Niemira M, Skwarska A, Chwialkowska K
… +12 more, Ostrowska A, Sokolowska G, Zeller A, Erol A, Eljaszewicz A, Hanczaruk B, Michalska-Falkowska A, Tarasik A, Reszec-Gielazyn J, Knapp P, Moniuszko M, Kretowski A
Deamination of adenosine to inosine (A-to-I) in double-stranded microRNAs (miRNAs) has been demonstrated to affect their function as suppressors or oncogenes in various cancers. Nevertheless, the functional impact of miR...Deamination of adenosine to inosine (A-to-I) in double-stranded microRNAs (miRNAs) has been demonstrated to affect their function as suppressors or oncogenes in various cancers. Nevertheless, the functional impact of miRNA editing in high-grade serous ovarian cancer (HGSOC) remains largely unexplored. Here, we identified A-to-I editing in miRNAs in 60 HGSOC tissues and 48 ovarian tissues received in nononcological procedures using small RNA sequencing (RNA-Seq). To investigate the functional impact of A-to-I modifications, we tested the effect of edited RNA mimics and small interfering RNA (siRNA)-mediated downregulation of the RNA-editing enzyme double-stranded RNA-specific editase Adar (ADAR1) on cell proliferation, migration and three-dimensional (3D) growth of HGSOC cells in vitro. Tumour suppressor miR-200b-3p was the most overedited miRNA in HGSOC tumours, and the increased editing level was associated with statistically significant worse overall survival (OS). Mechanistically, in contrast to wild-type miRNA, edited miR-200b-3p promoted cell proliferation, migration and formation of 3D spheroids. Loss of function of ADAR1 profoundly repressed proliferation, migration and 3D growth of HGSOC cells. RNA-Seq and Gene Set Enrichment Analysis (GSEA) analysis revealed that, whereas wild-type miR-200b-3p induced the apoptosis pathway, edited miR-200b-3p substantially inhibited cell-cycle-related pathways. Bioinformatic prediction revealed that edited miR-200b-3p gained the function to repress the expression of new targets, including tumour suppressor MAX interactor 1, dimerisation protein (MXI1), which was associated with a statistically significantly worse OS time in HGSOC patients. Our study reports the potential contribution of edited miR-200b-3p in HGSOC progression, and highlights its potential as a new therapeutic target.
Merkel cell carcinoma (MCC) is a highly aggressive disease with the poorest prognosis among skin cancers, originally posited to be derived from Merkel cells. Emerging evidence, however, suggests other potential origins f...Merkel cell carcinoma (MCC) is a highly aggressive disease with the poorest prognosis among skin cancers, originally posited to be derived from Merkel cells. Emerging evidence, however, suggests other potential origins for MCC, including hematological lineages. We utilized targeted and multi-omics approaches to explore gene expression patterns at protein and RNA levels of MCCs. Western blotting, immunofluorescence, and immunohistochemistry were performed using fresh and 92 FFPE samples of primary and metastatic MCC, and two MCC cell lines (MS-1, HaCaT). RNA sequencing of selected FFPE samples identified differentially expressed genes based on sex and Merkel cell polyomavirus (MCPyV) status. Finally, weighted gene correlation network analysis (WGCNA) and cell type enrichment analyses were employed to determine pathway and cell type enrichment, respectively. MCC patient samples heterogeneously expressed B-cell and neuroendocrine markers and novel molecular targets including BCMA, CD10, CD93, PAX5, TdT, IgA, and CD19. Transcriptome analysis demonstrated differentially expressed genes based on sex and MCPyV status. MCPyV+ tumors had significant upregulation of genes involved in immune cell function and downregulation of processes related to neuronal activity. WGCNA highlighted enrichment for pathways involved in immune function, including B-cell differentiation. Cell type enrichment analysis highlighted enrichment for multipotent stem cells, several immune cell types, and keratinocytes. Our findings support previous studies which confirm that MCC is unlikely to be derived from Merkel cells and instead from multiple or divergent cell types, including those of B-cell lineage. Our work highlights the need for a more personalized approach to diagnosis/characterization and treatment of MCCs, given the documented variability of novel potentially targetable pathways.
Tumor-associated macrophages (TAMs) in brain tumors contain two types of macrophages: tumor-associated microglia and infiltrating macrophages. This study explored whether these two populations have the same role in brain...Tumor-associated macrophages (TAMs) in brain tumors contain two types of macrophages: tumor-associated microglia and infiltrating macrophages. This study explored whether these two populations have the same role in brain tumor progression. In an in vitro coculture model using the astrocytoma cells ALTS1C1 with either the microglial cell line BV2 or the peripheral macrophage cell line RAW264.7, only BV2, not RAW264.7, gathers ALTS1C1 into tumor cell clusters. These BV2-associated clusters limited ALTS1C1 proliferation but not BV2 cell growth. The in vivo studies show that the survival time of mice co-inoculated with ALTS1C1 and BV2 was prolonged from 30.4 ± 3.1 days to more than 77 days in immune-competent mice but not in immune-compromised mice. Examining the tumor microenvironment (TME) by immunohistochemical staining revealed that the co-inoculation of BV2 increased the CD8 T cells' infiltration and the expression of Granzyme B. Mice bearing with BV2-containing ALTS1C1 tumor exhibited a reduced level of circulating myeloid-derived suppressor cells (MDSCs) and an elevated level of CD8 T cells in peripheral blood compared to the ALTS1C1 tumor-bearing group. This study suggests tumor-associated microglia restrict brain tumor development by limiting tumor cell proliferation and inducing T-cell-associated antitumor immunity.
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive and lethal malignancy with limited treatment options, a fact that underscores the urgent need for more effective therapies to improve patient outcomes. Preclin...Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive and lethal malignancy with limited treatment options, a fact that underscores the urgent need for more effective therapies to improve patient outcomes. Preclinical studies have shown promise for αV integrin-targeted therapies; however, clinical trials have been disappointing, highlighting the need for further research. In this study, we demonstrate that integrin subunit alpha V (ITGAV) signals through both mothers against decapentaplegic homolog 4 (SMAD4)-dependent or SMAD4-independent pathways, depending on the genetic context. In SMAD4-positive PDAC cells, ITGAV contributes to the transforming growth factor-beta (TGF-β) signaling pathway to regulate proliferation, migration, and invasion. Conversely, in SMAD4-negative PDAC cells, ITGAV influences only proliferation and migration via activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) pathway. High levels of ITGAV are also associated with poor prognostic outcomes in SMAD4 wild-type patients but are not prognostic in SMAD4 mutant patients. Thus, ITGAV contributes to different patterns of PDAC progression. These findings suggest that stratifying PDAC patients based on both SMAD4 status and ITGAV expression could inform more effective integrin-targeted treatment strategies.
There is a lack of genetic biomarkers for predicting response to immune checkpoint blockade (ICB) therapy in cancer. The recent discovery that loss-of-function mutations in the gene encoding the protein phosphatase 2A (P...There is a lack of genetic biomarkers for predicting response to immune checkpoint blockade (ICB) therapy in cancer. The recent discovery that loss-of-function mutations in the gene encoding the protein phosphatase 2A (PP2A) scaffold protein PPP2R1A confer sensitivity to immune checkpoint blockade in ovarian clear cell carcinoma, therefore represents a breakthrough. Mechanistically, mutations in the PPP2R1A gene induce a strong interferon gamma response in tumor cells, which enhances infiltration of activated CD8+ T cells into the tumor. The activity of these T cells is then fortified by ICB. Furthermore, preclinical studies have shown that PP2A inhibition leads to the generation of neoantigens by disrupting RNA splicing, and PP2A inhibition can remodel the immune microenvironment of tumors to enhance responses to ICB. The finding that loss-of-function PPP2R1A mutations predict benefit from immunotherapy also suggests that pharmacological inhibition of PP2A may act synergistically with ICB therapy.
Cisplatin is one of the most used anticancer chemotherapy agents; however, over time, patients develop resistance to the treatment, and survival rates drop dramatically. Investigation of tumor cell resistance mechanisms...Cisplatin is one of the most used anticancer chemotherapy agents; however, over time, patients develop resistance to the treatment, and survival rates drop dramatically. Investigation of tumor cell resistance mechanisms could increase sensitivity and prevent cancer progression. Here, we investigated the role of the E3 ubiquitin ligase SCF (β-TrCP) in cisplatin resistance in different tumor cell lines, analyzing its role in the stability of BRCA1 and CtIP, proteins involved in DNA damage repair by homologous recombination. We showed that SCF(β-TrCP) plays a key role in cisplatin response, as overexpression of wild-type β-TrCP increased DNA damage and cisplatin-induced apoptosis, while overexpression of a dominant-negative mutant or siRNA-mediated downregulation of β-TrCP decreased the damage and conferred treatment resistance. Furthermore, we demonstrated that BRCA1 and CtIP interacted with β-TrCP in vivo, and their levels changed when β-TrCP expression was modulated. We also described that β-TrCP-mediated BRCA1 degradation involves both lysosomal and proteasomal pathways. Mechanistically, the failure of β-TrCP to regulate the degradation of BRCA1 enables a more efficient DNA damage repair and thereby the acquisition of cisplatin resistance. Overall, β-TrCP overexpression sensitizes cisplatin-induced DNA damage by depleting BRCA1.
Lung adenocarcinoma (LUAD), the most common subtype of non-small-cell lung cancer (NSCLC), is often driven by mutations, particularly in epidermal growth factor receptor (EGFR), that guide targeted therapy choices. Howev...Lung adenocarcinoma (LUAD), the most common subtype of non-small-cell lung cancer (NSCLC), is often driven by mutations, particularly in epidermal growth factor receptor (EGFR), that guide targeted therapy choices. However, resistance to these treatments remains a major clinical challenge. Raf kinase inhibitory protein (RKIP), encoded by the PEBP1 gene, a metastasis suppressor, modulates key oncogenic pathways and may influence tumor aggressiveness and therapy response. Yet, its specific role in NSCLC remains unclear. This study investigates the influence of RKIP expression on NSCLC aggressiveness and explores its impact on therapy response, particularly to EGFR-targeted therapies. In silico analyses revealed that lower RKIP mRNA expression correlates with poorer survival outcomes in LUAD patients but not in other NSCLC subtypes. Genetic modulation of RKIP expression in LUAD cell lines demonstrated that its overexpression reduced migration, spheroid integrity, and suppressed tumor growth, whereas RKIP knockout had opposite effects, particularly in vivo. Expression profiling showed that RKIP overexpression impacts the activation of mitogen-activated protein kinase (MAPK), RAC serine/threonine-protein kinase (AKT), and signal transducer and activator of transcription 3 (STAT3) pathways, as well as processes related to extracellular matrix regulation and inflammatory responses. Importantly, in vitro and in vivo experiments demonstrated that RKIP overexpression sensitizes cells to anti-EGFR treatments, whereas RKIP knockout diminished their sensitivity. Overall, our findings indicate that RKIP modulates LUAD progression and response to EGFR-targeted therapies, although its clinical value as a biomarker requires further validation. These findings highlight RKIP's potential in overcoming therapeutic resistance and the need for further investigation into its regulatory mechanisms.
Spatial transcriptomics (ST) has emerged as a powerful tool to map gene expression patterns to the local tissue structure in cancer, enabling unprecedented insights into cellular heterogeneity and tumour microenvironment...Spatial transcriptomics (ST) has emerged as a powerful tool to map gene expression patterns to the local tissue structure in cancer, enabling unprecedented insights into cellular heterogeneity and tumour microenvironments. As the technology matures, developing new, spatially informed analytical frameworks will be essential to fully leverage its potential to elucidate the complex organisation and emerging properties of cancer tissues. Here, we highlight key challenges in cancer spatial transcriptomics, focusing on three emerging topics: (a) defining cell states, (b) delineating cellular niches and (c) integrating spatial data with other modalities that can pave the way towards clinical translation. We discuss multiple analytical approaches that are currently implemented or could be adapted in the future in order to tackle these challenges, including classical biostatistics methods as well as methods inherited from geospatial analytics or artificial intelligence. In the rapidly expanding landscape of ST, such methodologies lay the foundation for biological discoveries that conceptualise cancer as an evolving system of interconnected niches.
Quezada Urban R, Keerthikumar S, Clark A
… +10 more, Wang H, Phipson B, Bakshi A, Ryan A, Thorne H, Taylor RA, Lawrence MG, Risbridger GP, Toivanen R, Goode DL
Neuroendocrine prostate cancer (NEPC) tumours are classified by pathology into several distinct subtypes. Gene expression profiling has revealed transcriptional heterogeneity across NEPC, but this is rarely considered in...Neuroendocrine prostate cancer (NEPC) tumours are classified by pathology into several distinct subtypes. Gene expression profiling has revealed transcriptional heterogeneity across NEPC, but this is rarely considered in the context of variation between pathologies. Diagnosis typically relies on immunohistochemical markers (CHGA, SYP, NCAM1) and genomic alterations in RB1, PTEN and TP53. We hypothesized that NEPC pathologies have unique transcriptional features. Single-cell RNA sequencing of 18 632 tumour cells from nine patient-derived xenograft models representing five pathologies (small-cell and large-cell neuroendocrine carcinomas, focal neuroendocrine differentiation (Focal NED), low-grade neuroendocrine and amphicrine) demonstrated pathway-specific enrichment. Focal NED and amphicrine tumours exhibited cellular subpopulations enriched for KRAS, IL2-STAT5 and TNF signalling pathways, absent in small- and large-cell carcinomas, which were instead enriched for Myc and E2F pathways. Furthermore, focal NED cells exhibited minimal clonal divergence from adjacent adenocarcinoma cells, while small cell carcinoma cells were clonally distinct. These data underscore significant transcriptional variation among NEPC pathologies, highlighting focal NED's unique biological context and its clinical implications.
Kinase inhibitors have achieved great success in targeted cancer therapy, yet the evident limitations in their effectiveness persist due to therapeutic resistance. To gain insight into the molecular mechanisms and thwart...Kinase inhibitors have achieved great success in targeted cancer therapy, yet the evident limitations in their effectiveness persist due to therapeutic resistance. To gain insight into the molecular mechanisms and thwart resistance, we profiled the time-resolved nascent protein perturbations in response to drug therapy using metabolic labeling and facilitated the identification of 2238 proteins via liquid chromatography tandem mass spectrometry (LC-MS/MS). Among these, 51 proteins exhibited upregulation, whereas 105 proteins showed downregulation following a 24-h drug treatment. Clustering analysis revealed that the differential proteins were mainly enriched in metabolic-related pathways. Combined with changes in whole-protein levels, we noticed significant fluctuations in the metabolism-related protein nicotinamide N-methyltransferase (NNMT). Additionally, NNMT overexpression diminished drug effectiveness, whereas its inhibition enhanced therapeutic efficacy. An increase in NNMT was also found in drug-resistant cells, and the NNMT inhibitor JBSNF-000088 inhibited the proliferation of resistant cells. Subsequent phosphoproteomic analysis indicated that the effects of NNMT overexpression on transcription factors, proteins involved in the Rho GTPases cycle, and cell-cycle-related proteins may be related to tumor resistance. In summary, our study provides unique insights into nascent protein perturbations during the initial stages of drug therapy and identified NNMT as a promising target for delaying and overcoming therapeutic resistance.
Glioblastoma (GBM), the most aggressive brain tumor in adults, is characterized by its infiltrative growth along the perivascular space. Mural cells (MCs), encompassing pericytes and smooth muscle cells, are multifunctio...Glioblastoma (GBM), the most aggressive brain tumor in adults, is characterized by its infiltrative growth along the perivascular space. Mural cells (MCs), encompassing pericytes and smooth muscle cells, are multifunctional perivascular cells implicated in GBM progression. MCs not only facilitate vascular co-option but have also been suggested to contribute to the immunosuppressive tumor microenvironment, promoting tumor growth and migration. However, whether MC interactions with immune cells differ based on their proximity to the tumor remains unclear. Using single-cell RNA sequencing, we analyzed MC transcriptome profiles across distinct regions relative to the tumor mass in mouse and human GBM samples. Tumor-residing MCs exhibited profound phenotypic changes, showing upregulated gene expression and enhanced signaling activity toward immune cells, with region-specific ligand-receptor interactions. Conversely, border-residing MCs, despite their abundance, showed reduced activation and lacked distinct transcriptional profiles. These findings reveal spatially defined transcriptional heterogeneity in MCs within the GBM microenvironment, underscoring their dynamic role in the GBM microenvironment. This study provides novel insights into MC responses in GBM, identifying potential avenues for targeting MC-immune-cell interactions in therapeutic interventions.
Cardona-Benavides IJ, Cristobal-Vargas S, De Ramón C
… +11 more, Rojas EA, Misiewicz-Krzeminska I, Isidro I, Pérez JJ, Paiva B, Puig N, Alcoceba M, Mateos MV, Corchete LA, Cuadrado M, Gutiérrez NC
Cyclins D could be a unifying event in multiple myeloma (MM), even though MM is not typically considered a proliferative disease. In this study, we hypothesized that cyclins D might have additional roles in the pathogene...Cyclins D could be a unifying event in multiple myeloma (MM), even though MM is not typically considered a proliferative disease. In this study, we hypothesized that cyclins D might have additional roles in the pathogenesis of MM beyond cell cycle control. We showed that overexpression of CCND1 and CCND2 in MM cell lines lacking these proteins revealed a mutually exclusive expression pattern, with both cyclins D localized in the cytoplasm and no impact on proliferation. To investigate non-canonical roles of cyclin D1, we performed transcriptome analysis and multidimensional flow cytometry. Cyclin D1 overexpression led to upregulation of several key cell adhesion pathway proteins, including STAT1 and ZO-1, along with alterations in the actin cytoskeleton and decreased adhesion to certain matrices. Immunophenotypic analysis showed a significant reduction in CD56 expression following cyclin D1 overexpression, validated in a cohort of 85 MM patients, in which 73% with high cyclin D1 were CD56-negative. High cyclin D1 was also associated with increased circulating tumor cells (CTCs) (P < 0.001). Overall, we revealed novel functions of cyclin D1 in MM pathogenesis, particularly in cell adhesion and dissemination.
Ovarian cancer (OC) has the highest mortality rate of all gynaecological malignancies, partly attributable to its propensity for chemotherapy resistance. The most common subtype of OC is serous, of which high-grade serou...Ovarian cancer (OC) has the highest mortality rate of all gynaecological malignancies, partly attributable to its propensity for chemotherapy resistance. The most common subtype of OC is serous, of which high-grade serous ovarian cancer (HGSOC) is the most lethal subtype. Protein tyrosine phosphatase 4A3 (PTP4A3) overexpression is implicated in tumour cell invasion and metastasis by upregulating the PI3K/Akt/mTORC1 axis. Previously, we reported that PTP4A3 increased the survival of non-serous OC cells by activating the autophagy pathway. Here, we investigated the impact of PTP4A3 on cell proliferation, autophagy and chemoresistance in HGSOC cells and whether targeting PTP4A3 in HGSOC cells that overexpress this phosphatase would sensitise them to existing chemotherapeutic drugs. Gene silencing of PTP4A3 resulted in the upregulation of compensatory mechanisms that overcame the loss of PTP4A3 expression, but this was mitigated by pan-PTP4A inhibition with JMS-053 in HGSOC cells. Moreover, shRNA-mediated silencing of PTP4A3 sensitised HGSOC cells to clinically relevant chemotherapeutic drugs. Overall, we show that compensatory mechanisms from PTP4A1 and PTP4A2 can arise when specifically targeting PTP4A3 in HGSOC and that pan-PTP4A inhibition can overcome those effects.
Triple-negative breast cancer (TNBC) remains the breast cancer subtype with the poorest prognosis. While PARP inhibitors (PARPi) effectively target BRCA1/2-mutant TNBCs via synthetic lethality, most TNBCs are BRCA1/2 wil...Triple-negative breast cancer (TNBC) remains the breast cancer subtype with the poorest prognosis. While PARP inhibitors (PARPi) effectively target BRCA1/2-mutant TNBCs via synthetic lethality, most TNBCs are BRCA1/2 wild-type. Synergistic drug combinations may expand PARPi efficacy to BRCA-proficient TNBC. To identify new PARPi combinations, we screened a library of 166 FDA-approved oncology drugs for synergy with Olaparib in TNBC cells. We found that Exemestane, an aromatase inhibitor, synergized with Olaparib, significantly decreasing IC values and clonogenicity while increasing DNA damage and apoptosis. The mechanistic basis for this synergy was rationalized by the previously unreported ability of Exemestane to induce replication stress via reactive oxygen species (ROS) generation and oxidative stress. This combination had low cytotoxicity toward normal breast epithelial cells, and Exemestane has no reported severe toxicity as a monotherapy. The combination of Olaparib and Exemestane was able to achieve enhanced tumor growth inhibition in a murine xenograft model, greater than either drug employed as a single agent, and GO and KEGG enrichment analysis indicated alterations in pathways associated with cell death in response to Exemestane and Olaparib treatment.
Carcinoma-associated fibroblasts (CAFs), which are abundant in the tumor microenvironment, influence cancer hallmarks. We previously described transforming growth factor-β (TGF-β)-Smad2/3 signaling as being activated in...Carcinoma-associated fibroblasts (CAFs), which are abundant in the tumor microenvironment, influence cancer hallmarks. We previously described transforming growth factor-β (TGF-β)-Smad2/3 signaling as being activated in myofibroblastic CAFs (myCAFs) in an autocrine fashion by increasing TGF-β production. However, factors regulating such autocrine TGF-β signaling remain poorly understood. Herein, we show that the abundance of endoglin (ENG), a TGF-β superfamily coreceptor expressed on human breast myCAFs, is significantly associated with poorer outcomes of breast cancer patients. Inhibition of ENG expression on myCAFs not only suppressed the TGF-β-Smad2/3 pathway and TGF-β1 expression but also attenuated the ability of myCAF to promote primary tumor growth and metastasis. Mechanistically, ENG facilitates TGF-β-Smad2/3 signaling in myCAFs, presumably through association with a TGF-β ligand-receptor complex, leading to self-stimulating TGF-β1 production. Stromal TGF-β1, in turn, induces partial epithelial-mesenchymal transition in cancer cells in a paracrine manner, resulting in suppression of primary tumor growth and promotion of invasion and metastasis. ENG-primed TGF-β autocrine signaling also produces other factors that could mediate primary tumor growth promotion by myCAFs. Collectively, these findings suggest that ENG-primed TGF-β autocrine and paracrine signaling mediates tumor- and metastasis-promoting abilities of myCAFs.
Vychytilova-Faltejskova P, Pavlikova M, Pifkova L
… +24 more, Jugas R, Machackova T, Radova L, Sachlova M, Bartosova R, Samoilenko T, Feckova Z, Orlickova J, Slamova E, Ponechal Michu E, Al Tukmachi D, Ruckova M, Vodinska M, Kotoucek J, Catela Ivkovic T, Boudna M, Bohovicova L, Stanek T, Halamkova J, Svoboda M, Prochazka V, Grolich T, Kala Z, Slaby O
Early diagnosis of colorectal cancer (CRC) is crucial for successful treatment and mortality reduction. In this regard, blood-based tests play an indispensable role. Current research is focused on molecules actively secr...Early diagnosis of colorectal cancer (CRC) is crucial for successful treatment and mortality reduction. In this regard, blood-based tests play an indispensable role. Current research is focused on molecules actively secreted by tumor cells into small extracellular vesicles (EVs). This four-phase study included 613 CRC patients, 446 controls, and 120 precancerous lesions. High-throughput transcriptome profiling of small EVs was performed on samples from 100 CRC patients and 50 controls, followed by extensive validation using reverse transcription quantitative polymerase chain reaction. Diagnostic panels were developed via logistic regression and further characterized by enrolling samples from gastric cancer patients, CRC patients before/after surgery, and samples of tumor tissues/adjacent mucosa. We identified 17 molecules significantly elevated in CRC, with the highest levels in metastatic cases. Additionally, seven of them differentiated controls from precancerous lesions. Two diagnostic panels were developed, enabling early CRC detection with high sensitivity and specificity, outperforming the fecal occult blood test. Furthermore, six molecules were differentially expressed between tumor tissue and mucosa, while seven EV-enriched molecules decreased significantly after surgery. These findings highlight EVs as key reservoirs of CRC-associated molecules and a promising source of biomarkers.
The primary treatment for fatal pediatric-type diffuse high-grade glioma (PED-DHGG) which harbor the H3K27M or H3G34R/V mutation is radiation, but it provides only short-term relief. Inhibitors of phosphorylated eIF2α (P...The primary treatment for fatal pediatric-type diffuse high-grade glioma (PED-DHGG) which harbor the H3K27M or H3G34R/V mutation is radiation, but it provides only short-term relief. Inhibitors of phosphorylated eIF2α (PeIF2α) phosphatase-namely raphin-1 and salubrinal-decrease survival of PED-DHGG cell lines and sensitize them to radiation. However, although both drugs increase PeIF2α, they have different effects on common targets and different targets altogether. Here, we aimed to identify PeIF2α-phosphatase-dependent and PeIF2α-phosphatase-independent molecular targets. Raphin-1 but not salubrinal, decreased the level of BiP and CReP and increased that of DR5, in an ISRIB-independent manner. Raphin-1 induced similar changes in MEF cells and in irradiated PED-DHGG, suggesting a PeIF2α-independent contribution to raphin-1's radiosensitizing effect. Importantly, while the expression of [S51D] eIF2α decreased the survival of PED-DHGG and both raphin-1 and salubrinal decreased the survival of MEF cells, only raphin-1 decreased the survival of mutant MEF cells. Our results suggest that the sensitivity of PED-DHGG to raphin-1 is mediated by both PeIF2α-dependent and PeIF2α-independent processes. Elucidating these processes could reveal targets for the development of drugs to overcome radiotherapy resistance of PED-DHGG.
Genetic alterations in key oncogenes have been frequently identified in lung adenocarcinoma (LUAD), including genes encoding epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and...Genetic alterations in key oncogenes have been frequently identified in lung adenocarcinoma (LUAD), including genes encoding epidermal growth factor receptor (EGFR), Kirsten rat sarcoma viral oncogene homolog (KRAS), and anaplastic lymphoma kinase (ALK). In this pilot study, we aimed to characterize the differences in enriched biological pathways and phosphorylation events between LUAD tumors harboring EGFR, KRAS, or echinoderm microtubule-associated protein-like 4 (EML4)-ALK oncogenic alterations and triple wild-type LUAD tumors (WT, without EML4-ALK, KRAS, or EGFR alterations) by mass spectrometry (MS)-based quantitative proteomics and phosphoproteomics. We analyzed tumor regions of 82 formalin-fixed paraffin-embedded (FFPE) tissue sections with 6, 23, 31, and 22 samples from the EML4-ALK, EGFR, KRAS, and WT sample groups, respectively. A total of 1377 to 2189 proteins and 73 to 1781 phosphosites were quantified in these analyses. Based on the results, the samples clustered according to their genetic alteration type, and EGFR-mutated samples showed unique protein expression patterns. Membrane organization, vesicle organization, and vesicle-mediated transport Gene Ontology Biological Process (GOBP) terms were significantly downregulated in EGFR-mutated samples compared to the other sample groups. Changes in 36 proteins and 52 phosphosites were also identified as potentially specific to a given genetic alteration. Many of these proteins have previously been linked to EGFR or KRAS mutations [e.g., cathepsin L, stimulator of interferon genes protein (STING)], whereas several phosphoproteins are associated with RNA splicing [e.g., serine/arginine-rich splicing factor 1 (SRSF1), SRSF2, and SRSF7 proteins]. Kinase-substrate enrichment analysis indicated altered activities of 10 kinases, including mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs). For example, CDK2 activity was elevated in EML4-ALK samples compared to the other sample groups. Our results could provide significant insights into further studies that could contribute to developing improved diagnostic and therapeutic strategies for LUAD.