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A structural analysis of the isolated PsbO protein by FTIR spectroscopy, reporting detailed secondary structure quantification and changes in the secondary structure contents of the protein attributed to the effect of calcium (Ca2+), is presented. It is also shown that calcium modulates the structure of PsbO and this effect will be important for further functional studies.

Rationale: Efficient metastasis requires survival and adaptation of tumor cells to stringent conditions imposed by the extracellular milieu. Identification of critical survival signaling pathways in tumor cells might unveil novel targets relevant in disease progression.Objectives: To investigate the contribution of activated protein C (APC) and its receptor (endothelial protein C receptor [EPCR]) in animal models of lung cancer metastasis and in patients with lung adenocarcinoma.Methods: Signaling pathway triggered by APC/EPCR and its relevance in apoptosis was studied in vitro. Functional significance was assessed by silencing and blocking antibodies in several in vivo models of lung cancer metastasis in athymic nude Foxn1(nu) mice. We examined EPCR levels using a microarray dataset of 107 patients. Immunohistochemical analysis was performed in an independent cohort of 295 patients with lung adenocarcinoma.Measurements and Main Results: The effects of APC binding to EPCR rapidly triggered Akt and extracellular signal-regulated kinase signaling pathways, leading to attenuated in vitro apoptosis. In vivo, silencing of EPCR expression or blocking APC/EPCR interaction reduced infiltration in the target organ, resulting in impaired prometastatic activity. Moreover, overexpression of EPCR induced an increased metastatic activity to target organs. Analysis of clinical samples showed a robust association between high EPCR levels and poor prognosis, particularly in stage I patients.Conclusions: EPCR and its ligand APC promote cell survival that contributes to tumor cell endurance to stress favoring prometastatic activity of lung adenocarcinoma. EPCR/APC is a novel target of relevance in the clinical outcome of early-stage lung cancer. [ABSTRACT FROM AUTHOR]

Abstract: DNA copy number aberrations (CNAs) are genetic alterations common in cancer cells. Their transcriptional consequences are still poorly understood. Based on the fact that DNA copy number (CN) is highly correlated with the genomic position, we have applied a segmentation algorithm to gene expression (GE) to explore its relation with CN. We have found a strong correlation between segmented CN (sCN) and segmented GE (sGE), corroborating that CNAs have clear effects on genome-wide expression. We have found out that most of the recurrent regions of sGE are common to those obtained from sCN analysis. Results for two cancer datasets confirm the known targets of aberrations and provide new candidates to study. The suggested methodology allows to find recurrent aberrations specific to sGE, revealing loci where the expression of the genes is independent from their CNs. R code and additional files are available as supplementary material. [Copyright &y& Elsevier]

Assessment and improvement of the reliability of protein-protein interaction (ppi) data is critical for the progress of the currently active research on interactomes. Some interesting questions in this respect are: How three-dimensional (3D) protein structural data is present in known ppi data?, and How this kind of information can be used to validate and improve the interactomes? To address this problem, analysis and unification of six structural domain-domain interaction (sddi) datasets is presented; followed by a comparative study of these sddi data in three ppi reference sets produced at different levels of confidence. The results show that protein structural and interactomic data are partially complementary and that a larger proportion of structural information is observed in more confident interactomes. We also present, focused on the human interactome, an analysis of the domains that are more frequently present in: (i) an interactome based on validation by at least two experimental methods versus (ii) another interactome based on validation by 3D structural interaction data. These results allow to distinguish between domain pairs associated to protein interactions supported by 3D structures and domain pairs that at present are not supported by structural information. The domain pairs exclusive of interactions without associated 3D data reveal interacting conserved modules that are probably flexible, disordered, and difficult to crystallize; and which are often found in proteins involved in signaling pathways and DNA processing. Proteins 2010. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The structure and function of the photosystem II PsbO extrinsic protein is under intense research, being an essential part of the biomolecular engine that carries out water oxidation and oxygen production. This paper presents a structural analysis of the isolated PsbO protein by FTIR spectroscopy, reporting detailed secondary structure quantification and changes in the secondary structure content of the protein attributed to the effect of calcium (Ca[sup 2+]). Measurements in H[sub 2]O and D[sub 2]O have allowed us to see the effect of calcium on the conformation of the protein. The results indicate that (i) the protein presents a major content of β-structure (i.e., β-sheet, β-strands, β-turns) as detected by the infrared bands at 16241625, 1678-1679, 1688-1689 cm[sup -1], which account for about 38% in water and 33% in heavy water, in the presence of calcium; and (ii) the amount of this β-structure fraction increases 7-10% in the absence of calcium, with a concomitant decrease in loops and nonordered structure. The thermal denaturation profile of the protein in the presence of calcium showed low stability with T[sub m] ∼56 °C. This profile also shows a second phase of denaturation above 60 °C and the appearance of aggregation signals above 70 °C. Our observations indicate that calcium is able to modify the conformation of the protein at least in solution and confirm that PsbO is mainly a β-protein where β-sheet is the major ordered secondary structure element of the protein core. [ABSTRACT FROM AUTHOR]

Abstract: We used manual macrodissection or laser capture microdissection (LCM) to isolate tissue sections of the hippocampus area of Ras-GRF1 wild type and knockout mice brains, and analyzed their transcriptional patterns using commercial oligonucleotide microarrays. Comparison between the transcriptomes of macrodissected and microdissected samples showed that the LCM samples allowed detection of significantly higher numbers of differentially expressed genes, with higher statistical rates of significance. These results validate LCM as a reliable technique for in vivo genomic studies in the brain hippocampus, where contamination by surrounding areas (not expressing Ras-GRF1) increases background noise and impairs identification of differentially expressed genes. Comparison between wild type and knockout LCM hippocampus samples revealed that Ras-GRF1 elimination caused significant gene expression changes, mostly affecting signal transduction and related neural processes. The list of 36 most differentially expressed genes included loci concerned mainly with Ras/G protein signaling and cytoskeletal organization (i.e. 14-3-3γ/ζ, Kcnj6, Clasp2) or related, cross-talking pathways (i.e. jag2, decorin, strap). Consistent with the phenotypes shown by Ras-GRF1 knockout mice, many of these differentially expressed genes play functional roles in processes such as sensory development and function (i.e. Sptlc1, antiquitin, jag2) and/or neurological development/neurodegeneration processes affecting memory and learning. Indeed, potential links to neurodegenerative diseases such as Alzheimer disease (AD) or Creutzfeldt-Jacobs disease (CJD), have been reported for a number of differentially expressed genes identified in this study (Ptma, Aebp2, Clasp2, Hebp1, 14-3-3γ/ζ, Csnk1δ, etc.). These data, together with the previously described role of IRS and insulin (known Ras-GRF1 activators) in AD, warrant further investigation of a potential functional link of Ras-GRF1 to neurodegenerative processes. [Copyright &y& Elsevier]

The consensus molecular subtypes (CMSs) classification of colorectal cancer (CRC) is a system for patient stratification that can be potentially applied to therapeutic decisions. Hakai (CBLL1) is an E3 ubiquitin-ligase that induces the ubiquitination and degradation of E-cadherin, inducing epithelial-to-mesenchymal transition (EMT), tumour progression and metastasis. Using bioinformatic methods, we have analysed CBLL1 expression on a large integrated cohort of primary tumour samples from CRC patients. The cohort included survival data and was divided into consensus molecular subtypes. Colon cancer tumourspheres were used to analyse the expression of stem cancer cells markers via RT-PCR and Western blotting. We show that CBLL1 gene expression is specifically associated with canonical subtype CMS2. WNT target genes LGR5 and c-MYC show a similar association with CMS2 as CBLL1. These mRNA levels are highly upregulated in cancer tumourspheres, while CBLL1 silencing shows a clear reduction in tumoursphere size and in stem cell biomarkers. Importantly, CMS2 patients with high CBLL1 expression displayed worse overall survival (OS), which is similar to that associated with CMS4 tumours. Our findings reveal CBLL1 as a specific biomarker for CMS2 and the potential of using CMS2 with high CBLL1 expression to stratify patients with poor OS.

Background: Spinal Muscular Atrophy (SMA) and Amyotrophic Lateral Sclerosis (ALS) share phenotypic and molecular commonalities, including the fact that they can be caused by mutations in ubiquitous proteins involved in RNA metabolism, namely SMN, TDP-43 and FUS. Although this suggests the existence of common disease mechanisms, there is currently no model to explain the resulting motor neuron dysfunction. In this work we generated a parallel set of Drosophila models for adult-onset RNAi and tagged neuronal expression of the fly orthologues of the three human proteins, named Smn, TBPH and Caz, respectively. We profiled nuclear and cytoplasmic bound mRNAs using a RIP-seq approach and characterized the transcriptome of the RNAi models by RNA-seq. To unravel the mechanisms underlying the common functional impact of these proteins on neuronal cells, we devised a computational approach based on the construction of a tissue-specific library of protein functional modules, selected by an overall impact score measuring the estimated extent of perturbation caused by each gene knockdown., Results: Transcriptome analysis revealed that the three proteins do not bind to the same RNA molecules and that only a limited set of functionally unrelated transcripts is commonly affected by their knock-down. However, through our integrative approach we were able to identify a concerted effect on protein functional modules, albeit acting through distinct targets. Most strikingly, functional annotation revealed that these modules are involved in critical cellular pathways for motor neurons, including neuromuscular junction function. Furthermore, selected modules were found to be significantly enriched in orthologues of human neuronal disease genes., Conclusions: The results presented here show that SMA and ALS disease-associated genes linked to RNA metabolism functionally converge on neuronal protein complexes, providing a new hypothesis to explain the common motor neuron phenotype. The functional modules identified represent promising biomarkers and therapeutic targets, namely given their alteration in asymptomatic settings., (© 2023. BioMed Central Ltd., part of Springer Nature.)

The initial steps of B-cell acute lymphoblastic leukemia (B-ALL) development usually pass unnoticed in children. Several preclinical studies have shown that exposure to immune stressors triggers the transformation of preleukemic B cells to full-blown B-ALL, but how this takes place is still a longstanding and unsolved challenge. Here we show that dysregulation of innate immunity plays a driving role in the clonal evolution of pre-malignant Pax5 +/- B-cell precursors toward leukemia. Transcriptional profiling reveals that Myd88 is downregulated in immune-stressed pre-malignant B-cell precursors and in leukemic cells. Genetic reduction of Myd88 expression leads to a significant increase in leukemia incidence in Pax5 +/- Myd88 +/- mice through an inflammation-dependent mechanism. Early induction of Myd88-independent Toll-like receptor 3 signaling results in a significant delay of leukemia development in Pax5 +/- mice. Altogether, these findings identify a role for innate immunity dysregulation in leukemia, with important implications for understanding and therapeutic targeting of the preleukemic state in children., (© 2023. Springer Nature Limited.)

Newly growing evidence highlights the essential role that epitranscriptomic marks play in the development of many cancers; however, little is known about the role and implications of altered epitranscriptome deposition in prostate cancer. Here, we show that the transfer RNA N 7 -methylguanosine (m 7 G) transferase METTL1 is highly expressed in primary and advanced prostate tumours. Mechanistically, we find that METTL1 depletion causes the loss of m 7 G tRNA methylation and promotes the biogenesis of a novel class of small non-coding RNAs derived from 5'tRNA fragments. 5'tRNA-derived small RNAs steer translation control to favour the synthesis of key regulators of tumour growth suppression, interferon pathway, and immune effectors. Knockdown of Mettl1 in prostate cancer preclinical models increases intratumoural infiltration of pro-inflammatory immune cells and enhances responses to immunotherapy. Collectively, our findings reveal a therapeutically actionable role of METTL1-directed m 7 G tRNA methylation in cancer cell translation control and tumour biology., (© 2023. The Author(s).)

In the last two decades, many detailed full transcriptomic studies on complex biological samples have been published and included in large gene expression repositories. These studies primarily provide a bulk expression signal for each sample, including multiple cell-types mixed within the global signal. The cellular heterogeneity in these mixtures does not allow the activity of specific genes in specific cell types to be identified. Therefore, inferring relative cellular composition is a very powerful tool to achieve a more accurate molecular profiling of complex biological samples. In recent decades, computational techniques have been developed to solve this problem by applying deconvolution methods, designed to decompose cell mixtures into their cellular components and calculate the relative proportions of these elements. Some of them only calculate the cell proportions (supervised methods), while other deconvolution algorithms can also identify the gene signatures specific for each cell type (unsupervised methods). In these work, five deconvolution methods (CIBERSORT, FARDEEP, DECONICA, LINSEED and ABIS) were implemented and used to analyze blood and immune cells, and also cancer cells, in complex mixture samples (using three bulk expression datasets). Our study provides three analytical tools (corrplots, cell-signature plots and bar-mixture plots) that allow a thorough comparative analysis of the cell mixture data. The work indicates that CIBERSORT is a robust method optimized for the identification of immune cell-types, but not as efficient in the identification of cancer cells. We also found that LINSEED is a very powerful unsupervised method that provides precise and specific gene signatures for each of the main immune cell types tested: neutrophils and monocytes (of the myeloid lineage), B-cells, NK cells and T-cells (of the lymphoid lineage), and also for cancer cells.

Chromosome instability is a well-known hallmark of cancer, leading to increased genetic plasticity of tumoral cells, which favors cancer aggressiveness, and poor prognosis. One of the main sources of chromosomal instability are events that lead to a Whole-Genome Duplication (WGD) and the subsequently generated cell polyploidy. In recent years, several studies showed that WGD occurs at the early stages of cell transformation, which allows cells to later become aneuploid, thus leading to cancer progression. On the other hand, other studies convey that polyploidy plays a tumor suppressor role, by inducing cell cycle arrest, cell senescence, apoptosis, and even prompting cell differentiation, depending on the tissue cell type. There is still a gap in understanding how cells that underwent WGD can overcome the deleterious effect on cell fitness and evolve to become tumoral. Some laboratories in the chromosomal instability field recently explored this paradox, finding biomarkers that modulate polyploid cells to become oncogenic. This review brings a historical view of how WGD and polyploidy impact cell fitness and cancer progression, and bring together the last studies that describe the genes helping cells to adapt to polyploidy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sanz-Gómez, González-Álvarez, De Las Rivas and de Cárcer.)

Rheumatic diseases are high prevalence pathologies with different etiology and evolution and low sensitivity in clinical diagnosis. Therefore, it is necessary to develop an early diagnosis method which allows personalized treatment, depending on the specific pathology. The biology/disease initiative, at Human Proteome Project, is an integrative approach to identify relevant proteins in the human proteome associated with pathologies. A previously reported literature data mining analysis, which identified proteins related to osteoarthritis (OA), rheumatoid arthritis (RA), and psoriatic arthritis (PSA) was used to establish a systematic prioritization of potential biomarkers candidates for further evaluation by functional proteomics studies. The aim was to study the protein profile of serum samples from patients with rheumatic diseases such as OA, RA, and PSA. To achieve this goal, customized antibody microarrays (containing 151 antibodies targeting 121 specific proteins) were used to identify biomarkers related to early and specific diagnosis in a screening of 960 serum samples (nondepleted) (OA, n = 480; RA, n = 192; PSA, n = 288). This functional proteomics screening has allowed the determination of a panel (30 serum proteins) as potential biomarkers for these rheumatic diseases, displaying receiver operating characteristics curves with area under the curve values of 80-90%.

Motivation: Modern genomic technologies allow us to perform genome-wide analysis to find gene markers associated with the risk and survival in cancer patients. Accurate risk prediction and patient stratification based on robust gene signatures is a key path forward in personalized treatment and precision medicine. Several authors have proposed the identification of gene signatures to assign risk in patients with breast cancer (BRCA), and some of these signatures have been implemented within commercial platforms in the clinic, such as Oncotype and Prosigna. However, these platforms are black boxes in which the influence of selected genes as survival markers is unclear and where the risk scores provided cannot be clearly related to the standard clinicopathological tumor markers obtained by immunohistochemistry (IHC), which guide clinical and therapeutic decisions in breast cancer., Results: Here, we present a framework to discover a robust list of gene expression markers associated with survival that can be biologically interpreted in terms of the three main biomolecular factors (IHC clinical markers: ER, PR and HER2) that define clinical outcome in BRCA. To test and ensure the reproducibility of the results, we compiled and analyzed two independent datasets with a large number of tumor samples (1024 and 879) that include full genome-wide expression profiles and survival data. Using these two cohorts, we obtained a robust subset of gene survival markers that correlate well with the major IHC clinical markers used in breast cancer. The geneset of survival markers that we identify (which includes 34 genes) significantly improves the risk prediction provided by the genesets included in the commercial platforms: Oncotype (16 genes) and Prosigna (50 genes, i.e. PAM50). Furthermore, some of the genes identified have recently been proposed in the literature as new prognostic markers and may deserve more attention in current clinical trials to improve breast cancer risk prediction., Availability and Implementation: All data integrated and analyzed in this research will be available on GitHub (https://github.com/jdelasrivas-lab/breastcancersurvsign), including the R scripts and protocols used for the analyses., Supplementary Information: Supplementary data are available at Bioinformatics Advances online., (© The Author(s) 2023. Published by Oxford University Press.)

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Studies of CLL antibody reactivity have shown differential targets to autoantigens and antimicrobial molecular motifs that support the current hypothesis of CLL pathogenesis., Methods: In this study, we conducted a quantitative serum analysis of 7 immunoglobulins in CLL and monoclonal B-cell lymphocytosis (MBL) patients (bead-suspension protein arrays) and a serological profile (IgG and IgM) study of autoantibodies and antimicrobial antigens (protein microarrays)., Results: Significant differences in the IgA levels were observed according to disease progression and evolution as well as significant alterations in IgG1 according to IGHV mutational status. More representative IgG autoantibodies in the cohort were against nonmutagenic proteins and IgM autoantibodies were against vesicle proteins. Antimicrobial IgG and IgM were detected against microbes associated with respiratory tract infections., Conclusions: Quantitative differences in immunoglobulin serum levels could be potential biomarkers for disease progression. In the top 5 tumoral antigens, we detected autoantibodies (IgM and IgG) against proteins related to cell homeostasis and metabolism in the studied cohort. The top 5 microbial antigens were associated with respiratory and gastrointestinal infections; moreover, the subsets with better prognostics were characterized by a reactivation of Cytomegalovirus. The viral humoral response could be a potential prognosis biomarker for disease progression.

Background: Eltrombopag (EP) is a small molecule that acts directly on hematopoietic stem cells (HSCs) and megakaryocytes to stimulate the hematopoietic process. Mesenchymal stem/stromal cells (MSCs) are key hematopoietic niche regulators., Objectives: We aimed to determine whether EP has any effect on MSC function and properties (especially on their hematopoietic-supporting ability) and if so, what changes (e.g. genome-wide transcriptomic alterations) are induced in MSC after EP treatment., Design/methods: MSCs were isolated from 12 healthy donors and treated with 15 µM and 50 µM of EP for 24 h. The toxicity of the drug on MSCs and their differentiation ability were analyzed, as well as the transcriptomic profile, reactive oxygen species (ROS) and DNA damage and the changes induced in the clonogenic capacity of HSCs., Results: The results show that EP also modifies MSC functions, decreasing their adipogenic differentiation, increasing the expression of genes involved in hypoxia and other pathways related to oxygen homeostasis, and enhancing their ability to support hematopoiesis in vitro ., Conclusion: Our findings support the use of EP in cases where hematopoiesis is defective, despite its well-known direct effects on hematopoietic cells. Our findings suggest that further studies on the effects of EP on MSCs from patients with aplastic anemia are warranted., Competing Interests: FS-G has received honoraria and/or research support from Celgene/BMS, Kite/Gilead, Novartis, Pfizer, Roche and Takeda. MD-C has received honoraria and/or research support from Celgene/BMS, Novartis and Takeda. The remaining authors declare no conflict of interests., (© The Author(s), 2022.)

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBL hi )], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBL hi ) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBL hi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Landeira-Viñuela, Arias-Hidalgo, Juanes-Velasco, Alcoceba, Navarro-Bailón, Pedreira, Lecrevisse, Díaz-Muñoz, Sánchez-Santos, Hernández, García-Vaquero, Góngora, De Las Rivas, González, Orfao and Fuentes.)

Protein-protein interactions (PPI) play an essential role in the biological processes that occur in the cell. Therefore, the dissection of PPI networks becomes decisive to model functional coordination and predict pathological de-regulation. Cellular networks are dynamic and proteins display varying roles depending on the tissue-interactomic context. Thus, the use of centrality measures in individual proteins fall short to dissect the functional properties of the cell. For this reason, there is a need for more comprehensive, relational, and context-specific ways to analyze the multiple actions of proteins in different cells and identify specific functional assemblies within global biomolecular networks. Under this framework, we define Biological Interacting units (BioInt-U) as groups of proteins that interact physically and are enriched in a common Gene Ontology. A search strategy was applied on 33 tissue-specific (TS) PPI networks to generate BioInt libraries associated with each particular human tissue. The cross-tissue comparison showed that housekeeping assemblies incorporate different proteins and exhibit distinct network properties depending on the tissue. Furthermore, disease genes (DGs) of tissue-associated pathologies preferentially accumulate in units in the expected tissues, which in turn were more central in the TS networks. Overall, the study reveals a tissue-specific functional diversification based on the identification of specific protein units and suggests vulnerabilities specific of each tissue network, which can be applied to refine protein-disease association methods., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

Preventing development of childhood B-cell acute lymphoblastic leukemia (B-ALL), a disease with devastating effects, is a longstanding and unsolved challenge. Heterozygous germline alterations in the PAX5 gene can lead to B-ALL upon accumulation of secondary mutations affecting the JAK/STAT signaling pathway. Preclinical studies have shown that this malignant transformation occurs only under immune stress such as exposure to infectious pathogens. Here we show in Pax5+/- mice that transient, early-life administration of clinically relevant doses of ruxolitinib, a JAK1/2 inhibitor, significantly mitigates the risk of B-ALL following exposure to infection; 1 of 29 animals treated with ruxolitinib developed B-ALL versus 8 of 34 untreated mice. Ruxolitinib treatment preferentially targeted Pax5+/- versus wild-type B-cell progenitors and exerted unique effects on the Pax5+/- B-cell progenitor transcriptional program. These findings provide the first in vivo evidence for a potential strategy to prevent B-ALL development., Significance: JAK/STAT inhibition suppresses tumorigenesis in a B-ALL-susceptible mouse model, presenting a novel approach to prevent B-ALL onset., (©2022 The Authors; Published by the American Association for Cancer Research.)

The epithelial-mesenchymal transition (EMT) is associated with tumor aggressiveness and increased invasion, migration, metastasis, angiogenesis, and drug resistance. Although the HCT116 p21-/- cell line is well known for its EMT-associated phenotype, with high Vimentin and low E-cadherin protein levels, the gene signature of this rather intermediate EMT-like cell line has not been determined so far. In this work, we present a robust molecular and bioinformatics analysis, to reveal the associated gene expression profile and its correlation with different types of colorectal cancer tumors. We compared the quantitative signature obtained with the NanoString platform with the expression profiles of colorectal cancer (CRC) Consensus Molecular Subtypes (CMS) as identified, and validated the results in a large independent cohort of human tumor samples. The expression signature derived from the p21-/- cells showed consistent and reliable numbers of upregulated and downregulated genes, as evaluated with two machine learning methods against the four CRC subtypes (i.e., CMS1, 2, 3, and 4). High concordance was found between the upregulated gene signature of HCT116 p21-/- cells and the signature of the CMS4 mesenchymal subtype. At the same time, the upregulated gene signature of the native HCT116 cells was similar to that of CMS1. Using a multivariate Cox regression model to analyze the survival data in the CRC tumor cohort, we selected genes that have a predictive risk power (with a significant gene risk incidence score). A set of genes of the mesenchymal signature was proven to be significantly associated with poor survival, specifically in the CMS4 CRC human cohort. We suggest that the gene signature of HCT116 p21-/- cells could be a suitable metric for mechanistic studies regarding the CMS4 signature and its functional consequences in CRC. Moreover, this model could help to discover the molecular mechanisms of intermediate EMT, which is known to be associated with extraordinarily high stemness and drug resistance.

Hypoxia, a hallmark of solid tumors, determines the selection of invasive and aggressive malignant clones displaying resistance to radiotherapy, conventional chemotherapy or targeted therapy. The recent introduction of immunotherapy, based on immune checkpoint inhibitors (ICPIs) and chimeric antigen receptor (CAR) T-cells, has markedly transformed the prognosis in some tumors but also revealed the existence of intrinsic or acquired drug resistance. In the current review we highlight hypoxia as a culprit of immunotherapy failure. Indeed, multiple metabolic cross talks between tumor and stromal cells determine the prevalence of immunosuppressive populations within the hypoxic tumor microenvironment and confer upon tumor cells resistance to ICPIs and CAR T-cells. Notably, hypoxia-triggered angiogenesis causes immunosuppression, adding another piece to the puzzle of hypoxia-induced immunoresistance. If these factors concurrently contribute to the resistance to immunotherapy, they also unveil an unexpected Achille's heel of hypoxic tumors, providing the basis for innovative combination therapies that may rescue the efficacy of ICPIs and CAR T-cells. Although these treatments reveal both a bright side and a dark side in terms of efficacy and safety in clinical trials, they represent the future solution to enhance the efficacy of immunotherapy against hypoxic and therapy-resistant solid tumors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)