Redefining aberrant P53 expression of gastric cancer and its distinct clinical significance among molecular-histologic subtypes.

BACKGROUND: Numerous studies have demonstrated a correlation between p53 overexpression and diminished survival in gastric cancer patients. However, conflicting findings exist, and we hypothesize that these discrepancies arise from the cancer's complexity and heterogeneity, coupled with a lack of consensus on aberrant p53 expression. METHODS: We enrolled a cohort of 187 patients with surgically resected gastric cancer. Patient categorization was based on Epstein-Barr virus (EBV), microsatellite instability (MSI), and Lauren classification (intestinal, diffuse and mixed). Utilizing an incremental algorithm, we evaluated p53 immunohistochemical (IHC) patterns in all 187 cases, while next-generation sequencing was successfully performed on 152 cases to identify TP53 mutations (mutTP53). RESULTS: MutTP53 was identified in 32 % of the 152 cases, comprising 36 missense, 5 nonsense, and 7 frameshift alterations. Missense mutations predominantly correlated with p53 overexpression, while nonsense and frameshifting alterations related to null expression. Trial calculations indicated that null expression and a p53 IHC cutoff at >40 % offered the best prediction of mutTP53 (kappa coefficient, 0.427), with the highest agreement (0.524) observed in diffuse type and the lowest (0.269) in intestinal type. Null expression and a p53 IHC cutoff at >10 %, but not mutTP53 per se, provided the optimal prediction of survival outcome (p = 0.043), particularly in diffuse type (p = 0.044). Multivariate analysis showed that aberrant p53 IHC expression was not an independent prognostic factor. CONCLUSIONS: P53 IHC patterns are predictive biomarkers for mutTP53 and gastric cancer outcomes, where a prerequisite involves a nuanced approach considering cutoff values and molecular-histologic subtyping.

CAN008 prolongs overall survival in patients with newly diagnosed GBM characterized by high tumor mutational burden.

BACKGROUND: A previous phase 1 dose-escalation study in Taiwan indicated CAN008 (asunercept) with standard concurrent chemoradiotherapy (CCRT) improved progression-free survival (PFS) in newly diagnosed glioblastoma (GBM) patients. This study evaluates the efficacy of CAN008 in promoting overall survival (OS) and identifies genetic alterations associated with treatment responses. METHODS: We compared OS of 5-year follow-ups from 9 evaluable CAN008 cohort patients (6 received high-dose and 3 received low-dose) to a historical Taiwanese GBM cohort with 164 newly diagnosed patients. CAN008 treatment response-associated genetic alterations were identified by whole-exome sequencing and comparing variant differences between response groups. Associations among patient survival, tumor mutational burden (TMB), and genetic alterations were analyzed using CAN008 cohort and TCGA-GBM dataset. RESULTS: OS for high-dose CAN008 patients at 2 and 5 years was 83% and 67%, respectively, and 40.1% and 8.8% for the historical GBM cohort, respectively. Better OS was observed in the high-dose CAN008 cohort (without reaching the median survival) than the historical GBM cohort (median OS: 20 months; p=0.0103). Five high-dose CAN008 patients were divided into good and poor response groups based on their PFS. A higher variant count and TMB were observed in good response patients, whereas no significant association was observed between TMB and patient survival in the newly diagnosed TCGA-GBM dataset, suggesting TMB may modulate patient CAN008 response. CONCLUSION: CAN008 combined with standard CCRT treatment prolonged the PFS and OS of newly diagnosed GBM patients compared to standard therapy alone. Higher treatment efficacy was associated with higher TMB.

Association between hepatic angiosarcoma and end-stage renal disease: nationwide population-based evidence and enriched mutational signature of aristolochic acid exposure.

Hepatic angiosarcoma (HAS) is an aggressive mesenchymal malignancy that remains underexplored with respect to its etiology and mutational landscapes. To clarify the association between HAS and end-stage renal disease (ESRD), we used nationwide data of the National Health Insurance Research Database (NHIRD) in Taiwan, covering ~99% of the population, from 2001 to 2016. To investigate molecular signatures, we performed whole-exome sequencing (WES) in 27 surgical specimens, including nine ESRD-associated cases. The NHIRD analysis demonstrated that HAS ranked second among all angiosarcomas in Taiwan, with the incidence rates of HAS being 0.08, 2.49, and 5.71 per 100,000 person-years in the general population, chronic kidney disease (CKD), and ESRD patients, respectively. The standardized incidence ratios of HAS in CKD and ESRD patients were 29.99 and 68.77, respectively. In comparison with nonhepatic angiosarcoma, the multivariate regression analysis of our institutional cohort confirmed CKD/ESRD as an independent risk factor for HAS (odds ratio: 9.521, 95% confidence interval: 2.995-30.261, p < 0.001). WES identified a high tumor mutation burden (TMB; median: 8.66 variants per megabase) and dominant A:T-to-T:A transversion in HAS with frequent TP53 (81%) and ATRX (41%) mutations, KDR amplifications/gains (56%), and CDKN2A/B deletions (48%). Notably, ESRD-associated HAS had a significantly higher TMB (17.62 variants per megabase, p = 0.01) and enriched mutational signatures of aristolochic acid exposure (COSMIC SBS22, p < 0.001). In summary, a significant proportion of HAS in Taiwan is associated with ESRD and harbors a distinctive mutational signature, which concomitantly links nephrotoxicity and mutagenesis resulting from exposure to aristolochic acid or related compounds. A high TMB may support the eligibility for immunotherapy in treating ESRD-associated HAS. © 2023 The Pathological Society of Great Britain and Ireland.

Prognostic value of an APOBEC3 deletion polymorphism for glioma patients in Taiwan.

OBJECTIVE: The molecular pathogenesis of malignant gliomas, characterized by diverse tumor histology with differential prognosis, remains largely unelucidated. An APOBEC3 deletion polymorphism, with a deletion in APOBEC3B, has been correlated to risk and prognosis in several cancers, but its role in glioma is unclear. The authors aimed to examine the clinical relevance of the APOBEC3 deletion polymorphism to glioma risk and survival in a glioma patient cohort in Taiwan. METHODS: The authors detected deletion genotypes in 403 glioma patients and 1365 healthy individuals in Taiwan and correlated the genotypes with glioma risk, clinicopathological factors, patient survival, and patient sex. APOBEC3 gene family expression was measured and correlated to the germline deletion. A nomogram model was constructed to predict patient survival in glioma. RESULTS: The proportion of APOBEC3B-/- and APOBEC3B+/- genotypes was higher in glioblastoma (GBM) patients than healthy individuals and correlated with higher GBM risk in males. A higher percentage of cases with APOBEC3B- was observed in male than female glioma patients. The presence of APOBEC3B-/- was correlated with better overall survival (OS) in male astrocytic glioma patients. No significant correlation of the genotypes to glioma risk and survival was observed in the female patient cohort. Lower APOBEC3B expression was observed in astrocytic glioma patients with APOBEC3B-/- and was positively correlated with better OS. A 5-factor nomogram model was constructed based on male patients with astrocytic gliomas in the study cohort and worked efficiently for predicting patient OS. CONCLUSIONS: The germline APOBEC3 deletion was associated with increased GBM risk and better OS in astrocytic glioma patients in the Taiwan male population. The APOBEC3B deletion homozygote was a potential independent prognostic factor predicting better survival in male astrocytic glioma patients.

EV-miRome-wide profiling uncovers miR-320c for detecting metastatic colorectal cancer and monitoring the therapeutic response.

PURPOSE: Molecular composition of circulating small extracellular vesicles (EVs) does not merely reflect the cells of origin, but also is enriched in specific biomolecules directly associated with the cellular transformation. However, while most of the currently identified EV-miRs are only geared towards one-dimensional disease detection, their application for long-term tracking and treatment response monitoring has been largely elusive. METHODS: We established and optimized a rapid, sensitive and robust liquid biopsy sampling method, and further used small RNA sequencing to comprehensively catalogue EV-miRomes in association with the progression and outcome of metastatic colorectal cancer (mCRC). RESULTS: By cross-comparison of EV-miRomes (n = 290) from multi-stage and longitudinal cohorts, we uncovered a 15-EV-miR signature with dual detection and long-term monitoring of tumor size progression for mCRC. From this panel, EV-miR-320c was uncovered as a strong clinical marker - aside from its diagnostic power and a therapeutic monitoring performance superior to carcinoembryonic antigen (CEA), its high expression has also been linked to lower overall survival and a greater likelihood of disease recurrence. Further, integrative analyses of tissue transcriptomic and liquid biopsy implicated this 15-EV-miR signature in programming the mesenchymal-epithelial transition (MET) for distant localization of the metastasized cells and also in creating a tumor-favoring metastatic niche. CONCLUSION: Our clinically-oriented delineation of the mCRC-associated circulating EV-miRomes systematically revealed the functional significance of these liquid biopsy markers and further strengthen their translational potential in mCRC therapeutic monitoring.

Modular scaffolding by lncRNA HOXA10-AS promotes oral cancer progression.

Recent findings have implicated long noncoding RNAs (lncRNAs) as pivotal gene regulators for diverse biological processes, despite their lack of protein-coding capabilities. Accumulating evidence suggests the significance of lncRNAs in mediating cell signaling pathways, especially those associated with tumorigenesis. Consequently, lncRNAs have emerged as novel functional regulators and indicators of cancer development and malignancy. Recent transcriptomic profiling has recognized a tumor-biased expressed lncRNA, the HOXA10-AS transcript, whose expression is associated with patient survival. Functional cell-based assays show that the HOXA10-AS transcript is essential in the regulation of oral cancer growth and metastasis. LncRNA expression is also associated with drug sensitivity. In this study, we identify that HOXA10-AS serves as a modular scaffold for TP63 mRNA processing and that such involvement regulates cancer growth. These findings provide a functional interpretation of lncRNA-mediated molecular regulation, highlighting the significance of the lncRNA transcriptome in cancer biology.

Genomic and Molecular Signatures of Successful Patient-Derived Xenografts for Oral Cavity Squamous Cell Carcinoma.

Background: Oral cavity squamous cell carcinoma (OSCC) is an aggressive malignant tumor with high recurrence and poor prognosis in the advanced stage. Patient-derived xenografts (PDXs) serve as powerful preclinical platforms for drug testing and precision medicine for cancer therapy. We assess which molecular signatures affect tumor engraftment ability and tumor growth rate in OSCC PDXs. Methods: Treatment-naïve OSCC primary tumors were collected for PDX models establishment. Comprehensive genomic analysis, including whole-exome sequencing and RNA-seq, was performed on case-matched tumors and PDXs. Regulatory genes/pathways were analyzed to clarify which molecular signatures affect tumor engraftment ability and the tumor growth rate in OSCC PDXs. Results: Perineural invasion was found as an important pathological feature related to engraftment ability. Tumor microenvironment with enriched hypoxia, PI3K-Akt, and epithelial-mesenchymal transition pathways and decreased inflammatory responses had high engraftment ability and tumor growth rates in OSCC PDXs. High matrix metalloproteinase-1 (MMP1) expression was found that have a great graft advantage in xenografts and is associated with pooled disease-free survival in cancer patients. Conclusion: This study provides a panel with detailed genomic characteristics of OSCC PDXs, enabling preclinical studies on personalized therapy options for oral cancer. MMP1 could serve as a biomarker for predicting successful xenografts in OSCC patients.

Adult NTRK-rearranged spindle cell neoplasms of the viscera: with an emphasis on rare locations and heterologous elements.

NTRK-rearranged mesenchymal neoplasms mostly affect the soft tissues of pediatric patients. Given the responsiveness to selective NTRK inhibitors, it remains critical to identify those ultra-rare cases occurring in the viscera of adults. In five females and two males aged 18-53 years, we characterized visceral mesenchymal tumors harboring TPM3-NTRK1 [uterine cervix (N = 2), pleura, prostate], LMNA-NTRK1 (lung), SQSTM1-NTRK3 (heart), and NTRK3 rearrangement with unknown fusion partner (colon/mesocolon) with RNA sequencing, FISH, RT-PCR, and immunohistochemistry. The tumors exhibited spindled to ovoid/epithelioid or pleomorphic cells, often arranged in fascicles, and were low-to-intermediate-grade and high-grade in three and four cases, respectively. Keloid-like stromal collagen and perivascular hyalinization was noted in five. Adenosarcoma-like appearances were observed in two, manifesting frond-like protrusions in one cervical tumor and phyllodes-like architecture in the prostatic tumor. Abrupt high-grade transformation into pleomorphic liposarcoma was found in another cervical tumor, while the pleural tumor contained intermixed rhabdomyoblasts. Pan-TRK immunostaining was positive in all cases. All cases expressed CD34, while five were S100-positive. CDKN2A homozygous deletion with concomitant p16 loss occurred in 4/7. Whole-exome sequencing identified TP53 mutation (c.672+2T>C, involving a splice site, with concomitant protein loss) in a cervical sarcoma, limited to its heterologous liposarcomatous component. At least moderate pan-TRK immunoreactivity was present in varying proportions of potential pathologic mimics, with BCOR-positive sarcoma (56%, 5/9), undifferentiated uterine sarcoma (50%, 3/6), and spindle cell/sclerosing rhabdomyosarcoma (33%, 2/6) being among the most frequent. This underscored the unsatisfactory specificity of pan-TRK immunohistochemistry and warranted molecular confirmation in the diagnosis of adult NTRK-rearranged visceral mesenchymal neoplasms. The current report highlights the ever-expanding clinicopathologic and genetic spectrum of this entity by describing the unprecedented cardiac and pleural locations and heterologous differentiation, as well as the second NTRK-rearranged "prostatic stromal sarcoma," while substantiating CDKN2A deletion as a frequent occurrence.

Glioblastoma Primary Cells Retain the Most Copy Number Alterations That Predict Poor Survival in Glioma Patients.

Gliomas are solid tumors that originate from glial cells in the brain or spine and account for 74.6% of malignant primary central nervous system tumors worldwide. As patient-derived primary cells are important tools for drug screening and new therapy development in glioma, we aim to understand the genomic similarity of the primary cells to their parental tumors by comparing their whole-genome copy number variations and expression profile of glioma clinicopathologic factors. We found that the primary cells from grade II/III gliomas lost most of the gene copy number alterations (CNAs), which were mainly located on chromosome 1p and 19q in their parental tumors. The glioblastoma (GBM) primary cells preserved 83.7% of the gene CNAs in the parental GBM tumors, including chromosome 7 gain and 10q loss. The CNA gains of LINC00226 and ADAM6 and the chromosome 16p11 loss were reconstituted in primary cells from both grade II/III gliomas and GBMs. Interestingly, we found these CNAs were correlated to overall survival (OS) in glioma patients using the Merged Cohort LGG and GBM dataset from cBioPortal. The gene CNAs preserved in glioma primary cells often predicted poor survival, whereas the gene CNAs lost in grade II/III primary cells were mainly associated to better prognosis in glioma patients. Glioma prognostic factors that predict better survival, such as IDH mutations and 1p/19q codeletion in grade II/III gliomas, were lost in their primary cells, whereas methylated MGMT promoters as well as TERT promoter mutations were preserved in GBM primary cells while lost in grade II/III primary cells. Our results suggest that GBM primary cells tend to preserve CNAs in their parental tumors, and these CNAs are correlated to poor OS and predict worse prognosis in glioma patients.

The clinicopathological significance of SWI/SNF alterations in gastric cancer is associated with the molecular subtypes.

The clinicopathological significance of altered SWI/SNF complex has not been well evaluated in gastric cancer (GC). We examined SMARCA2, SMARCA4, SMARCB1 and ARID1A expression by immunohistochemistry in 1224 surgically resected GCs with subtyping into Epstein-Barr virus (EBV), microsatellite instability (MSI) and non-EBV/MSI Lauren histotypes. SWI/SNF mutations were investigated using the GC dataset of the TCGA Pan-Cancer Atlas. Clinicopathological association was assessed by statistical analysis. There were 427 cases (35%) of SWI/SNF-attenuated GC, including 344 SMARCA2 (28%), 28 SMARCA4 (2%), 11 SMARCB1 (1%) and 197 ARID1A (16%) cases. Simultaneous alterations of multiple subunits were observed. Compared to SWI/SNF-retained cases, SWI/SNF-attenuated GC exhibited a significant predilection to older ages, EBV and MSI genotypes, higher lymphatic invasion and less hematogenous recurrence (P < 0.05). SWI/SNF attenuation was an independent risk factor for short overall survival (P = 0.001, hazard ratio 1.360, 95% confidence interval 1.138-1.625). The survival impact stemmed from SMARCA2-attenuated GCs in stage III and non-EBV/MSI diffuse/mixed subtypes (P = 0.019 and < 0.001, respectively). ARID1A-lost/heterogeneous GCs were more aggressive in the EBV genotype (P = 0.016). SMARCB1 or SMARCA4 loss was not restricted to rhabdoid/undifferentiated carcinoma. In the TCGA dataset, 223 of 434 GCs (52%) harbored deleterious SWI/SNF mutations, including ARID1A (27%), SMARCA2 (9%), ARID2 (9%), ARID1B (8%), PBRM1 (7%), and SMARCA4 (7%). SWI/SNF-mutated GCs displayed a favorable outcome owing to the high percentage with the MSI genotype. In conclusion, SWI/SNF-altered GCs are common and the clinicopathological significance is related to the genotype.

Longitudinal High-Throughput Sequencing of the T-Cell Receptor Repertoire Reveals Dynamic Change and Prognostic Significance of Peripheral Blood TCR Diversity in Metastatic Colorectal Cancer During Chemotherapy.

Colorectal cancer (CRC) is a major cause of cancer mortality and morbidity. Despite advances in chemotherapy and targeted therapy, unsustainable clinical benefit was noted due to recurrence and therapy resistance. The immune status of the cancer patient may affect the effectiveness of disease treatments. The dynamic change in the T-cell receptor (TCR) repertoire might be a clinical parameter for monitoring treatment responses. In this study, we aimed to determine the characteristics and clinical significance of the TCR repertoire in patients with unresectable metastatic colorectal cancer (mCRC). Herein, we comprehensively profile 103 peripheral blood samples from 20 healthy controls and 16 CRC patients with a follow-up of 98 to 452 days to identify hypervariable rearrangements of the TCRα and TCRß repertoires using high-throughput sequencing. We found that TCRα repertoires, TCRß repertoires, and CDR3 clonotypes were altered in mCRC patients compared with healthy controls. The diversity of TCR repertoires and CDR3 clonotypes decreased in most mCRC patients after therapy. Furthermore, compared with baseline TCR diversity, patients whose TCR diversity dropped considerably during therapy had better treatment responses, including lower CEA and CA19-9 levels and smaller tumor sizes. TCR baseline diversity was also significantly associated with partial response (PR) status (odds ratio: 5.29, p = 0.04). In conclusion, the present study demonstrated the association between dynamic changes in TCR diversity during chemotherapy and clinical outcomes as well as the potential utility of the TCR repertoire in predicting the prognosis of cancer treatment.

ASC modulates HIF-1α stability and induces cell mobility in OSCC.

High-level expression of ASC (Apoptosis-associated speck-like protein containing a CARD) leads to lymph node metastasis in OSCC, but the underlying mechanism remains unclear. Here, we show that HIF-1α participates in ASC-induced metastasis. We identified 195 cell-motion-associated genes that were highly activated in ASC-overexpressed SAS_ASC cells; of them, 14 representative genes were found to be overexpressed in OSCC tissues in our previously reported RNA-seq dataset, OSCC-Taiwan. Nine of the 14 genes were also upregulated in OSCC-TCGA samples. Among the nine genes, RRAS2, PDGFA, and VEGFA, were correlated with poor overall survival of patients in OSCC-TCGA dataset. We further demonstrated that the promoters of these 14 ASC-induced genes contained binding motifs for the transcription-regulating factor, HIF-1α. We observed that ASC interacted with and stabilized HIF-1α in both the cytoplasm and the nucleus under normoxia. Molecules involved in the HIF-1α pathway, such as VHL and PHD2, showed no difference in their gene and protein levels in the presence or absence of ASC, but the expression of HIF-1α-OH, and the ubiquitination of HIF-1α were both decreased in SAS_ASC cells versus SAS_con cells. The migration and invasion activities of SAS_ASC cells were reduced when cells were treated with the HIF-1α synthesis inhibitor, digoxin. Taken together, our results demonstrate that the novel ASC-HIF-1α regulatory pathway contributes to lymph node metastasis in OSCC, potentially suggesting a new treatment strategy for OSCC.

Verification of Saliva Matrix Metalloproteinase-1 as a Strong Diagnostic Marker of Oral Cavity Cancer.

Oral squamous cell carcinoma (OSCC) accounts for >90% of cases of oral cancer, including cancer at the lip and oral cavity and cancer at the oropharynx. Most OSCCs develop from oral potentially malignant disorders (OPMDs), which consist of heterogeneous lesions with different malignant transformation potentials that make early detection of OSCC a challenge. Using a targeted mass spectrometry-based assay to compare multiple candidate proteins, we previously identified matrix metalloproteinase-1 (MMP-1) as one of the most promising salivary OSCC biomarkers. To explore the clinical utility of MMP-1 in OSCC detection, we developed an in-house, sensitive enzyme-linked immunosorbent assay (ELISA) for measuring MMP-1 content, and tested it on saliva samples from 1160 subjects (313 healthy controls, and 578 OPMD and 269 OSCC patients) collected at two medical centers. Salivary MMP-1 levels measured by our in-house ELISA significantly discriminated OSCC patients from non-cancerous groups. A receiver operating characteristic curve analysis showed that MMP-1 was effective in separating non-cancer groups from patients with OSCCs at the oral cavity. Additionally, salivary MMP-1 levels in oral cavity cancer patients were highly correlated with tumor progression (tumor size, lymph node metastasis, and overall stage). Collectively, our results indicate that salivary MMP-1 is an effective biomarker for OSCC that can be sensitively detected using our newly developed ELISA. The newly developed MMP-1 ELISA may be used as a new adjunctive tool to aid in detecting and monitoring OSCC.

circRNAome Profiling in Oral Carcinoma Unveils a Novel circFLNB that Mediates Tumour Growth-Regulating Transcriptional Response.

Deep sequencing technologies have revealed the once uncharted non-coding transcriptome of circular RNAs (circRNAs). Despite the lack of protein-coding potential, these unorthodox yet highly stable RNA species are known to act as critical gene regulatory hubs, particularly in malignancies. However, their mechanistic implications in tumor outcome and translational potential have not been fully resolved. Using RNA-seq data, we profiled the circRNAomes of tumor specimens derived from oral squamous cell carcinoma (OSCC), which is a prevalently diagnosed cancer with a persistently low survival rate. We further catalogued dysregulated circRNAs in connection with tumorigenic progression. Using comprehensive bioinformatics analyses focused on co-expression maps and miRNA-interaction networks, we delineated the regulatory networks that are centered on circRNAs. Interestingly, we identified a tumor-associated, pro-tumorigenic circRNA, named circFLNB, that was implicated in maintaining several tumor-associated phenotypes in vitro and in vivo. Correspondingly, transcriptome profiling of circFLNB-knockdown cells showed alterations in tumor-related genes. Integrated in silico analyses further deciphered the circFLNB-targeted gene network. Together, our current study demarcates the OSCC-associated circRNAome, and unveils a novel circRNA circuit with functional implication in OSCC progression. These systems-based findings broaden mechanistic understanding of oral malignancies and raise new prospects for translational medicine.

Cotargeting CHK1 and PI3K Synergistically Suppresses Tumor Growth of Oral Cavity Squamous Cell Carcinoma in Patient-Derived Xenografts.

Oral cavity squamous cell carcinomas (OSCCs) are aggressive tumors, and their recurrence leads to poor prognosis and reduced survival rates. This study aimed to identify therapeutic targets and to evaluate the efficacy of targeted inhibitors in OSCC patient-derived xenograft (PDX) models. Herein, we reported that OSCC PDXs recapitulated the genomic signatures of their paired primary tumors and the expression of CHEK1, PIK3CA, and PIK3CD was significantly upregulated in OSCC. The antitumor efficacy of CHK1 inhibitors (PF477736, AZD7762, LY2606368) and PI3K inhibitors (BYL719, GDC0941, GSK1059615) was investigated in OSCC cell lines and PDX models. Targeting either CHK1 or PI3K effectively inhibited cell proliferation and colony formation by inducing cell cycle arrest and apoptosis in in vitro cell-based assays. Cisplatin-based chemotherapy combined with CHK1 inhibitor treatment synergistically inhibited cell proliferation by suppressing CHK1 phosphorylation and inducing PARP cleavage. Furthermore, compared with monotherapy, cotreatment with CHK1 and PI3K inhibitors exerted synergistic anticancer effects by suppressing CHK1, AKT, and 4E-BP1 phosphorylation. In summary, our study identified CHK1 and PI3K as promising targets, especially in a dual treatment strategy combining a CHK1 inhibitor with cisplatin or a PI3K inhibitor as a novel therapeutic approach for OSCC patients with aberrant cell cycle regulation and PI3K signaling activation.

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

Assessment of candidate biomarkers in paired saliva and plasma samples from oral cancer patients by targeted mass spectrometry.

For oral cancer, numerous saliva- and plasma-derived protein biomarker candidates have been discovered and/or verified; however, it is unclear about the behavior of these candidates as saliva or plasma biomarkers. In this study, we developed two targeted assays, MRM and SISCAPA-MRM, to quantify 30 potential biomarkers in both plasma and saliva samples collected from 30 healthy controls and 30 oral cancer patients. Single point measurements were used for target quantification while response curves for assay metric determination. In comparison with MRM assay, SISCAPA-MRM effectively improved (>1.5 fold) the detection sensitivity of 11 and 21 targets in measurement of saliva and plasma samples, respectively. The integrated results revealed that the salivary levels of these 30 selected biomarkers weakly correlated (r < 0.2) to their plasma levels. Five candidate biomarkers (MMP1, PADI1, TNC, CSTA and MMP3) exhibited significant alterations and disease-discriminating powers (AUC = 0.914, 0.827, 0.813, 0.77, and 0.753) in saliva sample; nevertheless, no such targets could be found in plasma samples. Our data support the notion that saliva may be more suitable for the protein biomarker-based detection of oral cancer, and the newly developed SISCAPA-MRM assay could be applied to verify multiple oral cancer biomarker candidates in saliva samples. SIGNIFICANCE: In this work we systematically determined the abundance of 30 selected targets in the paired saliva and plasma samples to evaluate the utility of saliva and plasma samples for protein biomarker-based detection of oral cancer. Our study provides significant evidence to support the use of saliva, but not blood samples, offer more opportunity to achieve the success of protein biomarker discovery for oral cancer detection.

Identification of Salivary Biomarkers for Oral Cancer Detection with Untargeted and Targeted Quantitative Proteomics Approaches.

Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. In Taiwan, OSCC is the fifth leading cause of cancer-related mortality and leads to 2800 deaths per year. The poor outcome of OSCC patients is principally ascribed to the fact that this disease is often advanced at the time of diagnosis, suggesting that early detection of OSCC is urgently needed. Analysis of cancer-related body fluids is one promising approach to identify biomarker candidates of cancers. To identify OSCC biomarkers, salivary proteomes of OSCC patients, individuals with oral potentially malignant disorders (OPMDs), and healthy volunteers were comparatively profiled with isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry (MS). The salivary levels of 67 and 18 proteins in the OSCC group are elevated and decreased compared with that in the noncancerous group (OPMD and healthy groups), respectively. The candidate biomarkers were further selected using the multiple reaction monitoring (MRM)-MS and validated with the immunoassays. More importantly, the higher salivary level of three proteins, complement factor H (CFH), fibrinogen alpha chain (FGA), and alpha-1-antitrypsin (SERPINA1) was correlated with advanced stages of OSCC. Our results indicate that analysis of salivary proteome is a feasible strategy for biomarker discovery, and the three proteins are potential salivary markers for OSCC diagnosis.

Tumor-associated intronic editing of HNRPLL generates a novel splicing variant linked to cell proliferation.

Processing of the eukaryotic transcriptome is a dynamic regulatory mechanism that confers genetic diversity, and splicing and adenosine to inosine (A-to-I) RNA editing are well-characterized examples of such processing. Growing evidence reveals the cross-talk between the splicing and RNA editing, but there is a paucity of substantial evidence for its mechanistic details and contribution in a physiological context. Here, our findings demonstrate that tumor-associated differential RNA editing, in conjunction with splicing machinery, regulates the expression of variants of HNRPLL, a gene encoding splicing factor. We discovered an HNRPLL transcript variant containing an additional exon 12A (E12A), which is a substrate of ADAR1 and ADAR2. Adenosine deaminases acting on RNA (ADAR) direct deaminase-dependent expression of the E12A transcript, and ADAR-mediated regulation of E12A is largely splicing-based, and does not affect the stability or nucleocytoplasmic distribution of the transcript. Furthermore, ADAR-mediated modification of exon 12A generates an enhancer for the oncogenic splicing factor SRSF1 and consequently promotes the frequency of alternative splicing. Gene expression profiling by RNA-seq revealed that E12A acts distinctly from HNRPLL and regulates a set of growth-related genes, such as cyclin CCND1 and growth factor receptor TGFBR1 Accordingly, silencing E12A expression leads to impaired clonogenic ability and enhanced sensitivity to doxorubicin, thus highlighting the significance of this alternative isoform in tumor cell survival. In summary, we present the interplay of RNA editing and splicing as a regulatory mechanism of gene expression and also its physiological relevance. These findings extend our understanding of transcriptional dynamics and provide a mechanistic explanation to the link of RNA editors to tumorigenesis.

APOBEC3A is an oral cancer prognostic biomarker in Taiwanese carriers of an APOBEC deletion polymorphism.

Oral squamous cell carcinoma is a prominent cancer worldwide, particularly in Taiwan. By integrating omics analyses in 50 matched samples, we uncover in Taiwanese patients a predominant mutation signature associated with cytidine deaminase APOBEC, which correlates with the upregulation of APOBEC3A expression in the APOBEC3 gene cluster at 22q13. APOBEC3A expression is significantly higher in tumors carrying APOBEC3B-deletion allele(s). High-level APOBEC3A expression is associated with better overall survival, especially among patients carrying APOBEC3B-deletion alleles, as examined in a second cohort (n = 188; p = 0.004). The frequency of APOBEC3B-deletion alleles is ~50% in 143 genotyped oral squamous cell carcinoma -Taiwan samples (27A3B -/-:89A3B +/-:27A3B +/+), compared to the 5.8% found in 314 OSCC-TCGA samples. We thus report a frequent APOBEC mutational profile, which relates to a APOBEC3B-deletion germline polymorphism in Taiwanese oral squamous cell carcinoma that impacts expression of APOBEC3A, and is shown to be of clinical prognostic relevance. Our finding might be recapitulated by genomic studies in other cancer types.Oral squamous cell carcinoma is a prevalent malignancy in Taiwan. Here, the authors show that OSCC in Taiwanese show a frequent deletion polymorphism in the cytidine deaminases gene cluster APOBEC3 resulting in increased expression of A3A, which is shown to be of clinical prognostic relevance.