KRT5 Expression Research Articles

Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix

Small cell neuroendocrine carcinoma of the cervix (SCNEC/cervix) is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. In this study, a panel of organoid lines from patients with SCNEC/cervix was established and focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. By tracking single cells, the existence of cells with bipotential differentiation towards SCNEC and adenocarcinomas was revealed. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for KRT19 expression, changes in the differentiation of each cell were revealed to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

Differential efficacy of two small molecule PHLPP inhibitors to promote nucleus Pulposus cell health.

Intervertebral disc (IVD) degeneration is associated with chronic back pain. We previously demonstrated that the phosphatase pleckstrin homology domain and leucine-rich repeat protein phosphatase (PHLPP) 1 was positively correlated with IVD degeneration and its deficiency decelerated IVD degeneration in both mouse IVDs and human nucleus pulposus (NP) cells. Small molecule PHLPP inhibitors may offer a translatable method to alleviate IVD degeneration. In this study, we tested the effectiveness of the two PHLPP inhibitors NSC117079 and NSC45586 in promoting a healthy NP phenotype. Tail IVDs of 5-month-old wildtype mice were collected and treated with NSC117079 or NSC45586 under low serum conditions ex vivo. Hematoxylin & eosin staining was performed to examine IVD structure and NP cell morphology. The expression of KRT19 was analyzed through immunohistochemistry. Cell apoptosis was assessed by TUNEL assay. Human NP cells were obtained from patients with IVD degeneration. The gene expression of KRT19, ACAN, SOX9, and MMP13 was analyzed via real time qPCR, and AKT phosphorylation and the protein expression of FOXO1 was analyzed via immunoblot. In a mouse IVD organ culture model, NSC45586, but not NSC117079, preserved vacuolated notochordal cell morphology and KRT19 expression while suppressing cell apoptosis, counteracting the degenerative changes induced by serum deprivation, especially in males. Likewise, in degenerated human NP cells, NSC45586 increased cell viability and the expression of KRT19, ACAN, and SOX9 and reducing the expression of MMP13, while NSC117079 treatment only increased KRT19 expression. Mechanistically, NSC45586 treatment increased FOXO1 protein expression in NP cells, and inhibiting FOXO1 offset NSC45586-induced regenerative potential, especially in males. Our study indicates that NSC45586 was effective in promoting NP cell health, especially in males, suggesting that PHLPP plays a key role in NP cell homeostasis and that NSC45586 might be a potential drug candidate in treating IVD degeneration.

KRT17 from skin cells with high glucose stimulation promotes keratinocytes proliferation and migration.

Impaired diabetic wound healing is an important issue in diabetic complications. Proliferation and migration of keratinocytes are major processes of skin wound repair after injury. However, hyperkeratosis can affect the speed of wound healing. Based on the results of preliminary experiments on increased KRT17 expression after high glucose stimulation of human skin tissue cells, a cell model of human immortalized keratinocyte (HaCaT) stimulation with different concentrations of KRT17 was established in vitro, and the promotion in cell proliferation and migration were discovered. KRT17 silencing promoted diabetic wound healing in the db/db diabetic wound model. Transcriptome sequencing (RNA-seq) was performed on HaCaT cells after KRT17 stimulation, and analysis showed significant enrichment in the PI3K-AKT signaling pathway, in which the regulation of cell c-MYB mRNA, a key molecule regulating cell proliferation and migration, was significantly upregulated. In vitro assays showed increased c-MYB expression and enhanced pAKT activity after HaCaT cell stimulation by KRT17. We speculate that KRT17 is upregulated under high glucose and promotes keratinocyte proliferation and migration caused hyperkeratosis, through the c-MYB/PI3K-AKT pathway, contributing to delayed wound healing.

Feasibility and Impact of Immunohistochemistry-based Molecular Subtyping for Muscle-invasive Bladder Cancer in Patients Treated with Radiation-based Therapy

BackgroundDistinct molecular subtypes of muscle-invasive bladder cancer (MIBC) have been identified via gene expression profiling. ObjectiveWe investigated the feasibility of a simple immunohistochemistry (IHC)-based Lund subtyping method and the association of MIBC subtypes with oncological outcomes for patients after bladder-preserving radiation-based therapy. Design, setting, and participantsTransurethral resected tumor tissues from 104 patients treated with radiation-based therapy were sampled on tissue microarray blocks. Outcome measurements and statistical analysisThe expression of KRT5, GATA3, and p16 proteins was scored via digital image analysis. Hierarchical clustering was used to classify tumors as the basal subtype or one of two luminal subtypes: genomically unstable (GU) or urothelial-like (URO). Subtypes were evaluated for association with complete response (CR), recurrence-free survival (RFS), and overall survival (OS). Results and limitationsThe median OS was 43 mo (95% confidence interval 19–77) and median follow-up was 55 mo (interquartile range 39–75). Age and clinical stage had a significant impact on OS (p < 0.05). IHC-based subtype classification was feasible in most patients (89%). The subtype was basal in 23.6%, GU in 14.0%, URO in 31.2%, and unclassified in 31.2% of patients. No significant differences in CR, RFS, or OS were observed between the molecular subtypes. Limitations include the retrospective design and relatively small sample size. ConclusionsIHC-based molecular MIBC subtyping using a three-antibody algorithm is feasible in most patients treated with radiation-based therapy. MIBC subtype was not associated with response or survival. Further prospective studies are warranted to confirm the lack of association between molecular subtype and survival in patients treated with trimodal therapy. Patient summaryFor patients with invasive bladder cancer treated with radiation-based therapy, we classified tumors into different subtypes using just three molecular stains. This method is cheaper and more widely available than the usual approach. However, we did not find an association between different cancer subtypes and survival.

TRIM29 knockdown prevented the colon cancer progression through decreasing the ubiquitination levels of KRT5.

To investigate the specific role of TRIM29 in colon cancer progression, bioinformatic analysis was performed on TRIM29. Colon cancer tissues were collected and colon cancer cells were cultured for further experiments. Cell viability and proliferation were determined using CCK-8, colony formation, and EDU staining assays. The mRNA and protein levels of TRIM29 and KRT5 were determined using quantitative real-time PCR and western blotting, respectively. The interaction between TRIM29 and KRT5 was detected using a co-immunoprecipitation (CO-IP) assay. Cycloheximide treatment was performed to analyse the stability of KRT5. TRIM29 was upregulated in colon cancer tissues and cells. TRIM29 knockdown decreased the cell viability and proliferation and ubiquitination levels of KRT5 and enhanced the protein stability and expression of KRT5. The CO-IP assay confirmed that TRIM29 and KRT5 binded to each other. KRT5 knockdown neutralises the inhibitory effect of sh-TRIM29 on colon cancer cell growth and TRIM29 knockdown prevented the proliferation of colon cancer cells by decreasing ubiquitination of KRT5, which enhanced the protein stability and expression of KRT5 in cancer cells. Thus, targeting TRIM29-mediated ubiquitination levels of KRT5 might be a new direction for colon cancer therapy.

The Influence of Race/Ethnicity on the Transcriptomic Landscape of Uterine Fibroids.

The objective of this study was to determine if the aberrant expression of select genes could form the basis for the racial disparity in fibroid characteristics. The next-generation RNA sequencing results were analyzed as fold change [leiomyomas/paired myometrium, also known as differential expression (DF)], comparing specimens from White (n = 7) and Black (n = 12) patients. The analysis indicated that 95 genes were minimally changed in tumors from White (DF ≈ 1) but were significantly altered by more than 1.5-fold (up or down) in Black patients. Twenty-one novel genes were selected for confirmation in 69 paired fibroids by qRT-PCR. Among these 21, coding of transcripts for the differential expression of FRAT2, SOX4, TNFRSF19, ACP7, GRIP1, IRS4, PLEKHG4B, PGR, COL24A1, KRT17, MMP17, SLN, CCDC177, FUT2, MYO5B, MYOG, ZNF703, CDC25A, and CDCA7 was significantly higher, while the expression of DAB2 and CAV2 was significantly lower in tumors from Black or Hispanic patients compared with tumors from White patients. Western blot analysis revealed a greater differential expression of PGR-A and total progesterone (PGR-A and PGR-B) in tumors from Black compared with tumors from White patients. Collectively, we identified a set of genes uniquely expressed in a race/ethnicity-dependent manner, which could form the underlying mechanisms for the racial disparity in fibroids and their associated symptoms.

Abstract A011: Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples

Abstract Introduction: The study aims to identify the progenitor of clear-cell renal cell carcinoma (ccRCC). Background: Inactivation of tumor suppressor gene VHL is the driver for the pathogenesis of ccRCC, the majority subtype of kidney tumors. However, it has also been observed that not all VHL-deficient cells develop into tumors. It is therefore likely that additional characteristics are required for ccRCC formation. Accumulating evidence suggests that tumor formation is initiated by a small pool of cells with tumor-initiating capacity, which are termed tumor progenitor cells (TPCs). Yet, the concept of TPCs in ccRCC has not been fully validated. Methods: LSL-tdTomato reporter was used to trace Vhlh mutant cells in the prior pre-malignant ccRCC mouse model, in which Vhlh was conditionally knocked out (VhlhKO) in renal epithelial cells under the control of Hoxb7-Cre. We also utilized single-cell RNA sequencing (scRNA-seq) to investigate the diversity of Vhlh-deficient renal tubule cells. Results: We show that the tubule phenotypes (traced by the tdTomato reporter) are associated with Vhlh inactivation; the abnormal tdTomato+ cells in Vhlh KO mice exhibit clear-cell phenotype, hyperplasia and multi-layered epithelial cell clusters. scRNA-seq was then used to analyze isolated tdTomato+ cells from Vhlh KO and wild-type (WT) kidneys. The results reveal that inactivation of Vhlh generates an expanded population of principal collecting duct cells characterized by the expression of keratin 19 (Krt19). These Krt19+ cells highly express stem cell-related genes (Wnt4, Sox4, Klf4, Ly6a). They also express multiple tubule segment-specific markers, suggesting the process of dedifferentiation. The expression of Krt19 is strikingly increased and colocalized with a subpopulation of tdTomato+ cells in Vhlh KO kidney, compared with WT where Krt19+ cells are a rare population scattered in tubule epithelia. Colocalization of KRT19 and CAIX in ccRCC samples confirms the ccRCC identity of these KRT19+ cells. High expression of KRT19 is significantly associated with poor overall survival of ccRCC patients. On kidney tissue microarray, KRT19 is strongly expressed in the early-stage peri-tumor tissue with the presence of immune cell infiltration and lipid droplets in the vicinity. In the high-grade ccRCC, KRT19 is expressed in the typical ccRCC cells and in multi-layered adenomas. Notably, Krt19+ progenitor-like cells are found in the fibrotic region with immune cell infiltration in Vhlh mutant kidney. The direct contact between macrophages and Krt19+ cells in mutant kidney, but not in WT, implies that macrophages may be a component in the progenitor-cell niche when Vhlh is conditionally knocked out. Conclusion: We suspect that VHL loss-of-function in the rare tissue progenitor cells may generate a tumor progenitor subset. Elucidation of the role of KRT19 in the context of VHL inactivation and characterization of the stem/progenitor cell niche are of importance as they will shed new lights on early diagnosis and therapeutic intervention. Citation Format: Dinh-Xuan Pham, Chen-Yun Chen, Hieu-Huy Nguyen-Tran, Tien Hsu. Single-cell RNA sequencing reveals a potential renal tumor progenitor in conditional Vhlh knockout mouse model and ccRCC clinical samples [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr A011.

Epigenetic deregulation of MLF1 drives intrahepatic cholangiocarcinoma progression through EGFR/AKT and Wnt/β-catenin signaling.

Intrahepatic cholangiocarcinoma (iCCA) is an aggressive malignancy with multiple etiologies and is largely refractory to current treatment strategies. Myeloid leukemia factor 1 (MLF1) is associated with human cancer progression. Nevertheless, the function of MLF1 in iCCA remains unknown. We performed expression analyses of MLF1 in human iCCA. In vitro and in vivo experiments were conducted to investigate the role of MLF1 in iCCA progression. The upstream regulatory mechanism of MLF1 upregulation in iCCA was deciphered by luciferase and DNA methylation analyses. MLF1 was significantly upregulated in clinical iCCA tissue specimens and human iCCA cell lines. MLF1 was positively correlated with KRT19 and MUC1 expression and epithelial-mesenchymal transition (EMT) gene set enrichment score in clinical iCCA. High MLF1 expression was independently associated with worse prognoses in iCCA patients after curative resection. In addition, experimental knockdown of MLF1 attenuated, while overexpression of MLF1 promoted the proliferation, invasiveness, and growth of iCCA cells in vitro and in vivo. Mechanically, MLF1 comodulated EGFR/AKT and Wnt/β-catenin signalings through regulating EGFR, AKT, WNT3, and p-GSK3β expression. Promoter CpG sites' hypermethylation-induced downregulation of miR-29c-3p contributed to MLF1 upregulation in iCCA patients. The upregulation of DNA methyltransferase (DNMT)1, 3A, and 3B downregulated miR-29c-3p by dictating promoter DNA methylation pattern. MiR-29c-3p showed therapeutic potential by targeting MLF1 in iCCA. Our results demonstrated that hypermethylation-mediated miR-29c-3p downregulation contributes to MLF1 upregulation in iCCA, which resulted in tumor cells' proliferation and metastasis through comodulating EGFR/AKT and Wnt/β-catenin signalings.

P-430 Characterization of secondary follicles from cultured cryopreserved-thawed human ovarian cortical tissue

Abstract Study question Can we obtain secondary follicles from cultured cryopreserved-thawed human cortical tissue? Summary answer We obtained a comparable percentage of secondary follicles after culture of cryopreserved-thawed human cortical tissue to that reported after culture of fresh human cortical tissue. What is known already The complete in vitro maturation of oocytes starting from primary oocytes (present in unilaminar follicles) until mature MII oocytes has been achieved previously by two different multi-step culture protocols. These protocols were applied to culture fresh ovarian cortical tissue from adult cisgender donors. However, the efficiency of these two protocols for growing unilaminar follicles to secondary follicles starting from cryopreserved cortical tissue from cisgender patients has not been investigated. As the ovarian tissue available for fertility preservation is cryopreserved, it is important to understand the potential of cryopreserved unilaminar follicles to mature in vitro. Study design, size, duration Cryopreserved ovarian cortical tissue from 4 cisgender adult donors was used for in vitro culture. According to the existing culture protocols, cortical ovarian tissue was cultured either for 8 days using the first step medium (Telfer’s medium) reported by M.McLaughlin 2018 (doi: 10.1093/molehr/gay002) or for 7 and 21 days using the first step medium (Xu’s medium) reported by Xu 2021 (doi: 10.1093/humrep/deab003). Participants/materials, setting, methods Ovarian cortical tissue obtained from adult cisgender donors undergoing oophorectomy for fertility preservation purposes was cryopreserved before chemotherapy. The cryopreserved ovarian cortical tissue was thawed and cut into small pieces. Several pieces were fixed immediately (day 0), the others were either cultured in Telfer’s medium or Xu’s medium. After culture, follicle survival, growth and morphology were assessed by histology and immunofluorescence. Main results and the role of chance We performed quantification of the different follicular stages (primordial, primary, secondary and atretic) after culture and observed that the percentage of secondary follicles increased independently of the culture media used. However, the ovarian cortical tissue cultured using Telfer’s protocol resulted in a higher percentage of secondary follicles and lower percentage of atretic follicles compared to that using Xu’s protocol. After culture, the ovarian cortical tissue was further immunostained for TUNEL, PCNA, COLLIV, STAR, AMH and KRT19. We observed that secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium showed more proliferative (PCNA+) FOXL2+ granulosa cells and less apoptotic (TUNEL+) stromal cells. By contrast, ovarian cortical tissue cultured in Xu’s medium showed less proliferative (PCNA+) FOXL2+ granulosa cells and more apoptotic (TUNEL+) stromal cells. Moreover, the expression of AMH was high in granulosa cells of secondary follicles present in ovarian cortical tissue cultured in Telfer’s medium and low in Xu’s medium. Finally, in both Telfer’s and Xu’s medium cultures the expression of KRT19 was low in granulosa cells of secondary follicles present in ovarian cortical tissue. Limitations, reasons for caution The number of donors was limited and the study lacked fresh ovarian cortical tissue as control. Only the first step in Telfers’ and Xu’s protocol was performed, hence further culture is required to determine whether complete maturation of oocytes in vitro is possible starting from cryopreserved human cortical tissue. Wider implications of the findings Our study showed evidence of follicular growth in cryopreserved-thawed human ovarian cortical tissue after a period of in vitro culture. This is an important first step to achieve in vitro maturation of oocytes from cryopreserved-thawed human ovarian cortical tissue that could be used for clinical applications. Trial registration number NOT APPLICABLE

Clinical genomic implications of transcriptional subtypes in pancreatic cancer.

4145 Background: Transcriptional profiling of pancreatic cancers (PC) has defined classical and basal subtypes; basal tumors have worse outcomes. Mesenchymal (MES) and neural-like progenitor (NRP) subtypes are increasingly recognized and enriched post-therapy. Initial data suggests worse outcomes to FOLFIRINOX (FFX) compared with gemcitabine nab-Paclitaxel (GnP) in basal tumors. Several clinical trials are ongoing to investigate this. Here, we examined the clinical implications of transcriptional subtypes in a large, real-world dataset. Methods: Retrospective IRB exempt, deidentified data was examined from NextGen DNA and RNA sequencing performed on PCs at Caris Life Sciences (Phoenix, AZ). Classical and basal cell states were identified using RNA-seq and the PurIST algorithm in a genomic cohort or GATA6 and KRT5 expression levels in a clinical cohort. Tumor microenvironment immune cell composition on RNA seq was performed using QuantiSeq. Survival was obtained from insurance claims data and calculated from first treatment date to last known contact. Kaplan-Meier estimates were calculated for patient cohorts. P values were adjusted using Benjamini-Hochberg correction. Results: A total of 7,250 PCs were profiled in the genomic cohort. 3,063 tumors (42.2%) were strongly classical (SC), 2,015 tumors (27.8%) were strongly basal (SB) and the remaining had mixed phenotypes. MES and NRP marker genes were significantly co-expressed with each other, with basal genes, and anti-correlated with classical genes. When compared to SC, SB had significantly higher mutation rates in KRAS (93% vs. 88%), TP53 (83% vs. 72%) and ARID1A (12% vs. 8%), whereas SMAD4 (23% vs. 17%) mutations were more common in SC (all q &lt; 0.05). There were no differences in mutation rates in homologous recombination or mismatch repair genes. SB had a significantly higher fraction of M1 macrophages (fold change [FC]: 1.14) and neutrophils (FC 1.16), whereas SC tumors had higher M2 macrophages (FC 1.18), NK (FC 1.2), and dendritic cells. Overall proportions of CD4/8 T cells were low and not different. Interestingly, SB had higher levels of PD-L1 by IHC (4.8% vs. 35%) and higher expression of immune exhaustion genes including CTLA4 (FC 1.19), TIM3 (FC 1.22) and PD-1 (FC 1.43) (all q &lt; 0.05). The clinical cohort had 1,623 patients. Basal tumors had an inferior survival (median survival: 8.2 months (mo) vs 13.3 mo (Hazard Ratio (HR) 0.67, p &lt; 0.00001)) and showed a significant improvement in outcomes when treated with upfront FFX vs GnP (n = 80 vs 90, Median: 15.8 vs 7.4 mos., HR 0.68, p = 0.021). This difference between FFX vs GnP was less pronounced in classical tumors (n = 70 vs 89, Median: 17.3 vs 15.4 mos, HR 0.70, p = 0.049). Conclusions: Our work represents the largest known real world molecular comparison of transcriptional subtypes of PC. Differential outcomes for patients with basal tumors treated with FFX versus GnP warrants further investigation in prospective studies.

Plasma proteomic profiling reveals KRT19 could be a potential biomarker in patients with anti-MDA5+ dermatomyositis.

To investigate the immune response-related protein profiling in plasma of patients with idiopathic inflammatory myopathies (IIMs), especially in anti-MDA5+ dermatomyositis (DM). A total of 166 IIM patients and 107 healthy controls (HCs) were enrolled in our study. Ninety-two plasma immune response-related proteins were detected by Olink proteomics in 36 IIM patients and 25 HCs. The expression of plasma KRT19 was validated in another 130 IIM patients, 82 HCs, and 55 other rheumatic diseases. A total of 46 differentially expressed proteins were detected, including 12 upregulated proteins and 34 downregulated proteins in IIM patients compared with HCs. Pathway analysis revealed lactoferrin danger signal response pathway, TLR4 signaling and tolerance, infection, and IL-10 signaling pathway were activated. The immune response-related protein profiling significantly altered in anti-MDA5+ DM patients, with LAMP3, HSD11B1, and KRT19 significantly increased, while SH2D1A, ITGA11, TRIM21, CD28, ITGB6, and HEXIM1 tremendously decreased. In addition, KRT19 was significantly increased in IIM patients, especially in anti-MDA5+ DM patients with the diagnostic value of a significant area under the ROC curve of 0.881. Immune response-related proteins are significantly altered in patients with anti-MDA5+ DM patients. KRT19 could be a potential biomarker for anti-MDA5+ DM patients. Key Points • What is already known on this topic? Anti-MDA5+ DM is a distinctive subtype of IIM. Plasma immune response-related proteins panel needs to be investigated. • What this study adds? Plasma protein profiling of immune response-related proteins significantly altered in patients with idiopathic inflammatory myopathies (IIM), especially in anti-MDA5+ DM patients. • How this study might affect research, practice, or policy? KRT19 could be a potential biomarker in patients with anti-MDA5+ dermatomyositis.

KRT17 Promotes T-lymphocyte Infiltration Through the YTHDF2-CXCL10 Axis in Colorectal Cancer.

Poor infiltration of T lymphocytes has been regarded as a crucial mechanism of tumor immune escape. Here, we demonstrate a protective role of KRT17 in colorectal cancer, where KRT17 reversed the tumor immunosuppressive microenvironment by increasing T-lymphocyte infiltration. High-throughput RNA sequencing suggested that KRT17 was significantly upregulated in deficient mismatch repair (dMMR) tumors compared with proficient mismatch repair (pMMR) tumors. In a colorectal cancer cohort of 446 cases, KRT17 expression positively correlated with better clinical outcomes. Krt17 overexpression decreased xenograft tumor growth in immune-competent mice. T-cell depletion in a murine model showed that the presence of T lymphocytes was necessary for Krt17-mediated disruption of tumorigenesis. Mass spectrometry and coimmunoprecipitation assays suggested KRT17 caused YTHDF2 degradation through the ubiquitin-proteasome system. Through high-throughput RNA immunoprecipitation sequencing, we found that CXCL10 was the target gene of the N6-methyladenosine (m6A) "reader" YTHDF2. KRT17 synergized with anti-PD-1 for better tumor control in an immunotherapy-resistant murine model. In a cohort of patients with colorectal cancer receiving pembrolizumab, high KRT17 expression was found within the tumors of responders. Collectively, we elucidated a critical role of KRT17 in colorectal cancer to prevent immune escape. These findings present new insights into potential therapeutic strategies and effective markers of immunotherapy reactivity against pMMR tumors.

Abstract 296: Clinical significance of glycolytic metabolic activity in liver cancer and its implication to immunotherapy

Abstract Background &amp; Aims: High metabolic activity is a hallmark of cancers including hepatocellular carcinoma (HCC), a major form of liver cancer. However, the molecular features of HCC with high metabolic activity contributing to the clinical outcomes and their therapeutic implications are poorly understood. We aimed to uncover the metabolic characteristics of HCC that are associated with clinical relevance such as overall survival and response to immunotherapy. Methods: By applying cross-species comparison of genomic data, we integrated gene expression profile data from well-defined mouse models and human HCC tumor tissues to stratify HCC tumors according to their intrinsic metabolic activity. For stratification of HCC tumors, we developed a robust genomic predictor and validated it in 5 HCC cohorts (n=1038). Statistics and informatics approaches were performed to assess clinical significance such as overall survival and response to immunotherapy in metabolic subtypes. Genomic data from patient derived xenograft (PDX) models were integrated to the analysis. Results: Analysis of systematically integrated genomic data revealed three distinct metabolic subtypes of HCC (high, moderate and low metabolic subtypes). The high metabolic subtype is characterized by poor survival, the strongest stem cell features, high genomic instability, high expression of alpha-fetoprotein and KRT19, and activation of EPCAM and SALL4, and poor response to immunotherapy. Importantly, analysis of immune cell population in HCC tumors showed that immune-suppressive regulatory T-cells (Treg) are highly enriched in high metabolic HCC tumors, suggesting that high metabolic activity of HCC cells may trigger activation or infiltration of Treg cells, leading to evasion of HCC cells from anti-cancer immune cells. Interestingly, recent study showed that Treg cells can effectively utilize lactic acid as metabolic fuel for its proliferation. Because high metabolic activity of HCC tumors eventually leads to accumulation of glycolysis by-product lactic acid in tumor microenvironment, we postulate that accumulated lactic acid in tumor microenvironment might account for aggregation of Treg cells in high metabolic HCC tumors, leading to resistance to immunotherapy. In agreement with this, 18F-FDG PET/CT imaging data showed that high metabolic activity of tumors reflected in SUVmax is significantly associated with poorer survival of cancer patients after immunotherapy. Conclusions: We identified clinically and metabolically distinct subtypes of HCC tumors, potential biomarkers associated with these subtypes, and potential mechanism of metabolism-mediated evasion of HCC cells to anti-cancer immunotherapy. Moreover, the metabolic subtypes are well conserved in PDX models, offering a tool for selecting the best preclinical models for future study. Citation Format: Sowon Grace Park, Joann Jung, Yeonwoo Grace Jang, Sung Hwan Lee, Yun-Seong Jeong, Sun Young Yim, Ju-Seog Lee. Clinical significance of glycolytic metabolic activity in liver cancer and its implication to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 296.

The endocannabinoid system promotes hepatocyte progenitor cell proliferation and maturation by modulating cellular energetics

The proliferation and differentiation of hepatic progenitor cells (HPCs) drive the homeostatic renewal of the liver under diverse conditions. Liver regeneration is associated with an increase in Axin2+Cnr1+ HPCs, along with a marked increase in the levels of the endocannabinoid anandamide (AEA). But the molecular mechanism linking AEA signaling to HPC proliferation and/or differentiation has not been explored. Here, we show that in vitro exposure of HPCs to AEA triggers both cell cycling and differentiation along with increased expression of Cnr1, Krt19, and Axin2. Mechanistically, we found that AEA promotes the nuclear localization of the transcription factor β-catenin, with subsequent induction of its downstream targets. Systemic analyses of cells after CRISPR-mediated knockout of the β-catenin-regulated transcriptome revealed that AEA modulates β-catenin-dependent cell cycling and differentiation, as well as interleukin pathways. Further, we found that AEA promotes OXPHOS in HPCs when amino acids and glucose are readily available as substrates, but AEA inhibits it when the cells rely primarily on fatty acid oxidation. Thus, the endocannabinoid system promotes hepatocyte renewal and maturation by stimulating the proliferation of Axin2+Cnr1+ HPCs via the β-catenin pathways while modulating the metabolic activity of their precursor cells.

Transcription factors TP63 facilitates malignant progression of thyroid cancer by upregulating KRT17 expression and inducing epithelial-mesenchymal transition

Thyroid cancer (TC) is a relatively prevalent endocrine tumor among women, the incidence of which is rapidly rising. In this present study, we aimed to provide new therapeutic targets from the aspect of transcription factor-target gene interaction. TP63 and KRT17 were both highly expressed in TC tissues and cells. The results of ChIP and dual-luciferase assays confirmed TP63 to bind the KRT17 promoter. Cell function assays revealed that knockdown of TP63 could repress TC cell progression. Furthermore, the rescue assay verified that TP63 could facilitate KRT17 expression to activate the AKT signaling pathway, which in turn stimulated TC cell invasion and migration, and induced EMT. All these results verified that TP63 facilitates TC malignant progression by promoting KRT17 expression and inducing EMT.

KRT17 promotes endometrial cancer cell migration as well as angiogenesis by regulating HIF-1α/VEGF pathway

Endometrial cancer (EC) is a common type malignant tumors in women. To combat this type of cancer, more effective treatments are still needed. EC cell growth and metastasis depend on angiogenesis. Keratin (KRT) is a family of proteins which are essential for hair formation. KRT17 affected a variety of cancers. Here, we reported that KRT17 expression was high in human EC. The depletion of KRT17 suppressed EC cell growth. We further showed the ablation of KRT17 led to the suppression of cell motility. Also, its depletion resulted in the inhibition of angiogenesis. We further demonstrated that KRT17 contributed to the progression of EC via regulating the HIF-1α/VEGF axis through a tumor growth assay in mice. In conclusion, KRT17 could serve as an EC target.

A novel lncRNA-mediated epigenetic regulatory mechanism in periodontitis.

Periodontitis is a highly prevalent chronic inflammatory disease with an exaggerated host immune response, resulting in periodontal tissue destruction and potential tooth loss. The long non-coding RNA, LncR-ANRIL, located on human chromosome 9p21, is recognized as a genetic risk factor for various conditions, including atherosclerosis, periodontitis, diabetes, and cancer. LncR-APDC is an ortholog of ANRIL located on mouse genome chr4. This study aims to comprehend the regulatory role of lncR-APDC in periodontitis progression. Our experimental findings, obtained from lncR-APDC gene knockout (KO) mice with induced experimental periodontitis (EP), revealed exacerbated bone loss and disrupted pro-inflammatory cytokine regulation. Downregulation of osteogenic differentiation occurred in bone marrow stem cells harvested from lncR-APDC-KO mice. Furthermore, single-cell RNA sequencing of periodontitis gingival tissue revealed alterations in the proportion and function of immune cells, including T and B cells, macrophages, and neutrophils, due to lncR-APDC silencing. Our findings also unveiled a previously unidentified epithelial cell subset that is distinctively presenting in the lncR-APDC-KO group. This epithelial subset, characterized by the positive expression of Krt8 and Krt18, engages in interactions with immune cells through a variety of ligand-receptor pairs. The expression of Tff2, now recognized for its role in chronic inflammatory conditions, exhibited a notable increase across various tissue and cell types in lncR-APDC deficient mice. Additionally, our investigation revealed the potential for a direct binding interaction between lncR-APDC and Tff2. Intra-gingival administration of AAV9-lncR-APDC was shown to have therapeutic effects in the EP model. In conclusion, our results suggest that lncR-APDC plays a critical role in the progression of periodontal disease and holds therapeutic potential for periodontitis. Furthermore, the presence of the distinctive epithelial subpopulation and significantly elevated Tff2 levels in the lncR-APDC-silenced EP model offer new perspectives on the epigenetic regulation of periodontitis pathogenesis.

Keratin 18 Depletion as a Possible Mechanism for the Induction of Apoptosis and Ferroptosis in the Rat Hippocampus After Hypobaric Hypoxia

Memory impairment is one of the neuropsychological effects of hypobaric hypoxia (HH), which can be associated with programmed cell death, such as apoptosis and ferroptosis. Emerging evidence indicates crosstalk between apoptosis and ferroptosis, while the crosstalk between HH-induced apoptosis and ferroptosis in the hippocampus has not been clarified. Here, microarray profiles were extracted to analyze the differentially expressed genes with and without HH exposure, and keratin 18 (Krt18) was found to be a potential gene related to both apoptosis and ferroptosis. Then, we conducted morphological observations that showed that apoptosis and ferroptosis coexisted in the rat hippocampus after HH exposure. Combined with the real-time q-PCR analysis, the mRNA expression of Krt18 decreased significantly after HH exposure for 1 day and 3 days, and Mapk10 (JNK3) was upregulated at the corresponding time points. After exposure for 7 days, Krt18 and JNK3 showed no significant change. In conclusion, Krt18 may regulate apoptosis and ferroptosis simultaneously, possibly via the JNK signaling pathway, which might provide a potential central target for apoptosis and ferroptosis in hippocampal injury after HH exposure.

Cyclin B1 expression as an independent prognostic factor for lung adenocarcinoma and its potential pathways.

Although great progress has been made in the early diagnosis and targeted therapy of lung adenocarcinoma (LUAD), the survival of patients with LUAD remains unsatisfactory. There is an urgent requirement for new biomarkers to guide the diagnosis, prognosis and treatment of LUAD. Following an initial bioinformatics screen, the present study focused on cyclin B1 (CCNB1) in LUAD. A total of 94 patients with LUAD from a single hospital were included in the study. CCNB1 protein expression was detected and scored in 94 LUAD samples and 30 normal tissue samples by immunohistochemistry. The associations between CCNB1 expression and the clinicopathological features of the patients with LUAD were analyzed. Furthermore, the relationship between prognosis and the CCNB1 expression level was analyzed using Cox regression and survival analyses. Weighted gene co-expression network analysis and RNA-sequencing were also applied to identify the potential molecular mechanisms of CCNB1 in LUAD. CCNB1 was highly expressed in patients with LUAD and was associated with poor prognosis. It may affect the expression of CPLX1, PPIF, SRPK2, KRT8, SLC20A1 and CBX2 genes and function via different pathways. CCNB1 has the potential to become a novel prognostic target for LUAD.

The Transcriptional Regulator Prdm1 Is Essential for the Early Development of the Sensory Whisker Follicle and Is Linked to the Beta-Catenin First Dermal Signal.

Prdm1 mutant mice are one of the rare mutant strains that do not develop whisker hair follicles while still displaying a pelage. Here, we show that Prdm1 is expressed at the earliest stage of whisker development in clusters of mesenchymal cells before placode formation. Its conditional knockout in the murine soma leads to the loss of expression of Bmp2, Shh, Bmp4, Krt17, Edar, and Gli1, though leaving the β-catenin-driven first dermal signal intact. Furthermore, we show that Prdm1 expressing cells not only act as a signaling center but also as a multipotent progenitor population contributing to the several lineages of the adult whisker. We confirm by genetic ablation experiments that the absence of macro vibrissae reverberates on the organization of nerve wiring in the mystacial pads and leads to the reorganization of the barrel cortex. We demonstrate that Lef1 acts upstream of Prdm1 and identify a primate-specific deletion of a Lef1 enhancer named Leaf. This loss may have been significant in the evolutionary process, leading to the progressive defunctionalization and disappearance of vibrissae in primates.