74 [-1.16; -0.32]) in synovial biopsies from patients treated for 4-12 weeks with bDMARDs. Reduction in CD3+ mostly correlated with clinical response. Despite heterogeneity among the biomarkers evaluated, the reduction in CD3+/CD68+sl cells during the first 3 months of treatment with TNF inhibitors represents the most consistent variation reported in the literature.

Bowel ischemia as onset of COVID-19 in otherwise asymptomatic patients with persistently negative swab.

BACKGROUND: Asymptomatic patients with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can develop hypercoagulable conditions and acute vascular events. The objective of this study is to determine whether SARS-CoV-2 was present in resected specimens from patients with acute bowel ischemia, but asymptomatic for Coronavirus Disease 2019 (COVID-19) and with persistently real-time polymerase chain reaction negative pharyngeal swab. METHODS: Three consecutive patients presented severe abdominal symptoms due to extensive ischemia and necrosis of the bowel, with co-existent thrombosis of abdominal blood vessels. None had the usual manifestations of COVID-19, and repeated pharyngeal swabs tested negative. They underwent emergency surgery with intestinal resection. Immunohistochemical testing for SARS-CoV-2 on resected tissue was performed. RESULTS: All tested samples were strongly positive for SARS-CoV-2. CONCLUSIONS: This is the first case report in which patients with severe intestinal symptoms presented a marked SARS-CoV-2 positivity in the resected tissues, without any usual clinical manifestations of COVID-19. These results suggest that the patients might be infected with SARS-CoV-2 presenting acute abdominal distress but without respiratory or constitutional symptoms.

Relevance of VEGF and CD147 in different SARS-CoV-2 positive digestive tracts characterized by thrombotic damage.

Several evidence suggests that, in addition to the respiratory tract, also the gastrointestinal tract is a main site of severe acute respiratory syndrome CoronaVirus 2 (SARS-CoV-2) infection, as an example of a multi-organ vascular damage, likely associated with poor prognosis. To assess mechanisms SARS-CoV-2 responsible of tissue infection and vascular injury, correlating with thrombotic damage, specimens of the digestive tract positive for SARS-CoV-2 nucleocapsid protein were analyzed deriving from three patients, negative to naso-oro-pharyngeal swab for SARS-CoV-2. These COVID-19-negative patients came to clinical observation due to urgent abdominal surgery that removed different sections of the digestive tract after thrombotic events. Immunohistochemical for the expression of SARS-CoV-2 combined with a panel of SARS-CoV-2 related proteins angiotensin-converting enzyme 2 receptor, cluster of differentiation 147 (CD147), human leukocyte antigen-G (HLA-G), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 was performed. Tissue samples were also evaluated by electron microscopy for ultrastructural virus localization and cell characterization. The damage of the tissue was assessed by ultrastructural analysis. It has been observed that CD147 expression levels correlate with SARS-CoV-2 infection extent, vascular damage and an increased expression of VEGF and thrombosis. The confirmation of CD147 co-localization with SARS-CoV-2 Spike protein binding on gastrointestinal tissues and the reduction of the infection level in intestinal epithelial cells after CD147 neutralization, suggest CD147 as a possible key factor for viral susceptibility of gastrointestinal tissue. The presence of SARS-CoV-2 infection of gastrointestinal tissue might be consequently implicated in abdominal thrombosis, where VEGF might mediate the vascular damage.

Detection of disease-causing mutations in prostate cancer by NGS sequencing.

Gene mutations may affect the fate of many tumors including prostate cancer (PCa); therefore, the research of specific mutations associated with tumor outcomes might help the urologist to identify the best therapy for PCa patients such as surgical resection, adjuvant therapy or active surveillance. Genomic DNA (gDNA) was extracted from 48 paraffin-embedded PCa samples and normal paired tissues. Next, gDNA was amplified and analyzed by next-generation sequencing (NGS) using a specific gene panel for PCa. Raw data were refined to exclude false-positive mutations; thus, variants with coverage and frequency lower than 100× and 5%, respectively were removed. Mutation significance was processed by Genomic Evolutionary Rate Profiling, ClinVar, and Varsome tools. Most of 3000 mutations (80%) were single nucleotide variants and the remaining 20% indels. After raw data elaboration, 312 variants were selected. Most mutated genes were KMT2D (26.45%), FOXA1 (16.13%), ATM (15.81%), ZFHX3 (9.35%), TP53 (8.06%), and APC (5.48%). Hot spot mutations in FOXA1, ATM, ZFHX3, SPOP, and MED12 were also found. Truncating mutations of ATM, lesions lying in hot spot regions of SPOP and FOXA1 as well as mutations of TP53 correlated with poor prognosis. Importantly, we have also found some germline mutations associated with hereditary cancer-predisposing syndrome. gDNA sequencing of 48 cancer tissues by NGS allowed to detect new tumor variants as well as confirmed lesions in genes linked to prostate cancer. Overall, somatic and germline mutations linked to good/poor prognosis could represent new prognostic tools to improve the management of PCa patients.

Monitoring Somatic Genetic Alterations in Circulating Cell-Free DNA/RNA of Patients with "Oncogene-Addicted" Advanced Lung Adenocarcinoma: A Real-World Clinical Study.

Liquid biopsy has advantages over tissue biopsy, but also some technical limitations that hinder its wide use in clinical applications. In this study, we aimed to evaluate the usefulness of liquid biopsy for the clinical management of patients with advanced-stage oncogene-addicted non-small-cell lung adenocarcinomas. The investigation was conducted on a series of cases-641 plasma samples from 57 patients-collected in a prospective consecutive manner, which allowed us to assess the benefits and limitations of the approach in a real-world clinical context. Thirteen samples were collected at diagnosis, and the additional samples during the periodic follow-up visits. At diagnosis, we detected mutations in ctDNA in 10 of the 13 cases (77%). During follow-up, 36 patients progressed. In this subset of patients, molecular analyses of plasma DNA/RNA at progression revealed the appearance of mutations in 29 patients (80.6%). Mutations in ctDNA/RNA were typically detected an average of 80 days earlier than disease progression assessed by RECIST or clinical evaluations. Among the cases positive for mutations, we observed 13 de novo mutations, responsible for the development of resistance to therapy. This study allowed us to highlight the advantages and disadvantages of liquid biopsy, which led to suggesting algorithms for the use of liquid biopsy analyses at diagnosis and during monitoring of therapy response.

Late-onset intrauterine growth restriction and HHV-6 infection: A pilot study.

Late-onset Intrauterine growth restriction (IUGR) refers to impaired growth and development of the fetus, characterized by placental morphological abnormalities that affect the fetus's supply of nutrients. Human leukocyte antigen-G (HLA-G) is physiologically expressed during pregnancy, but decreased in normal placenta during the last weeks of gestation possibly inducing childbirth. Several viruses involved in congenital infection, such as herpesviruses, exploit HLA-G expression as an immune-escape mechanism. To date, despite different congenital herpetic infections having been associated with late IUGR, no direct implication of Human herpesvirus 6 (HHV-6) infection has been reported. We evaluated HLA-G expression and HHV-6 infection in 11 placentas from late-onset IUGR newborns and 11 placentas from uncomplicated pregnancies by histopathological and immunohistochemistry analysis. We found higher levels of HLA-G expression and HHV-6 presence in IUGR placenta samples compared with control placenta samples. We report HHV-6 staining in IUGR placenta samples, characterized by high HLA-G expression. These preliminary data suggest a possible involvement of HHV-6 infection in HLA-G deregulation that might affect vessel remodeling and prevent the correct pregnancy outcome in the IUGR condition.

The P2X7 Receptor Is Overexpressed in the Lesional Skin of Subjects Affected by Hidradenitis Suppurativa: A Preliminary Study.

BACKGROUND: P2X receptors (P2XRs) are plasma membrane channels involved in the modulation of immune responses. The role of the P2X7 receptor (P2X7R) has never been investigated in hidradenitis suppurativa (HS), which is a recurrent skin disease characterized by inflammatory nodules, scarring, and suppuration. OBJECTIVE: Our aim was to investigate by immunohistochemistry (IHC) P2X7R, NLRP3 (NOD-like receptor family, pyrin domain-containing 3), and interleukin-1ß (IL-1ß) expression in HS lesions compared to healthy control (HC) skin. METHOD: The intensity of IHC immunostaining was semi-quantitatively graded for keratinocytes, neutrophils, lymphocytes, and monocytes. Statistical significance was assessed by the Mann-Whitney U test, Cohen's κ coefficient, and χ2 test. RESULTS: A total of 59 samples, 31 from HS and 28 from HC, were collected and analysed. In skin keratinocytes, lymphocytes, and monocytes, but not in neutrophils, P2X7R and NLRP3 protein expression was significantly increased in HS versus the HC group. IL-1ß protein expression was also higher in HS versus the HC group both in skin keratinocytes and in the inflammatory infiltrate. Cohen's κ correlation coefficients for the expression of P2X7R versus NLRP3 or IL-1ß in skin keratinocytes were significant (κ = 0.43 and 0.34, respectively). The same association between P2X7R and NLRP3 or IL-1ß was confirmed by χ2 tests. CONCLUSION: P2X7R, NLRP3, and IL-1ß are overexpressed, and therefore the entire P2X7R/NLRP3/IL-1ß pro-inflammatory axis is likely overactive in the skin of HS patients. This observation might provide clues to the pathogenesis of this disease and suggest novel therapies and markers of disease activity.

The histomorphological and molecular landscape of colorectal adenomas and serrated lesions.

The 2019 WHO classification of digestive system tumors significantly reformed the classificatory definition of serrated lesions of the colorectal mucosa and added new essential diagnostic criteria for both conventional adenomas and hereditary gastrointestinal polyposis syndromes. Histopathological examination of colorectal adenocarcinoma precursors lesions represents an important segment of daily clinical practice in a pathology department and is essential for the implementation of current colorectal adenocarcinoma secondary prevention strategies. This overview will focus on a schematic histopathological and molecular classification of precursor lesions arising within colorectal mucosa.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

Molecular biomarkers predicting early development of endometrial carcinoma: A pilot study.

OBJECTIVE: Endometrial carcinoma represents the most common gynaecological cancer and the sixth most frequent cancer among women worldwide. The 5-year survival of patients with stage I endometrial carcinoma is 75%-88% versus 50% for stage III or 15% for stage IV disease. Therefore, early detection could improve survival rates. Specifically, in the most prevalent, type 1 endometrial cancer develops from hyperplastic endometrium. The aim of the study was to evaluate the utility of cancer gene mutations from endometrial biopsies towards predicting synchronous or metachronous development of malignant lesions. The aim of the study was to evaluate whether endometrial biopsies could already carry mutations in cancer genes useful for predicting or anticipating subsequent cancer development. METHODS: Patients with a previous endometrial biopsy negative for cancer, followed by a subsequent biopsy positive for cancer, were included in the study. A fifty cancer genes targeted next-generation sequencing panel were used to investigate mutations in matched non-cancerous and malignant samples. RESULTS: All biopsies from cancer tissues harboured mutations in one or more of the following genes: APC, CTNNB1, FBXW7, HNF1A, KRAS, MTOR, NRAS, PIK3CA, PTEN, RB1 and TP53. Additionally, 50% of the biopsies from matched non-cancerous tissues exhibited mutations in PTEN, KRAS or PIK3CA genes. CONCLUSIONS: These results suggest that detecting pathogenic mutations in oncogenes or tumour suppressor genes in an otherwise benign condition is associated with a risk of developing a malignant disease. Given the identification of mutations several months or years before the appearance of a malignancy, our finding suggests that a closer monitoring of patients who present such molecular alterations in non-cancerous uterine mass is warranted.

Double inhibition of cAMP and mTOR signalling may potentiate the reduction of cell growth in ADPKD cells.

BACKGROUND: ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients. METHODS: The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting. RESULTS: The activation of A3AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 flox/-: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost. CONCLUSION: The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.

The clinical and biological significance of MIR-224 expression in colorectal cancer metastasis.

OBJECTIVE: MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis. DESIGN: We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression. RESULTS: MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175). CONCLUSIONS: MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer.

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR-17-5p, and miR-20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk.

Gene expression changes in progression of cervical neoplasia revealed by microarray analysis of cervical neoplastic keratinocytes.

To evaluate the gene expression changes involved in neoplastic progression of cervical intraepithelial neoplasia. Using microarray analysis, large-scale gene expression profile was carried out on HPV16-CIN2, HPV16-CIN3, and normal cervical keratinocytes derived from two HPV16-CIN2, two HPV-CIN3 lesions, and two corresponding normal cervical tissues, respectively. Differentially expressed genes were analyzed in normal cervical keratinocytes compared with HPV16-CIN2 keratinocytes and in HPV16-CIN2 keratinocytes compared with HPV16-CIN3 keratinocytes; 37 candidate genes with continuously increasing or decreasing expression during CIN progression were identified. One of these genes, phosphoglycerate dehydrogenase, was chosen for further characterization. Quantitative reverse transcription-polymerase chain reaction and immunohistochemical analysis confirmed that expression of phosphoglycerate dehydrogenase consistently increases during progression of CIN toward cancer. Gene expression changes occurring during CIN progression were investigated using microarray analysis, for the first time, in CIN2 and CIN3 keratinocytes naturally infected with HPV16. Phosphoglycerate dehydrogenase is likely to be associated with tumorigenesis and may be a potential prognostic marker for CIN progression.