Monocytes are recruited to tumors and undergo transcriptional reprogramming resulting in tumor-promoting functions. Epigenomic features, such as post-translational modification of histones and chromatin accessibility, are key determinants of transcription factor binding and thereby play an important role in controlling transcriptional responses to the tissue environment. It remains unknown whether systemic tumor-associated signals could alter the epigenomic landscape of peripheral monocytes before they reach the tumor, thus shaping their subsequent response to the tumor microenvironment.

We used a combination of genome-wide assays for chromatin accessibility and multiple histone modifications (H3K4me1, H3K4me3, H3K27ac) in a mouse tumor model to investigate changes in the epigenomic landscape of peripheral monocytes. We then integrated these epigenomic data with transcriptomic data to link altered regulatory elements to gene expression changes in monocytes occurring in the periphery or during tumor infiltration.

We found that tumor-induced systemic inflammation was associated with transcriptional and epigenomic preconditioning of peripheral monocytes. The distal tumor caused extensive remodeling of both H3K4me3⁺ promoters and H3K4me1⁺ enhancers. Specifically, this involved the repression of interferon-responsive regulatory elements as well as the establishment of enhancers harboring binding motifs for transcription factor families downstream of pro-inflammatory signaling, such as C/EBP, AP-1, and STAT. Reprogrammed enhancers in peripheral monocytes were linked to sustained gene expression changes that persisted after tumor infiltration. In addition, key pro-tumor genes upregulated in tumor-infiltrating monocytes showed epigenetic priming already in the circulation.

These results suggest that cancer-associated remodeling of the epigenomic landscape in peripheral monocytes can shape the gene expression programs they acquire in the tumor, highlighting the role of the epigenome in redirecting monocyte function to support cancer progression.

Nucleoside analogues are crucial in treating non-small cell lung cancer (NSCLC), but resistance hampers patient outcomes. The cytidine analogue RX-3117 shows promise in gemcitabine-resistant cancers, yet mechanisms underlying acquired resistance to this drug remain unexplored. This study includes a comprehensive investigation into RX-3117 resistance mechanisms by leveraging new preclinical models and cutting-edge genomic tools, including a CRISPR-Cas9 knockout screen and transcriptomics.

NSCLC cell lines A549 and SW1573 were exposed to stepwise increasing concentrations of RX-3117 to establish stable resistant subclones, confirmed by SRB and clonogenic assays. Intracellular RX-3117 nucleotide levels were measured via LC/MS-MS, prompting the evaluation and modulation of the expression of key metabolic enzymes by Western blot and siRNA. A CRISPR-Cas9 screen identified genes whose loss increased RX-3117 sensitivity, while RNA-sequencing with differential expression analyses revealed resistance-related pathways, further investigated through cell cycle distribution, knock-out, and ELISA assays.

Resistant clones exhibited decreased accumulation of RX-3117 nucleotides, which however, was not associated to reduced expression of activation enzymes (UCK2, UMPK, CMPK, NME1/NDPK, RR1 and RR2). Instead, increased expression was observed in certain DNA repair and deactivation enzymes (NT5C3) but pharmacological inhibition and silencing of the latter did not circumvent resistance. Remarkably, a comprehensive approach with CRISPR-Cas9 screen highlighted DNA-repair and cell cycle determinants as key sensitizing genes. XL-PCR and RNA-sequencing confirmed aberrations in DNA-repair and pathways involved in cell cycle regulation. Knock-out and pharmacological inhibition validated the role of PKMYT1, a protein kinase involved in G2/M transition and genomic stability. RX-3117-resistant A549 cells showed enhanced sensitivity to the PKMYT1 inhibitor lunresertib and its synergism with RX-3117, suggesting further studies, especially in patients with high PKMYT1 expression who have significantly shorter survival rates, as observed in public databases and validated in an internal cohort of NSCLC patients.

By integrating CRISPR-Cas9 with functional assays and transcriptomics, our study established a framework for decoding resistance mechanisms and highlights potential therapeutic strategies to enhance RX-3117 efficacy in NSCLC. We demonstrated for the first time that aberrant DNA repair and cell cycle dysregulation led resistance, identifying PKMYT1 as a promising target.

Homeobox C8 (HOXC8) is a transcription factor preferentially overexpressed in a large percentage of non-small cell lung carcinoma (NSCLC). To investigate the function of HOXC8 in NSCLC, we showed that knockdown of HOXC8 led to massive NSCLC cell death in a mechanism of pyroptosis because both YVAD, a caspase-1 (CASP1) inhibitor, and disulfiram, which prevents gasdermin D (GSDMD) pore formation, blocked cell death caused by HOXC8 depletion. Intriguingly, ASC, a key component of canonic inflammasome, was dispensable for pyroptosis occurring in HOXC8-depleted cells. Instead, we detected greatly elevated levels of both CASP1 protein and mRNA in HOXC8-knockdown cells. As forced expression of CASP1 is sufficient to induce CASP1 activation and pyroptosis, we reason that pyroptosis led by HOXC8 depletion results from massive increase in the abundance of CASP1. To uncover the functional connection between HOXC8 and CASP1 expression, we revealed that HDAC1/2 was involved in augmented CASP1 transcription induced by HOXC8 knockdown. Moreover, we found that HOXC8 and HDAC1 were in the same immunocomplex and the presence of HOXC8 is required for the recruitment of HDAC1 to CASP1 promoter. Since HOXC8 also binds CASP1 promoter, we conclude that HOXC8 negatively regulates CASP1 expression by drafting HDAC1/2 to CASP1 gene. Finally, we demonstrated that cholesterol-conjugated HOXC8 siRNA was able to slow down NSCLC tumorigenesis. This study suggests that HOXC8 participates NSCLC development by controlling CASP1 expression and pyroptosis.

The pH gradient inversion is one of the characteristic features of cancer cells. The acidic environment outside the tumor and the alkaline inside cancer cells significantly affect its growth and ability to metastasize and reduce the effectiveness of therapy. In this study, we examined the effect of the alkaline pH on the viability of colorectal cancer (CRC) cells Caco-2 and Ht-29, and normal human fibroblasts BJ-CRL-2522 as a control cell line. Moreover, we analyzed the expression level of 3 genes at different pH: pro-apoptotic BAX, proliferation marker MKI67, and pH-sensitive receptor GPR4 using the RT-qpCR method. We demonstrated increased Ht-29 cell viability at alkaline pH, with simultaneous morphological changes, and decreased Caco-2 cell viability at pH above 9. The expression of BAX, GPR4 was upregulated in Ht-29 and BJ cells cultured at alkaline pH, whereas in Caco-2 cells the expression was significantly different between conditions. MKI67 expression was slightly increased in HT-29 and BJ, while decreased in Caco-2 cells. Our study shed new light on the cellular processes occurring under the influence of alkalization, but it is difficult to assess what role alkalization may play in the potential therapy of CRC, therefore, further studies are necessary.

Breast cancer remains a significant global health concern, with its molecular intricacies and the mechanistic role of tissue inhibitor of metalloproteinases-1 (TIMP1) still poorly understood. This study employed an integrated approach combining bioinformatic analyses and primary experimental validations to discover the complexities surrounding TIMP1 in breast cancer. Bioinformatic tools such as pan-cancer view, mRNA expression analysis, immune infiltrations, pathway enrichment, and functional annotations provided a clear perspective on TIMP1 in breast cancer. Gene expression by qPCR analysis for TIMP1 were conducted in MCF-7 and MDA-MB-231 and T47D cells and compared to normal breast cells, MCF-10A. Bioinformatic platform, The University of Alabama at Birmingham Cancer (UALCAN) data analysis underscored the diagnostic relevance of TIMP1, showing its upregulated mRNA expression across different stages and metastatic properties. Notably, the impact of breast cancer on immune cells was explored, revealing a direct influence of TIMP1 on CD4⁺, CD8⁺, and B cells, with strong correlations were observed. Kaplan-Meier (KM) survival analysis revealed that high TIMP1 expression correlates with poor prognosis in breast cancer patients, reinforcing its oncogenic potential. Furthermore, immunohistochemistry supported these findings, and protein-protein interaction analysis through STRING and CYTOSCAPE identified interconnected genes linked to TIMP1 in breast cancer. Enriched pathway analysis using KEGG pathways unveiled the potential involvement of TIMP1 in the phosphatidylinositol 3-kinases (PI3K) pathway and cell cycle regulation, further substantiating its oncogenic role. Experimental validation through siRNA silencing TIMP1 reduces cell growth and promotes G1 phase cell cycle arrest in MDA-MB-231 cells. In conclusion, this comprehensive study suggests that targeting TIMP1 in breast cancer could present a promising avenue for therapeutic development, highlighting its potential as a crucial player in the molecular landscape of breast cancer progression.

Defining the mechanisms that promote development and progression of myeloproliferative neoplasms (MPNs) is important for understanding the mechanisms of malignant hematopoiesis and critical development of new treatment approaches. We provide evidence for a key and essential role of the kinase ULK1 in MPN pathophysiology. Our studies demonstrate that genetic or pharmacological targeting of ULK1 delays substantially disease development in Jak2^V617F-mutant MPN models in vivo and establish that ULK1 activity is required for transcription of genes that control hematopoietic stem cell differentiation. Pharmacological targeting of ULK1 exhibits potent therapeutic effects, resulting in reduction of early stage erythroid progenitors in spleen and bone marrow, decreased levels of hemoglobin, and reduced spleen size in MPN mouse models in vivo. Taken together, these findings provide the first evidence for a novel protumorigenic role for ULK1 downstream of the hyperactive JAK2 signaling in MPNs and raise the potential of ULK1 as a new therapeutic target for the treatment of MPNs.

CircRNAs can be applied as tumor biomarkers and potential therapeutic targets. Nevertheless, the function of circ_PSD3 in papillary thyroid carcinoma (PTC) has not been thoroughly explored. The current project attempts to analyze it. RT-qPCR was adopted for measuring the expression of circ_PSD3, HMGB3, miR-145-5p as well as miR-338-3p in PTC tissues and cells. Through performing cell counting kit-8 and transwell experiments, the biological effects of circ_PSD3, HMGB3, miR-145-5p and miR-338-3p on PTC cells were detected. Besides, a dual-luciferase reporter gene was employed to identify the underlying mechanism of circ_PSD3. The Western blot analysis assay was utilized to assess the expression levels of molecular marker proteins associated with epithelial-mesenchymal transition. According to the results, the expressions of circ_PSD3 and HMGB3 showed obvious upregulation in PTC tissues, whereas knockdown of circ_PSD3 or HMGB3 notably hindered cell proliferation, migration as well as invasion in PTC. Mechanistically, it could be discovered that miR-145-5p and miR-338-3p served as the targets of circ_PSD3, and HMGB3 was a target of miR-145-5p and miR-338-3p. Moreover, miR-145-5p and miR-338-3p were discovered to play the role of tumor suppressors in PTC. More importantly, the findings showed that cell proliferation, migration, invasion together with EMT processes were attenuated by circ_PSD3 knockdown, but partially counteracted by miR-338-3p (miR-145-5p) inhibitor or HMGB3 overexpression. Based on the obtained data, circ_PSD3 promotes PTC cell proliferation, migration, invasion and EMT by regulating the miR-145-5p/miR-338-3p/HMGB3 axis. The current work revealed the mechanism of action of circ_PSD3 in PTC and may play the role of a new medical target for PTC.

Hypoxia serves as a fundamental component of the tumor microenvironment, exerting a crucial influence on tumor advancement. Nonetheless, a comprehensive examination of a prognostic signature linked to hypoxia in pancreatic cancer is notably absent, presenting an urgent necessity. Therefore, our objective was to create and authenticate a robust prognostic signature capable of predicting outcomes for pancreatic cancer. Initially, the Gene Set Enrichment Analysis (GSEA) database was used to obtain hypoxia-related genes, and prognostic genes were analyzed. Following this, we utilized the Lasso Cox regression model to construct the hypoxia risk score model. Pancreatic cancer patients were subsequently categorized into high- and low-risk groups according to the median risk score. Finally, the CIBERSORT technique was used to assess immune cell infiltration while examining the relationship between hypoxia and immune-related genes. Applying the Lasso Cox regression model, we pinpointed 2 significant genes, GYS1 and ALDOB. Following this, patients were categorized into hypoxia high-risk and low-risk groups. Notably, the low-risk cohort demonstrated a substantially heightened survival rate relative to the high-risk group. Further investigation into the immune microenvironment unveiled a greater prevalence of resting mast cells, monocytes, plasma cells, and naive CD4+ T cells in the low-risk category. In addition, we detected differences in the expression of 39 immune-related genes between the 2 groups. In summary, our study has established a predictive signature comprising molecular markers for forecasting the prognosis of pancreatic cancer patients.

MicroRNA (miR)-148a-3p is most frequently upregulated in solid tumors, such as colorectal cancer (CRC). This study aimed to elucidate the role of miR-148a-3p in CRC cell proliferation and immune escape and its potential mechanism. miR-148a-3p and Kruppel-like transcription factor 4 (KLF4) expressions were quantified by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and immune evasion abilities of CRC cells were evaluated with the cell counting kit-8 assay, Transwell, western blot, and enzyme-linked immunosorbent assays. The proliferation or apoptosis of CD8+ and CD4+ T cells after coculture with CRC cells was assessed by flow cytometry. Dual-luciferase reporter gene testing was used to validate the targeting association between KLF4 and miR-148a-3p. A nude mouse subcutaneous graft tumor model was constructed, and CD8+ T cell infiltration was detected by immunohistochemistry and flow cytometry. miR-148a-3p exhibited a high level, while KLF4 was under-expressed in CRC cells; miR-148a-3p negatively regulated the KLF4 level. Overexpression of miR-148a-3p enhanced CRC cell proliferation, migration, invasion, EMT, and immune escape; silencing miR-148a-3p caused the opposite trend; moreover, the said biological functions of CRC cells were weakened with overexpression of KLF4 but enhanced with silencing of KLF4; silencing KLF4 weakened the influences of dampened miR-148a-3p on CRC development. Silencing miR-148a-3p promoted the infiltration of CD8+ T cells and inhibited tumor growth. In summary, miR-148a-3p promotes CRC cell proliferation and immune evasion by regulating the expression of KLF4. This finding can be used for reference when developing a new way of CRC treatment.

After chemotherapy or radiation therapy, autophagy activity increases in tumor cells for the adaptation of the tumor cells to stress. Thus, disturbance in autophagy can enhance the effectiveness of anticancer drugs. On the other hand, recent findings highlight the importance of microRNAs (miRs) in autophagy, including miR-146a-5p. In gastric and breast cancer miR-146a-5p is frequently reduced, and more precise identification of its function in these cancers is needed. The aim of this study was to evaluate the relationship between miR-146a-5p and autophagy in MKN-45 (human stomach cancer cell line) and MCF-7(breast cancer cell line). The expression of miR-146a-5p in MKN-45 and MCF-7 cell lines was measured before and after induction of autophagy using real-time polymerase chain reaction (PCR). A flow cytometry assay was used for the apoptosis assay, and autophagy induction was approved. Also, the formation of autophagic vacuoles was ensured in cells by western blotting and fluorescence microscopy. Real-time PCR showed that miR-146a-5p level in starvation groups, during autophagy, was significantly lower than in control groups, and also tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) level, a key target of miR-146a-5p, in starvation groups, during autophagy, was more than control groups but it was significant only in the MCF-7 group. According to previous studies and the results of the present study, miR-146a-5p may be considered a negative regulator of autophagy. However, to confirm this, further studies are needed on different cancer cell lines.