Comprehensive pan-cancer analysis identifies the RNA-binding protein LRPPRC as a novel prognostic and immune biomarker.

AIMS: RNA-binding proteins (RBPs) play pivotal roles in carcinogenesis and immunotherapy. Leucine-rich pentapeptide repeat-containing protein (LRPPRC) is crucial for RNA polyadenylation, transport, and stability. Although recent studies have suggested LRPPRC's potential role in tumor progression, its significance in tumor prognosis, diagnosis, and immunology remains unclear. MAIN METHODS: We comprehensively analyzed LRPPRC expression in tumors using various databases, including Human Transcriptome Cell Atlas (HTCA), University of California Santa Cruz (UCSC), Human Protein Atlas (HPA), Sangerbox, TISIDB, GeneMANIA, GSCALite, and CellMiner. We examined the correlation between LRPPRC expression level and prognosis, immune infiltration, immunotherapy, methylation, biological function, and drug sensitivity. Single-cell analysis was performed using Tumor Immune Single Cell Hub (TISCH) and CancerSEA software. Patients with acute myeloid leukemia (AML) were categorized based on LRPPRC levels for functional and immune infiltration analyses. The role of LRPPRC in cancer was validated using in vitro experiments. KEY FINDINGS: Our findings revealed that LRPPRC was highly expressed in almost all cancer types, indicating its significant prognostic and diagnostic potential. Notably, LRPPRC was associated with diverse immune features, such as immune cell infiltration, immune checkpoint genes, tumor mutational burden, and microsatellite instability, suggesting its value in guiding immunotherapy strategies. Within AML, the high-expression group had lower levels of immune cells, including CD8+ T cells. In vitro experiments confirmed the inhibitory effects of LRPPRC knockdown on AML cell proliferation. SIGNIFICANCE: This study highlights LRPPRC as a reliable pan-cancer prognostic and immune biomarker, particularly in AML. It lays the groundwork for future research on LRPPRC-targeted cancer therapies.

Pyrroline-5-carboxylate reductase 1 reprograms proline metabolism to drive breast cancer stemness under psychological stress.

Cancer stem-like cells (CSCs) contribute to cancer metastasis, drug resistance and tumor relapse, yet how amino acid metabolism promotes CSC maintenance remains exclusive. Here, we identify that proline synthetase PYCR1 is critical for breast cancer stemness and tumor growth. Mechanistically, PYCR1-synthesized proline activates cGMP-PKG signaling to enhance cancer stem-like traits. Importantly, cGMP-PKG signaling mediates psychological stress-induced cancer stem-like phenotypes and tumorigenesis. Ablation of PYCR1 markedly reverses psychological stress-induced proline synthesis, cGMP-PKG signaling activation and cancer progression. Clinically, PYCR1 and cGMP-PKG signaling components are highly expressed in breast tumor specimens, conferring poor survival in breast cancer patients. Targeting proline metabolism or cGMP-PKG signaling pathway provides a potential therapeutic strategy for breast patients undergoing psychological stress. Collectively, our findings unveil that PYCR1-enhanced proline synthesis displays a critical role in maintaining breast cancer stemness.

Cancer cell employs a microenvironmental neural signal trans-activating nucleus-mitochondria coordination to acquire stemness.

Cancer cell receives extracellular signal inputs to obtain a stem-like status, yet how tumor microenvironmental (TME) neural signals steer cancer stemness to establish the hierarchical tumor architectures remains elusive. Here, a pan-cancer transcriptomic screening for 10852 samples of 33 TCGA cancer types reveals that cAMP-responsive element (CRE) transcription factors are convergent activators for cancer stemness. Deconvolution of transcriptomic profiles, specification of neural markers and illustration of norepinephrine dynamics uncover a bond between TME neural signals and cancer-cell CRE activity. Specifically, neural signal norepinephrine potentiates the stemness of proximal cancer cells by activating cAMP-CRE axis, where ATF1 serves as a conserved hub. Upon activation by norepinephrine, ATF1 potentiates cancer stemness by coordinated trans-activation of both nuclear pluripotency factors MYC/NANOG and mitochondrial biogenesis regulators NRF1/TFAM, thereby orchestrating nuclear reprograming and mitochondrial rejuvenating. Accordingly, single-cell transcriptomes confirm the coordinated activation of nuclear pluripotency with mitochondrial biogenesis in cancer stem-like cells. These findings elucidate that cancer cell acquires stemness via a norepinephrine-ATF1 driven nucleus-mitochondria collaborated program, suggesting a spatialized stemness acquisition by hijacking microenvironmental neural signals.

High expression of cytoplasmic FOXO3 protein associated with poor prognosis of rectal cancer patients: A study from Swedish clinical trial of preoperative radiotherapy to big database analysis.

Introduction: Accumulating evidence has implicated a pivotal role for FOXO3, FOXM1 and SIRT6 in cancer progression. The majority of researches focused on the functions of these proteins in drug resistance, but their relationships with radiotherapy (RT) response remain unclear. In this study, we examined protein expression of FOXO3, FOXM1 and SIRT6 and their clinical significance in a Swedish rectal cancer trial of preoperative RT. Methods: Expression of FOXO3, FOXM1 and SIRT6 protein was examined by immunohistochemistry in patient samples. Genetic analysis of FOXO3, FOXM1 and SIRT6 were performed by cBioportal and MEXPRESS database. Gene-gene network analysis was conducted using GeneMANIA. Functional enrichment analysis was performed based on LinkedOmics and Metascape online software. Results: FOXO3 and FOXM1were mainly expressed in the cytoplasm in both normal and tumour tissues, and SIRT6 in both the cytoplasm and nucleus in normal and tumour tissues. FOXO3 and FOXM1 expression increased from normal mucosa to primary cancer (P < 0.001), while SIRT6 expression decreased from normal mucosa to primary cancer (P < 0.001). High FOXO3 expression correlated with late TNM stage (P = 0.040), distant metastasis (P = 0.032) and independently with disease free survival (DFS) in the RT patients (HR = 7.948; P = 0.049; 95% CI = 1.002-63.032) but not in non-RT patients (P > 0.05). Genetic analysis indicated that DNA methylation status contributed to FOXO3 overexpression. Functional enrichment analysis demonstrated that FOXO3 was closely related to metabolism-related signalling pathway which in turn associated with cancer radioresistance. Moreover, there were strong gene-gene interactions between FOXO3 and metabolism-related signalling. Conclusions: Our findings suggest that FOXO3 may be a prognostic factor in rectal cancer patients with RT.

Smad3 is essential for polarization of tumor-associated neutrophils in non-small cell lung carcinoma.

Neutrophils are dynamic with their phenotype and function shaped by the microenvironment, such as the N1 antitumor and N2 pro-tumor states within the tumor microenvironment (TME), but its regulation remains undefined. Here we examine TGF-ß1/Smad3 signaling in tumor-associated neutrophils (TANs) in non-small cell lung carcinoma (NSCLC) patients. Smad3 activation in N2 TANs is negatively correlate with the N1 population and patient survival. In experimental lung carcinoma, TANs switch from a predominant N2 state in wild-type mice to an N1 state in Smad3-KO mice which associate with enhanced neutrophil infiltration and tumor regression. Neutrophil depletion abrogates the N1 anticancer phenotype in Smad3-KO mice, while adoptive transfer of Smad3-KO neutrophils reproduces this protective effect in wild-type mice. Single-cell analysis uncovers a TAN subset showing a mature N1 phenotype in Smad3-KO TME, whereas wild-type TANs mainly retain an immature N2 state due to Smad3. Mechanistically, TME-induced Smad3 target genes related to cell fate determination to preserve the N2 state of TAN. Importantly, genetic deletion and pharmaceutical inhibition of Smad3 enhance the anticancer capacity of neutrophils against NSCLC via promoting their N1 maturation. Thus, our work suggests that Smad3 signaling in neutrophils may represent a therapeutic target for cancer immunotherapy.

Targeting epiregulin in the treatment-damaged tumor microenvironment restrains therapeutic resistance.

The tumor microenvironment (TME) represents a milieu enabling cancer cells to develop malignant properties, while concerted interactions between cancer and stromal cells frequently shape an "activated/reprogramed" niche to accelerate pathological progression. Here we report that a soluble factor epiregulin (EREG) is produced by senescent stromal cells, which non-cell-autonomously develop the senescence-associated secretory phenotype (SASP) upon DNA damage. Genotoxicity triggers EREG expression by engaging NF-κB and C/EBP, a process supported by elevated chromatin accessibility and increased histone acetylation. Stromal EREG reprograms the expression profile of recipient neoplastic cells in a paracrine manner, causing upregulation of MARCHF4, a membrane-bound E3 ubiquitin ligase involved in malignant progression, specifically drug resistance. A combinational strategy that empowers EREG-specific targeting in treatment-damaged TME significantly promotes cancer therapeutic efficacy in preclinical trials, achieving response indices superior to those of solely targeting cancer cells. In clinical oncology, EREG is expressed in tumor stroma and handily measurable in circulating blood of cancer patients post-chemotherapy. This study establishes EREG as both a targetable SASP factor and a new noninvasive biomarker of treatment-damaged TME, thus disclosing its substantial value in translational medicine.

Chronic Inflammation Pathway NF-κB Cooperates with Epigenetic Reprogramming to Drive the Malignant Progression of Glioblastoma.

Without an effective strategy for targeted therapy, glioblastoma is still incurable with a median survival of only 15 months. Both chronic inflammation and epigenetic reprogramming are hallmarks of cancer. However, the mechanisms and consequences of their cooperation in glioblastoma remain unknown. Here, we discover that chronic inflammation governs H3K27me3 reprogramming in glioblastoma through the canonical NF-κB pathway to target EZH2. Being a crucial mediator of chronic inflammation, the canonical NF-κB signalling specifically directs the expression and redistribution of H3K27me3 but not H3K4me3, H3K9me3 and H3K36me3. Using RNA-seq screening to focus on genes encoding methyltransferases and demethylases of histone, we identify EZH2 as a key methyltransferase to control inflammation-triggered epigenetic reprogramming in gliomagenesis. Mechanistically, NF-κB selectively drives the expression of EZH2 by activating its transcription, consequently resulting in a global change in H3K27me3 expression and distribution. Furthermore, we find that co-activation of NF-κB and EZH2 confers the poorest clinical outcome, and that the risk for glioblastoma can be accurately molecularly stratified by NF-κB and EZH2. It is notable that NF-κB can potentially cooperate with EZH2 in more than one way, and most importantly, we demonstrate a Synergistic effect of cancer cells induced by combinatory inhibition of NF-κB and EZH2, which both are frequently over-activated in glioblastoma. In summary, we uncover a functional cooperation between chronic inflammation and epigenetic reprogramming in glioblastoma, combined targeting of which by inhibitors guaranteed in safety and availability furnishes a potent strategy for effective treatment of this fatal disease.

Single-cell RNA sequencing uncovers a neuron-like macrophage subset associated with cancer pain.

Tumor innervation is a common phenomenon with unknown mechanism. Here, we discovered a direct mechanism of tumor-associated macrophage (TAM) for promoting de novo neurogenesis via a subset showing neuronal phenotypes and pain receptor expression associated with cancer-driven nocifensive behaviors. This subset is rich in lung adenocarcinoma associated with poorer prognosis. By elucidating the transcriptome dynamics of TAM with single-cell resolution, we discovered a phenomenon "macrophage to neuron-like cell transition" (MNT) for directly promoting tumoral neurogenesis, evidenced by macrophage depletion and fate-mapping study in lung carcinoma models. Encouragingly, we detected neuronal phenotypes and activities of the bone marrow-derived MNT cells (MNTs) in vitro. Adoptive transfer of MNTs into NOD/SCID mice markedly enhanced their cancer-associated nocifensive behaviors. We identified macrophage-specific Smad3 as a pivotal regulator for promoting MNT at the genomic level; its disruption effectively blocked the tumor innervation and cancer-dependent nocifensive behaviors in vivo. Thus, MNT may represent a precision therapeutic target for cancer pain.

Sp1 induced gene TIMP1 is related to immune cell infiltration in glioblastoma.

Tumor immune microenvironment exerts a profound effect on the population of infiltrating immune cells. Tissue inhibitor of matrix metalloproteinase 1 (TIMP1) is frequently overexpressed in a variety of cells, particularly during inflammation and tissue injury. However, its function in cancer and immunity remains enigmatic. In this study, we find that TIMP1 is substantially up-regulated during tumorigenesis through analyzing cancer bioinformatics databases, which is further confirmed by IHC tissue microarrays of clinical samples. The TIMP1 level is significantly increased in lymphocytes infiltrating the tumors and correlated with cancer progression, particularly in GBM. Notably, we find that the transcriptional factor Sp1 binds to the promoter of TIMP1 and triggers its expression in GBM. Together, our findings suggest that the Sp1-TIMP1 axis can be a potent biomarker for evaluating immune cell infiltration at the tumor sites and therefore, the malignant progression of GBM.

Smad3 Promotes Cancer-Associated Fibroblasts Generation via Macrophage-Myofibroblast Transition.

Cancer-associated fibroblasts (CAFs) are important in tumor microenvironment (TME) driven cancer progression. However, CAFs are heterogeneous and still largely underdefined, better understanding their origins will identify new therapeutic strategies for cancer. Here, the authors discovered a new role of macrophage-myofibroblast transition (MMT) in cancer for de novo generating protumoral CAFs by resolving the transcriptome dynamics of tumor-associated macrophages (TAM) with single-cell resolution. MMT cells (MMTs) are observed in non-small-cell lung carcinoma (NSCLC) associated with CAF abundance and patient mortality. By fate-mapping study, RNA velocity, and pseudotime analysis, existence of novel macrophage-lineage-derived CAF subset in the TME of Lewis lung carcinoma (LLC) model is confirmed, which is directly transited via MMT from M2-TAM in vivo and bone-marrow-derived macrophages (BMDM) in vitro. Adoptive transfer of BMDM-derived MMTs markedly promote CAF formation in LLC-bearing mice. Mechanistically, a Smad3-centric regulatory network is upregulated in the MMTs of NSCLC, where chromatin immunoprecipitation sequencing(ChIP-seq) detects a significant enrichment of Smad3 binding on fibroblast differentiation genes in the macrophage-lineage cells in LLC-tumor. More importantly, macrophage-specific deletion and pharmaceutical inhibition of Smad3 effectively block MMT, therefore, suppressing the CAF formation and cancer progression in vivo. Thus, MMT may represent a novel therapeutic target of CAF for cancer immunotherapy.

MCF-7 Drug Resistant Cell Lines Switch Their Lipid Metabolism to Triple Negative Breast Cancer Signature.

Obesity and adipose tissue have been closely related to a poor cancer prognosis, especially in prostate and breast cancer patients. The ability of transferring lipids from the adipose tissue to the tumor cells is actively linked to tumor progression. However, different types of breast tumor seem to use these lipids in different ways and metabolize them in different pathways. In this study we have tracked by mass spectrometry how palmitic acid from the adipocytes is released to media being later incorporated in different breast cancer cell lines (MDA-MB-231, SKBR3, BT474, MCF-7 and its resistant MCF-7 EPIR and MCF-7 TAXR). We have observed that different lines metabolize the palmitic acid in a different way and use their carbons in the synthesis of different new lipid families. Furthermore, we have observed that the lipid synthesis pattern varied according to the cell line. Surprisingly, the metabolic pattern of the resistant cells was more related to the TNBC cell line compared to their sensitive cell line MCF-7. These results allow us to determine a specific lipid pattern in different cell lines that later might be used in breast cancer diagnosis and to find a better treatment according to the cancer molecular type.

Modulation of oxidative phosphorylation augments antineoplastic activity of mitotic aurora kinase inhibition.

Uncontrolled mitosis is one of the most important features of cancer, and mitotic kinases are thought to be ideal targets for anticancer therapeutics. However, despite numerous clinical attempts spanning decades, clinical trials for mitotic kinase-targeting agents have generally stalled in the late stages due to limited therapeutic effectiveness. Alisertib (MLN8237) is a promising oral mitotic aurora kinase A (AURKA, Aurora-A) selective inhibitor, which is currently under several clinical evaluations but has failed in its first Phase III trial due to inadequate efficacy. In this study, we performed genome-wide CRISPR/Cas9-based screening to identify vulnerable biological processes associated with alisertib in breast cancer MDA-MB-231 cells. The result indicated that alisertib treated cancer cells are more sensitive to the genetic perturbation of oxidative phosphorylation (OXPHOS). Mechanistic investigation indicated that alisertib treatment, as well as other mitotic kinase inhibitors, rapidly reduces the intracellular ATP level to generate a status that is highly addictive to OXPHOS. Furthermore, the combinational inhibition of mitotic kinase and OXPHOS by alisertib, and metformin respectively, generates severe energy exhaustion in mitotic cells that consequently triggers cell death. The combination regimen also enhanced tumor regression significantly in vivo. This suggests that targeting OXPHOS by metformin is a potential strategy for promoting the therapeutic effects of mitotic kinase inhibitors through the joint targeting of mitosis and cellular energy homeostasis.

USMB-shMincle: a virus-free gene therapy for blocking M1/M2 polarization of tumor-associated macrophages.

Mincle is essential for tumor-associated macrophage (TAM)-driven cancer progression and represents a potential immunotherapeutic target for cancer. Nevertheless, the lack of a specific inhibitor has largely limited its clinical translation. Here, we successfully developed a gene therapeutic strategy for silencing Mincle in a virus-free and tumor-specific manner by combining RNA interference technology with an ultrasound-microbubble-mediated gene transfer system (USMB). We identified a small hairpin RNA (shRNA) sequence shMincle that can silence not only Mincle expression but also the protumoral effector production in mouse bone marrow- and human THP-1-derived macrophages in the cancer setting in vitro. By using our well-established USMB system (USMB-shMincle), the shMincle-expressing plasmids were delivered in a tissue-specific manner into xenografts of human lung carcinoma A549 and melanoma A375 in vivo. Encouragingly, we found that USMB-shMincle effectively inhibited the protumoral phenotypes of TAMs as well as the progression of both A549 and A375 xenografts in a dose-dependent manner in mice without significant side effects. Mechanistically, we identified that USMB-shMincle markedly enhanced the anticancer M1 phenotype of TAMs in the A549 and A375 xenografts by blocking the protumoral Mincle/Syk/nuclear factor κB (NF-κB) signaling axis. Thus, USMB-shMincle may represent a clinically translatable novel and safe gene therapeutic approach for cancer treatment.

Altered Serum Metabolic Profile Assessed by Advanced 1H-NMR in Breast Cancer Patients.

BACKGROUND: Altered lipid metabolism has been described in some types of cancer. To analyse in depth the metabolic modifications in breast cancer patients, advanced 1H-nuclear magnetic resonance was performed in these patients. The main objective of this paper was to define a specific lipidomic signature for these cancer patients. MATERIALS AND METHODS: Serum from 240 women (171 breast cancer patients and 69 control women) were studied and analysed by nuclear magnetic resonance. RESULTS: Triglyceride-enriched particles, specifically very low-density lipoprotein triglycerides, intermediate-density lipoprotein triglycerides, low-density lipoprotein triglycerides, and high-density lipoprotein triglycerides, were positively associated with breast cancer. Moreover, alanine, tyrosine, and branched amino acids were also associated with increased risk of breast cancer. CONCLUSIONS: Breast cancer patients showed a modified metabolome, giving a very interesting tool to draw different radar charts between control women and breast cancer patients. To our knowledge, this is the first time that advanced nuclear magnetic resonance profiling has been used to identify relevant and specifically altered lipid or amino acid metabolites in BC serum samples. The altered metabolic signature could be analysed for early and reliable BC patient diagnosis and prognosis.

Targeting cancer cell plasticity by HDAC inhibition to reverse EBV-induced dedifferentiation in nasopharyngeal carcinoma.

Application of differentiation therapy targeting cellular plasticity for the treatment of solid malignancies has been lagging. Nasopharyngeal carcinoma (NPC) is a distinctive cancer with poor differentiation and high prevalence of Epstein-Barr virus (EBV) infection. Here, we show that the expression of EBV latent protein LMP1 induces dedifferentiated and stem-like status with high plasticity through the transcriptional inhibition of CEBPA. Mechanistically, LMP1 upregulates STAT5A and recruits HDAC1/2 to the CEBPA locus to reduce its histone acetylation. HDAC inhibition restored CEBPA expression, reversing cellular dedifferentiation and stem-like status in mouse xenograft models. These findings provide a novel mechanistic epigenetic-based insight into virus-induced cellular plasticity and propose a promising concept of differentiation therapy in solid tumor by using HDAC inhibitors to target cellular plasticity.

KDM4 Orchestrates Epigenomic Remodeling of Senescent Cells and Potentiates the Senescence-Associated Secretory Phenotype.

Cellular senescence restrains the expansion of neoplastic cells through several layers of regulation. We report that the histone H3-specific demethylase KDM4 is expressed as human stromal cells undergo senescence. In clinical oncology, upregulated KDM4 and diminished H3K9/H3K36 methylation correlate with poorer survival of prostate cancer patients post-chemotherapy. Global chromatin accessibility mapping via ATAC-seq, and expression profiling through RNA-seq, reveal global changes of chromatin openness and spatiotemporal reprogramming of the transcriptomic landscape, which underlie the senescence-associated secretory phenotype (SASP). Selective targeting of KDM4 dampens the SASP of senescent stromal cells, promotes cancer cell apoptosis in the treatment-damaged tumor microenvironment (TME), and prolongs survival of experimental animals. Our study supports dynamic changes of H3K9/H3K36 methylation during senescence, identifies an unusually permissive chromatin state, and unmasks KDM4 as a key SASP modulator. KDM4 targeting presents a novel therapeutic avenue to manipulate cellular senescence and limit its contribution to age-related pathologies including cancer.

CRISPR/Cas9 screening identifies a kinetochore-microtubule dependent mechanism for Aurora-A inhibitor resistance in breast cancer.

BACKGROUND: Overexpression of Aurora-A (AURKA) is a feature of breast cancer and associates with adverse prognosis. The selective Aurora-A inhibitor alisertib (MLN8237) has recently demonstrated promising antitumor responses as a single agent in various cancer types but its phase III clinical trial was reported as a failure since MLN8237 did not show an apparent effect in prolonging the survival of patients. Thus, identification of potential targets that could enhance the activity of MLN8237 would provide a rationale for drug combination to achieve better therapeutic outcome. METHODS: Here, we conducted a systematic synthetic lethality CRISPR/Cas9 screening of 507 kinases using MLN8237 in breast cancer cells and identified a number of targetable kinases that displayed synthetic lethality interactions with MLN8237. Then, we performed competitive growth assays, colony formation assays, cell viability assays, apoptosis assays, and xenograft murine model to evaluate the synergistic therapeutic effects of Haspin (GSG2) depletion or inhibition with MLN8237. For mechanistic studies, immunofluorescence was used to detect the state of microtubules and the localization of Aurora-B and mitotic centromere-associated kinesin (MCAK). RESULTS: Among the hits, we observed that Haspin depletion or inhibition marginally inhibited breast cancer cell growth but could substantially enhance the killing effects of MLN8237. Mechanistic studies showed that co-treatment with Aurora-A and Haspin inhibitors abolished the recruitment of Aurora-B and mitotic centromere-associated kinesin (MCAK) to centromeres which were associated with excessive microtubule depolymerization, kinetochore-microtubule (KT-MT) attachment failure, and severe mitotic catastrophe. We further showed that the combination of MLN8237 and the Haspin inhibitor CHR-6494 synergistically reduced breast cancer cell viability and significantly inhibited both in vitro and in vivo tumor growth. CONCLUSIONS: These findings establish Haspin as a synthetic lethal target and demonstrate CHR-6494 as a potential combinational drug for promoting the therapeutic effects of MLN8237 on breast cancer.