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BackgroundDNA polymerase theta (POLQ) is a translesion synthesis polymerase essential for the repair of double strand breaks by the error-prone TMEJ (Theta Mediated End Joining) pathway. Although POLQ participates in maintaining genome stability, several studies have shown that its overexpression correlates with cancer progression and poor prognosis. Due to the fact that its role as a biomarker in pancreatic cancer remains unexplored, we aimed to study the usefulness of POLQ H-score as a prognostic factor in a pancreatic cancer patient cohort.MethodsWe evaluated POLQ gene expression using a web-based tool to deliver gene expression profiling and interactive analyses based on TCGA and GTEx (GEPIA) and we examined the POLQ immunostaining in 152 biliopancreatic cancer surgical specimens using tissue microarrays. Association with survival was evaluated by Kaplan Meier curves and uni-multivariate Cox regression.ResultsGEPIA analysis showed statistical differences according to POLQ mRNA levels in Disease Free Survival (DFS) (log rank 0.023, HR 2.8, p=0.029) and Overall Survival (OS) (log rank 0.011, HR 3.1, p=0.016). For immunohistochemistry (IHC) evaluation, POLQ H-score was calculated, and showed statistical differences for OS in Kaplan Meier curves (log rank 0.001) and uni-multivariate analysis (HR 2.27; 95% CI 1.24-4.15, p=0.008).ConclusionsOur results indicate that POLQ is an independent prognostic factor in pancreatic cancer when analyzed by immunostaining, which is in agreement with the results shown by the POLQ gene expression analysis (GEPIA).

Posttranslational modifications of epigenetic modifiers provide a flexible and timely mechanism for rapid adaptations to the dynamic environment of cancer cells. SIRT1 is an NAD+-dependent epigenetic modifier whose activity is classically associated with healthy aging and longevity, but its function in cancer is not well understood. Here, we reveal that 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol), the active metabolite of vitamin D (VD), promotes SIRT1 activation through auto-deacetylation in human colon carcinoma cells, and identify lysine 610 as an essential driver of SIRT1 activity. Remarkably, our data show that the post-translational control of SIRT1 activity mediates the antiproliferative action of 1,25(OH)2D3. This effect is reproduced by the SIRT1 activator SRT1720, suggesting that SIRT1 activators may offer new therapeutic possibilities for colon cancer patients who are VD deficient or unresponsive. Moreover, this might be extrapolated to inflammation and other VD deficiency-associated and highly prevalent diseases in which SIRT1 plays a prominent role.

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7’s role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. The five-year survival rate of CRC patients depends on the stage at diagnosis, being higher than 80% when CRC is diagnosed in the early stages but lower than 10% when CRC is diagnosed in advanced stages. Autoantibodies against specific CRC autoantigens (tumor-associated antigens (TAAs)) in the sera of patients have been widely demonstrated to aid in early diagnosis. Thus, we herein aim to identify autoantigens target of autoantibodies specific to CRC that possess a significant ability to discriminate between CRC patients and healthy individuals by means of liquid biopsy. To that end, we examined the protein content of the exosomes released by five CRC cell lines and tissue samples from CRC patients by means of immunoprecipitation coupled with mass spectrometry analysis. A total of 103 proteins were identified as potential autoantigens specific to CRC. After bioinformatics and meta-analysis, we selected 15 proteins that are more likely to be actual CRC autoantigens in order to evaluate their role in CRC prognosis by Western blot (WB) and immunohistochemistry (IHC). We found dysregulation at the protein level for 11 of these proteins in both tissue and plasma exosome samples from patients, along with an association of nine of these proteins with CRC prognosis. After validation, all but one showed a statistically significant high diagnostic ability to distinguish CRC patients and individuals with premalignant lesions from healthy individuals, either by luminescence Halotag-based beads, or by a multiplexed biosensing platform involving the use of magnetic microcarriers as solid support modified with covalently immobilized Halotag fusion proteins constructed for CRC detection. Taken together, our results highlight the usefulness of the approach defined here to identify the TAAs specific to chronic diseases; they also demonstrate that the measurement of autoantibody levels in plasma against the TAAs identified here could be integrated into a point-of-care (POC) device for CRC detection with high diagnostic ability.

Coordination of gene expression with nutrient availability supports proliferation and homeostasis and is shaped by protein acetylation. Yet how physiological/pathological signals link acetylation to specific gene expression programs and whether such responses are cell-type-specific is unclear. AMP-activated protein kinase (AMPK) is a key energy sensor, activated by glucose limitation to resolve nutrient supply-demand imbalances, critical for diabetes and cancer. Unexpectedly, we show here that, in gastrointestinal cancer cells, glucose activates AMPK to selectively induce EP300, but not CREB-binding protein (CBP). Consequently, EP300 is redirected away from nuclear receptors that promote differentiation towards β-catenin, a driver of proliferation and colorectal tumorigenesis. Importantly, blocking glycogen synthesis permits reactive oxygen species (ROS) accumulation and AMPK activation in response to glucose in previously nonresponsive cells. Notably, glycogen content and activity of the ROS/AMPK/EP300/β-catenin axis are opposite in healthy versus tumor sections. Glycogen content reduction from healthy to tumor tissue may explain AMPK switching from tumor suppressor to activator during tumor evolution.

Metastasis is the primary cause of colorectal cancer (CRC) death. The liver and lung, besides adjacent lymph nodes, are the most common sites of metastasis. Here, we aimed to study the lymph nodes, liver, and lung CRC metastasis by quantitative spatial proteomics analysis using CRC cell-based models that recapitulate these metastases. The isogenic KM12 cell system composed of the non-metastatic KM12C cells, liver metastatic KM12SM cells, and liver and lung metastatic KM12L4a cells, and the isogenic non-metastatic SW480 and lymph nodes metastatic SW620 cells, were used. Cells were fractionated to study by proteomics five subcellular fractions corresponding to cytoplasm, membrane, nucleus, chromatin-bound proteins, and cytoskeletal proteins, and the secretome. Trypsin digested extracts were labeled with TMT 11-plex and fractionated prior to proteomics analysis on a Q Exactive. We provide data on protein abundance and localization of 4710 proteins in their different subcellular fractions, depicting dysregulation of proteins in abundance and/or localization in the most common sites of CRC metastasis. After bioinformatics, alterations in abundance and localization for selected proteins from diverse subcellular localizations were validated via WB, IF, IHC, and ELISA using CRC cells, patient tissues, and plasma samples. Results supported the relevance of the proteomics results in an actual CRC scenario. It was particularly relevant that the measurement of GLG1 in plasma showed diagnostic ability of advanced stages of the disease, and that the mislocalization of MUC5AC and BAIAP2 in the nucleus and membrane, respectively, was significantly associated with poor prognosis of CRC patients. Our results demonstrate that the analysis of cell extracts dilutes protein alterations in abundance in specific localizations that might only be observed studying specific subcellular fractions, as here observed for BAIAP2, GLG1, PHYHIPL, TNFRSF10A, or CDKN2AIP, which are interesting proteins that should be further analyzed in CRC metastasis.

Muscular atrophy is a complex catabolic condition that develops due to several inflammatory-related disorders, resulting in muscle loss. Tumor necrosis factor alpha (TNF-α) is believed to be one of the leading factors that drive inflammatory response and its progression. Until now, the link between inflammation and muscle wasting has been thoroughly investigated, and the non-coding RNA machinery is a potential connection between the candidates. This study aimed to identify specific miRNAs for muscular atrophy induced by TNF-α in the C2C12 murine myotube model. The difference in expression of fourteen known miRNAs and two newly identified miRNAs was recorded by next-generation sequencing between normal muscle cells and treated myotubes. After validation, we confirmed the difference in the expression of one novel murine miRNA (nov-mmu-miRNA-1) under different TNF-α-inducing conditions. Functional bioinformatic analyses of nov-mmu-miRNA-1 revealed the potential association with inflammation and muscle atrophy. Our results suggest that nov-mmu-miRNA-1 may trigger inflammation and muscle wasting by the downregulation of LIN28A/B, an anti-inflammatory factor in the let-7 family. Therefore, TNF-α is involved in muscle atrophy through the modulation of the miRNA cellular machinery. Here, we describe for the first time and propose a mechanism for the newly discovered miRNA, nov-mmu-miRNA-1, which may regulate inflammation and promote muscle atrophy.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplasm with very poor patient survival outcomes despite available treatments. There is an urgent need for new potential treatment options and novel biomarkers for these patients. Delta-like canonical Notch ligand 3 (DLL3) interacts with the Notch receptor and causes inhibition of Notch signaling, which confers a survival advantage to PDAC cells. Thus, DLL3 expression could affect cell survival, and its inhibition could increase a patient’s survival. To test this hypothesis, a survival analysis was conducted using the progression-free and overall survival from two independent datasets of PDAC patients, with one using mRNA z-score levels and the other using the Hscore protein expression level; both were carried out using a log-rank test and plotted using Kaplan–Meier curves. DLL3 at the mRNA expression level showed an association between high mRNA expression and both a longer progression-free survival (PFS) and overall survival (OS) of patients. Then, we designed a retrospective study with resected PDAC samples. Our primary objective with this dataset was to assess the relationship between PFS and OS and DLL3 protein expression. The secondary assessment was to provide a rationale for the use of anti-DLL3-based treatments in combination with immunotherapy that is supported by the link between DLL3 and other factors that are involved in immune checkpoints. The survival analyses revealed a protective effect of high DLL3 protein expression levels in both PFS and OS. Interestingly, high DLL3 protein expression levels were significantly correlated with PD-L1/2 and negatively correlated with NOTCH1. Therefore, DLL3 could be considered a biomarker for better prognosis in resectable PDAC patients as well as a therapeutic biomarker for immunotherapy response. These facts set a rationale for testing anti-DLL3-based treatments either alone or combined with immunotherapy or other NOTCH1 inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating tumor type where a very high proportion of people diagnosed end up dying from cancer. Surgical resection is an option for only about 20% of patients, where the 5-year survival increase ranges from 10 to 25%. In addition to surgical resection, there are adjuvant chemotherapy schemes, such as FOLFIRINOX (a mix of Irinotecan, oxaliplatin, 5-Fluorouraci and leucovorin) or gemcitabine-based treatment. These last two drugs have been compared in the NAPOLI-3 clinical trial, and the NALIRIFOX arm was found to have a higher overall survival (OS) (11.1 months vs. 9.2 months). Despite these exciting improvements, PDAC still has no effective treatment. An interesting approach would be to drive ferroptosis in PDAC cells. A non-apoptotic reactive oxygen species (ROS)-dependent cell death, ferroptosis was first described by Dixon et al. in 2012. ROS are constantly produced in the tumor cell due to high cell metabolism, which is even higher when exposed to chemotherapy. Tumor cells have detoxifying mechanisms, such as Mn-SOD or the GSH-GPX system. However, when a threshold of ROS is exceeded in the tumor cell, the cell’s antioxidant systems are overwhelmed, resulting in lipid peroxidation and, ultimately, ferroptosis. In this review, we point out ferroptosis as an approach to consider in PDAC and propose that altering the cellular ROS balance by combining oxidizing agents or with inhibitors of the main cellular detoxifiers triggers ferroptosis in PDAC.

BackgroundEndoscopic submucosal dissection has become the primary option of treatment for early gastric cancer. However, lymph node metastasis may lead to poor prognosis. We analyzed factors related to lymph node metastasis in EGC patients, and we developed a construction prediction model with machine learning using data from a retrospective series.MethodsTwo independent cohorts’ series were evaluated including 305 patients with EGC from China as cohort I and 35 patients from Spain as cohort II. Five classifiers obtained from machine learning were selected to establish a robust prediction model for lymph node metastasis in EGC.ResultsThe clinical variables such as invasion depth, histologic type, ulceration, tumor location, tumor size, Lauren classification, and age were selected to establish the five prediction models: linear support vector classifier (Linear SVC), logistic regression model, extreme gradient boosting model (XGBoost), light gradient boosting machine model (LightGBM), and Gaussian process classification model. Interestingly, all prediction models of cohort I showed accuracy between 70 and 81%. Furthermore, the prediction models of the cohort II exhibited accuracy between 48 and 82%. The areas under curve (AUC) of the five models between cohort I and cohort II were between 0.736 and 0.830.ConclusionsOur results support that the machine learning method could be used to predict lymph node metastasis in early gastric cancer and perhaps provide another evaluation method to choose the suited treatment for patients.

KRAS mutation is a confirmed predictive biomarker for anti-EGFR monoclonal antibody therapy response for metastatic colorectal cancer. However, its prognosis impact and the predictive potential for first-line standard chemotherapy remains unclear. On the other hand, V600E mutation is the most frequent and studied mutation in the BRAF gene, and it has been associated with a poor outcome of patients and a low response to anti-EGFR treatment. Thus, the aim of this study is to evaluate the role of KRAS and BRAF mutations as prognosis factors and predictive biomarkers for 1st line standard chemotherapy in metastatic colorectal cancer. KRAS mutations and BRAF V600E mutations exhibited a poor outcome (p = 0.021 and p < 0.0001, respectively). Cox multivariate analysis showed that the presence of liver metastasis (HR = 1.595; 95% CI: 1.086−2.343; p = 0.017), KRAS mutation (HR = 1.643; 95% CI: 1.110−2.431; p = 0.013) and BRAF V600E mutation (HR = 5.861; 95% CI: 2.531−13.570; p < 0.0001) were statistically significant co-variables for progression-free survival. Interestingly, patients with KRAS mutations were associated with a poor response to first line standard chemotherapy (p = 0.008). In contrast, the BRAF V600E mutation did not have any impact on the first line standard chemotherapy response (p = 0.540). Therefore, in the present study, we provide new insight on the role of KRAS and BRAF, not only as prognosis biomarkers, but also as first line standard chemotherapy response biomarkers in metastatic colorectal cancer.

Pancreatic ductal adenocarcinoma is an aggressive form of pancreatic cancer and the fourth leading cause of cancer-related death. When possible, curative approaches are based on surgical resection, though not every patient is a candidate for surgery. There are clinical guidelines for the management of these patients that offer different treatment options depending on the clinical and pathologic characteristics. However, the survival rates seen in this kind of patients are still low. The CDSE1 gene is located upstream of NRAS and encodes an RNA-binding protein termed UNR. The aim of this study was to analyze UNR expression and its correlation with outcome in patients with resectable pancreatic ductal adenocarcinoma (PDAC). For this, samples from resectable PDAC patients who underwent duodenopancreatectomy were used to evaluate UNR protein expression by immunohistochemistry using a tissue microarray. Here, we observed that low UNR expression was significantly associated with shorter progression-free survival after surgery (P = 0.010). Moreover, this prognostic marker remained significant after Cox proportional hazards model (P = 0.036). We further studied the role of CDSE1 expression in patient's prognosis using data from public repositories (GEO and TGCA), confirming our results. Interestingly, CDSE1 expression correlated with that of genes characteristic of an immunogenic molecular subtype of pancreatic cancer. Based on these findings, UNR may be considered a potential prognostic biomarker for resectable PDAC and may serve to guide subsequent adjuvant treatment decisions.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of tumours, and its incidence is rising worldwide. Although survival can be improved by surgical resection when these tumours are detected at an early stage, this cancer is usually asymptomatic, and disease only becomes apparent after metastasis. Several risk factors are associated with this disease, the most relevant being chronic pancreatitis, diabetes, tobacco and alcohol intake, cadmium, arsenic and lead exposure, certain infectious diseases, and the mutational status of some genes associated to a familial component. PDAC incidence has increased in recent decades, and there are few alternatives for chemotherapeutic treatment. Endoplasmic reticulum (ER) stress factors such as GRP78/BiP (78 kDa glucose-regulated protein), ATF6α (activating transcription factor 6 isoform α), IRE1α (inositol-requiring enzyme 1 isoform α), and PERK (protein kinase RNA-like endoplasmic reticulum kinase) activate the transcription of several genes involved in both survival and apoptosis. Some of these factors aid in inducing a non-proliferative state in cancer called dormancy. Modulation of endoplasmic reticulum stress could induce dormancy of tumour cells, thus prolonging patient survival. In this systematic review, we have compiled relevant results concerning those endoplasmic reticulum stress factors involved in PDAC, and we have analysed the mechanism of dormancy associated to endoplasmic reticulum stress and its potential use as a chemotherapeutic target against PDAC.