NPTX2 accelerates cell cycle progression, activates EMT, and regulates glycolytic metabolic pathways to promote clear cell renal cell carcinoma progression.

To investigate the expression and clinical significance of integrin subunit alpha 9 (ITGA9) and neuronal pentraxin 2 (NPTX2) in acute myeloid leukemia (AML), and to evaluate their potential diagnostic and prognostic value. Differentially expressed genes (DEGs) associated with AML prognosis were identified from the Gene Expression Omnibus (GEO) dataset (GSE12417), followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The expression and prognostic relevance of candidate genes were further validated using The Cancer Genome Atlas (TCGA) dataset and the Xiantao Academic online platform. Correlations of ITGA9 and NPTX2 with immune-regulatory genes, immune checkpoint molecules, and immune cell infiltration were analyzed to explore their potential immune associations. Peripheral blood samples from 60 newly diagnosed AML patients and 50 healthy controls were collected for quantitative real-time PCR validation. Diagnostic and prognostic performances were assessed using receiver operating characteristic (ROC) curve and Kaplan-Meier survival analyses. Seven prognosis-related genes were identified, among which ITGA9 and NPTX2 were significantly upregulated in AML. ITGA9 and NPTX2 were predominantly involved in modulating signaling receptor functions and were primarily associated with the phosphatidylinositol 3-kinase-AKT (PI3K-Akt) signaling. Immune correlation analysis revealed that ITGA9 and NPTX2 were associated with multiple immune-regulatory and checkpoint molecules, indicating potential involvement in AML immune microenvironment modulation. Clinical validation confirmed that ITGA9 and NPTX2 were markedly overexpressed in the AML patients. ROC curve analysis demonstrated strong diagnostic efficacy for ITGA9 and NPTX2. Among the 60 AML patients followed up, 29 died, and the deceased group exhibited significantly higher ITGA9 and NPTX2 expression. High expression of ITGA9 and NPTX2 was associated with reduced relapse-free survival. ITGA9 and NPTX2 are significantly overexpressed in AML and are closely associated with poor prognosis. Their strong diagnostic performance suggests that these two genes may serve as potential biomarkers and therapeutic targets for AML.

Accumulating evidence from clinical and epidemiological studies has highlighted the close correlation between the individual risk of cancer and nervous system diseases. The expression of neuronal pentraxin 2 (NPTX2) is absent in Alzheimer’s disease, anxiety, and depression. Herein, we found that NPTX2 mRNA and protein expression was significantly upregulated in colorectal carcinoma (CRC). NPTX2 expression level gradually increased with CRC progression and was closely associated with poor prognosis. In vitro and in vivo studies demonstrated that NPTX2 promoted CRC proliferation and metastasis through the activation of the Wnt/β-catenin signaling pathway. As NPTX2 receptors are absent on CRC cells, NPTX2 was shown to physically interact with frizzled class receptor 6 (FZD6) to promote β-catenin translocation into the cell nucleus, resulting in an increase in the expression of MYC, cyclin D1, snail, and N-cadherin along with a decrease in the expression of E-cadherin. Knockdown of FZD6 expression with a small-interfering RNA almost completely reversed the proliferative effects of NPTX2 on CRC development. In conclusion, NPTX2, a molecule related to nervous system diseases, promotes CRC cell proliferation and metastasis through the activation of the Wnt/β-catenin pathway via direct interaction with FZD6.

Objective: A previous study by this group demonstrated the feasibility of RNA sequencing (RNAseq) technology for capturing disease biology of clear cell renal cell carcinoma (ccRCC), and presented initial results for carbonic anhydrase-9 (CA9) and tumor necrosis factor-α-induced protein-6 (TNFAIP6) as possible biomarkers of ccRCC (discovery set) [Eikrem et al. PLoS One 2016;11:e0149743]. To confirm these results, the previous study is expanded, and RNAseq data from additional matched ccRCC and normal renal biopsies are analyzed (confirmation set).Materials and methods: Two core biopsies from patients (n = 12) undergoing partial or full nephrectomy were obtained with a 16 g needle. RNA sequencing libraries were generated with the Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using linear modeling (voom/Limma; R Bioconductor).Results: The formalin-fixed and paraffin-embedded discovery and confirmation data yielded 8957 and 11,047 detected transcripts, respectively. The two data sets shared 1193 of differentially expressed genes with each other. The average expression and the log2-fold changes of differentially expressed transcripts in both data sets correlated, with R² = .95 and R² = .94, respectively. Among transcripts with the highest fold changes were CA9, neuronal pentraxin-2 and uromodulin. Epithelial–mesenchymal transition was highlighted by differential expression of, for example, transforming growth factor-β1 and delta-like ligand-4. The diagnostic accuracy of CA9 was 100% and 93.9% when using the discovery set as the training set and the confirmation data as the test set, and vice versa, respectively. These data further support TNFAIP6 as a novel biomarker of ccRCC. TNFAIP6 had combined accuracy of 98.5% in the two data sets.Conclusions: This study provides confirmatory data on the potential use of CA9 and TNFAIP6 as biomarkers of ccRCC. Thus, next-generation sequencing expands the clinical application of tissue analyses.

Colon cancer is a malignant tumor of the digestive tract associated with high morbidity and mortality. Currently, recurrence after surgery is often the ultimate cause of death in colon cancer. Gambogic acid (GA) has demonstrated anti-tumor efficacy against multiple cancers. Moreover, the expression of carbonic anhydrase 9 (CA9) expression is correlated with poor prognosis in various malignancies. Therefore, this study aims to investigate the roles and mechanisms of GA and CA9 in the progression and metastasis of colon cancer. The cellular morphology was assessed by the microscope. Cell migration and invasion were examined using wound healing and transwell assays, respectively. Angiogenesis was measured using an angiogenesis assay. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and western blot were performed to examine the expression of protein and mRNA. Lung metastases were monitored using an in vivo live imaging system . In vivo experiments, the number of pulmonary metastatic lesions was quantified by counting metastatic nodules on the lung surface and further examined by hematoxylin and eosin (H&E) staining. GA suppressed transforming growth factor-β1 (TGF-β1)-induced colon cancer cell migration, invasion, and epithelial-mesenchymal transition (EMT), meanwhile, CA9 expression was up-regulated in colon cancer tissues and TGF-β1-stimulated colon cancer cells. Additionally, CA9 knockdown suppressed the TGF-β1-stimulated migration, invasion, and EMT by colon cancer cells. Mechanistically, GA impeded colon cancer progression and metastasis by silencing CA9 regulated small mother against decapentaplegic (SMAD) signal pathway in vivo or in vitro . GA inhibits colon cancer progression and metastasis by silencing CA9 mediated SMAD pathway. The GA may be a potential therapeutic agent for the treatment of colon cancer by regulating CA9. Gambogic acid attenuates TGF-β1-stimulated colon cancer cell migration, invasion, angiogenesis, EMT and lung metastasis by silencing the CA9 via regulating the SMAD signal pathway. • GA hampers the progression and metastasis of TGF-β1-stimulated colon cancer cells. • In the colon cancer and colon cancer cells, CA9 levels are increased. • CA9 facilitates progression and metastasis of TGF-β1-stimulated colon cancer cells. • GA inhibits the colon cancer progression and metastasis via down-regulated CA9. • GA inhibits colon cancer progression and metastasis by inhibiting CA9-regulated SMAD signal pathway.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an underused resource for molecular analyses. This proof of concept study aimed to compare RNAseq results from FFPE biopsies with the corresponding RNAlater® (Qiagen, Germany) stored samples from clear cell renal cell carcinoma (ccRCC) patients to investigate feasibility of RNAseq in archival tissue. From each of 16 patients undergoing partial or full nephrectomy, four core biopsies, such as two specimens with ccRCC and two specimens of adjacent normal tissue, were obtained with a 16g needle. One normal and one ccRCC tissue specimen per patient was stored either in FFPE or RNAlater®. RNA sequencing libraries were generated applying the new Illumina TruSeq® Access library preparation protocol. Comparative analysis was done using voom/Limma R-package. The analysis of the FFPE and RNAlater® datasets yielded similar numbers of detected genes, differentially expressed transcripts and affected pathways. The FFPE and RNAlater datasets shared 80% (n = 1106) differentially expressed genes. The average expression and the log2 fold changes of these transcripts correlated with R2 = 0.97, and R2 = 0.96, respectively. Among transcripts with the highest fold changes in both datasets were carbonic anhydrase 9 (CA9), neuronal pentraxin-2 (NPTX2) and uromodulin (UMOD) that were confirmed by immunohistochemistry. IPA revealed the presence of gene signatures of cancer and nephrotoxicity, renal damage and immune response. To simulate the feasibility of clinical biomarker studies with FFPE samples, a classifier model was developed for the FFPE dataset: expression data for CA9 alone had an accuracy, specificity and sensitivity of 94%, respectively, and achieved similar performance in the RNAlater dataset. Transforming growth factor-ß1 (TGFB1)-regulated genes, epithelial to mesenchymal transition (EMT) and NOTCH signaling cascade may support novel therapeutic strategies. In conclusion, in this proof of concept study, RNAseq data obtained from FFPE kidney biopsies are comparable to data obtained from fresh stored material, thereby expanding the utility of archival tissue specimens.

BackgroundPostoperative delirium (POD) is a common complication with poor prognosis in the elderly, but its mechanism has not been fully elucidated. There is evidence that the changes in synaptic activity in the brain are closely related to the occurrence of POD. And neuronal pentraxin 2 (NPTX2) can regulate synaptic activity in vivo.AimsThis study aims to explore whether decreased NPTX2 levels affects POD and whether the cerebrospinal fluid (CSF) biomarkers of POD mediate this association.MethodsIn this prospective cohort study, we interviewed patients with knee/hip replacement 1 day before surgery to collect patient information and assess their cognitive function. CSF was extracted for measuring the CSF levels of NPTX2 and other POD biomarkers on the day of surgery. And postoperative follow-up visits were performed 1–7 days after surgery.ResultsFinally, 560 patients were included in the study. The patients were divided into POD group and NPOD (non-POD) group. The POD group had a median age of 80 years, a female proportion of 45%, a median BMI of 24.1 kg/m2, and a median years of education of 9 years. The Mann–Whitney U test showed that CSF NPTX2 levels were significantly lower in POD group, compared with the NPOD group (P < 0.05). Univariate binary logistic regression analysis showed that reduced CSF levels of NPTX2 protected against POD (crude OR = 0.994, 95% CI 0.993–0.995, P < 0.001). The receiver-operating characteristic (ROC) curve indicated that CSF NPTX2 level had high predictive value for POD. Mediation analyses showed that CSF T-tau (mediating proportion = 21%) and P-tau (mediating proportion = 29%) had significant mediating effects on the association between CSF NPTX2 and POD.ConclusionCSF NPTX2 levels were associated with the occurrence of POD. Low CSF NPTX2 levels may be an independent protective factor for POD. CSF T-tau and P-tau could mediate the association between CSF NPTX2 and POD occurrence.Clinical trial registrationThe trial registration number (TRN): ChiCTR2200064740, Date of Registration: 2022-10-15.

Hepatocellular carcinoma (HCC) is correlated with a poor prognosis and high mortality worldwide. Neuronal pentraxin 1 (NPTX1) has been reported to play an oncogenic role in several types of tumors. However, its expression and function in HCC is not yet fully understood. In the present study, we aimed to investigate the clinicopathological significance of NPTX1 in HCC and the underlying mechanisms. We observed that the expression of NPTX1 was decreased significantly in HCC and was associated with tumor size and metastasis in patients. Gain-of-function approaches revealed that NPTX1 suppressed the growth ability of HCC cells and contributed to mitochondria- related apoptosis. Furthermore, mechanistic investigations showed that the AKT (AKT serine/threonine kinase) pathway can regulate the effects of NPTX1 in HCC cells. After blocking the AKT pathway, the action of NPTX1 was greatly increased. In summary, we demonstrated that NPTX1 inhibited growth and promoted apoptosis in HCC via an AKT-mediated signaling mechanism. These findings indicate that NPTX1 is a potential clinical therapeutic target.

Carbonic anhydrase 9 (CA9) is a plasma membrane-associated isoenzyme that catalyzes pH regulation under hypoxic conditions. CA9 is transcriptionally regulated by hypoxia-inducible factor 1. Recent studies reported that hypoxia also promoted the epithelial-mesenchymal transition (EMT) in various cancers. In the present study, we evaluated the relationship between CA9 expression and EMT invitro with two hepatoma cell lines. We also examined the clinical significance of CA9 expression in 117consecutive patients that underwent hepatectomies for hepatocellular carcinoma (HCC). We evaluated CA9 expression and EMT induction under hypoxia with quantitative RT-PCR, western blot analysis and immunofluorescence staining, in HuH7 and HepG2 cells. We knocked down CA9 expression with small interfering RNA to evaluate the relationship between CA9 and EMT. We found that hypoxia induced CA9 expression in HCC cells and promoted EMT, evidenced by a loss of E-cadherin and an increase in N-cadherin. Twist, a transcriptional regulator of EMT, was also upregulated with hypoxia. The CA9 deficiency attenuated hypoxia-induced changes in E-cadherin and N-cadherin. Immunohistochemical evaluations of patient samples showed that CA9 was expressed in 50.4% of patients (59/117). However, patients with and without CA9 expression were not significantly different in clinicopathological factors. Nevertheless, a multivariate analysis showed that CA9 expression was an independent factor for both recurrence and prognosis among patients that underwent curative surgery for HCC. In conclusion, this study revealed that CA9 expression was a pivotal predictive factor for poor prognosis after radical surgery for HCC. Moreover, the CA9 regulation of the expression of EMT-related molecules represented a mechanism that enhanced malignant potential.

Hepatocellular carcinoma (HCC) is a common malignant tumor. Histone lactylation, a novel epigenetic modification, plays a crucial role in various cancers. However, the functional role and underlying mechanism of histone lactylation in HCC progression have not yet been investigated. Histone lactylation levels in HCC tissues and cells were assessed using a densitometric kit and western blot analysis. The role of histone lactylation in cell malignant phenotypes was determined through functional assays in vitro, and a xenograft tumor model was established to verify the function of histone lactylation in vivo. ChIP assay was performed to explore the interaction between histone lactylation and endothelial cell-specific molecule 1 (ESM1). Additionally, gain-and-loss-of-function assays were conducted to investigate the regulatory role of ESM1 in HCC pathogenesis. Histone lactylation levels were increased in HCC tissues and cells, and H3K9 lactylation (H3K9la) and H3K56 lactylation (H3K56la) were identified as the histone modification sites. We observed that H3K9la and H3K56la caused abnormal histone lactylation and were associated with poor prognosis. Functionally, histone lactylation was found to promote HCC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) process in vitro. However, histone lactylation inhibition with 2-deoxy-d-glucose (2-DG) reduced the malignant phenotypes of HCC cells. In vivo, 2-DG treatment reduced tumor growth and metastasis in the HCC mouse model. Mechanistically, it was revealed that histone lactylation activated ESM1 transcription in HCC cells. ESM1 was expressed at a high level in HCC and exerted a carcinogenic role. Histone lactylation facilitates cell malignant phenotypes, tumor growth, and metastasis by upregulating ESM1 expression in HCC, which reveals the downstream molecular mechanism of histone lactylation and might provide a novel therapeutic target for HCC therapy.

Background: Epithelial-mesenchymal transition (EMT) is a tumor progression-related process by which epithelial cells lose their epithelial characteristics and acquire mesenchymal properties. EMT promotes the malignant phenotypes including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Therefore, to prevent tumor progression, EMT targeted therapy is required. Oncolytic virotherapy is a promising strategy for anticancer therapy. Although a variety of oncolytic adenoviruses have been developed to induce tumor-selective cell death, the effect of oncolytic adenovirus on the EMT-induced tumor progression remains unclear. In this study, we investigated the biological effect of oncolytic adenovirus on EMT in human cancer cells. Methods: We previously generated a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. Transforming growth factor-β (TGF-β) was used to induce EMT in human lung (A549) and pancreatic (Panc-1) cancer cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT in these cells, western blot and real-time PCR analysis for EMT-related markers were performed. We also examined cell mobility using transwell migration and invasion assays. In vitro antitumor effect of OBP-301 and chemotherapeutic agents in TGF-β-treated cancer cells were assessed by trypan blue exclusion assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-like cell morphology, down-regulation of E-cadherin, up-regulation of vimentin and high motility, in A549 and Panc-1 cells. When TGF-β-treated cancer cells were infected with OBP-301, suppression of E-cadherin expression was attenuated. Conversely, the expression levels of mesenchymal markers, vimentin and N-cadherin, and EMT-inducing transcription factors, Snail, Slug and ZEB1, were reduced. OBP-301 infection also inhibited TGF-β-induced enhancement of migration and invasion in cancer cells. Moreover, OBP-301 efficiently killed TGF-β-treated cancer cells, whereas TGF-β-treated cancer cells were resistant to cisplatin and docetaxel. Conclusion: Our results suggest that telomerase-specific oncolytic adenovirus OBP-301 inhibits TGF-β-induced EMT in human cancer cells. OBP-301 has therapeutic potential against cancer cells that have undergone EMT. Therefore, OBP-301 might be a promising anticancer agent to suppress EMT-mediated cancer progression and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 343. doi:1538-7445.AM2012-343

Epithelial to mesenchymal transition (EMT) induces cell migration, invasion, and drug resistance, and consequently, contributes to cancer metastasis and disease aggressiveness. This study attempted to address crucial biological parameters to correlate EMT and drug-treated cancer cells traversing through microcapillaries, reminiscent of metastatic conditions. MDA-MB-468 breast cancer cells induced to undergo EMT by treatment with 20 ng/mL of epidermal growth factor (EGF) were initially passed through several blockages and then through a constricted microchannel, mimicking the flow of invasive metastatic cells through constricted blood microcapillaries. EMT cells acquired enhanced migratory properties and retained 50% viability, even after migration through wells 10–15 μm in size and a constricted passage of 7 μm and 150 μm in length at a constant flow rate of 50 μL/h. The hydrodynamic properties revealed cellular deformation with a deformation index, average transit velocity, and entry time of 2.45, 12.3 mm/s, and 31,000 μs, respectively for a cell of average diameter 19 μm passing through one of the 7 μm constricted sections. Interestingly, cells collected at the channel outlet regained epithelial character, undergoing reverse transition (mesenchymal to epithelial transition, MET) in the absence of EGF. Remarkably, real-time polymerase chain reaction (PCR) analysis confirmed increases of 2- and 2.7-fold in the vimentin and fibronectin expression in EMT cells, respectively; however, their expression reduced to basal level in the MET cells. A scratch assay revealed the pronounced migratory nature of EMT cells compared with MET cells. Furthermore, the number of colonies formed from EMT cells and paclitaxel-treated EMT cells after passing through a constriction were found to be 95 ± 10 and 79 ± 4, respectively, confirming that the EMT cells were more drug resistant with a concomitant two-fold higher expression of the multi-drug resistance (MDR1) gene. Our results highlight the hydrodynamic and drug-evading properties of cells that have undergone an EMT, when passed through a constricted microcapillary that mimics their journey in blood circulation.

Cancer is one of the leading causes of death. A primary tumor forms when cells start to proliferate in an uncontrolled way and stop reacting to restraining signals. Tumors that reach a critical volume induce angiogenesis, a vascular remodeling process that provides nutrients and oxygen to the degenerated cell mass. Upon further tumor progression to a malignant cancer, cells acquire the ability to invade the surrounding tissue. In order to do so, formerly epithelial tumor cells undergo an epithelial to mesenchymal transition (EMT). This process of cell-cell detachment, breaching through the basement membrane and gaining migratory capabilities is the first step of the metastatic cascade. Metastasis is a process that allows tumor cells to leave the primary lesion and disseminate via the vascular system to secondary sites. Metastases, as the fatal feature of cancer, lead in most of the cases to patients’ death. Furthermore, metastasis, as the end stage of malignant disease, until now is incurable. This is because metastases are spread systemically throughout the body. Moreover, they even can establish from a disseminated tumor cell months after the primary tumor has already been surgically removed. Another barely controllable feature of cancer is tumor relapse. Even after a thorough surgery with the aim to completely remove the primary tumor and eventual draining lymph nodes combined with chemotherapy, patients relapse. Resistance to chemotherapy and establishment of metastases has both been accounted for to the abundance of cancer stem cells (CSC) within the tumor mass. These CSCs are endowed with the ability to evade chemotherapeutic drugs, they are mesenchymal in nature, migratory and invasive, and, most importantly, they are able to establish cancer and metastasis de novo. The work of my thesis has been dedicated to investigate the process of metastasis and the function of cancer initiation. Tumor initiation has recently been associated with EMT. To understand the functional circumstances how EMT cells gain the ability to form tumors, I used cellular murine breast cancer models. These model systems allowed me to study cells’ behavior before and after EMT in vitro and in vivo. In vitro experiments validated the observation of others that EMT cells indeed resemble cancer stem cells by being able to form hollow spheres and being susceptible to the CSC-specific drug salinomycin. In vivo studies revealed that EMT cells initiate tumors with a much earlier onset and with a higher efficiency when limited amounts of cells were injected orthotopically into mice compared to their epithelial counterpart. Moreover, EMT cell-generated cancers are highly vascularized already in the early phase of tumor establishment. Knockdown studies of the main pro-angiogenic factor VEGF-A revealed that it is required for early tumor onset. Thus, tumor angiogenesis is not only an effect of early and fast EMT-tumor progression. Further supporting this notion are limiting dilution experiments, which suggest that tumor initiation in EMT cells is a multifactorial event. This concept was validated by the observation that 10 EMT cells could initiate a tumor, whereas VEGF‑A knockdown cells could not. Hence, EMT-induced tumor initiation is achieved by the ability of promoting angiogenesis. EMT can be induced by the cytokine TGFβ. Normally, cells that experience TGFβ signaling become quiescent or die due to induction of apoptosis. Cancer cells can overcome these effects and react to TGFβ by undergoing EMT. As part of my thesis, I could show that the transcription factor Dlx2 is an important switch that allows cells to react to TGFβ by undergoing EMT without facing apoptosis induction. Dlx2 exerts its anti-apoptotic, pro-survival function by directly reducing TGFβRI expression and inducing the expression of the epidermal growth factor receptor ligand betacellulin. Another feature of EMT is the gain of cell motility. Cell migration is extremely important for cancer cells in order to leave their primary site and to disseminate. I have found ephrinB2 expression upregulated during EMT. EphrinB2 is a member of the Eph-ephrin signaling network that is known to be crucial for cell-cell communication. Thereby, cells that do not belong to the same entity repulse each other, restricting intermingling of tissue. Furthermore, ephrinB2 is required for neuronal axon guidance and angiogenesis. In my thesis, I showed that EMT cells need ephrinB2 to efficiently migrate. As an explanation for this phenotype I described that a knockdown for ephrinB2 led to an over-stabilization of focal adhesions. Cells normally use focal adhesions to hold on to an extracellular matrix (ECM) surface. Over-stabilization of focal adhesions attaches cells too firmly to the ECM and, hence, cells cannot retract their rear-end anymore which decreases cell motility. In summary, I succeeded in gathering further insights into tumor initiation, EMT, and with this, the metastatic process.

The sub-population of tumor cells termed 'cancer stem cells' (CSCs) possess the capability to generate tumors, undergo epithelial-mesenchymal transition (EMT) and are implicated in metastasis, making treatments to specifically target CSCs an attractive therapeutic strategy. Tumor hypoxia plays a key role in regulating EMT and cancer stem cell function. Carbonic anhydrase IX (CAIX) is a hypoxia-inducible protein that regulates cellular pH to promote cancer cell survival and invasion in hypoxic microenvironments and is a biomarker of poor prognosis for breast cancer metastasis and survival. Here, we demonstrate that inhibition of CAIX expression or activity with novel small-molecule inhibitors in breast cancer cell lines, or in primary metastatic breast cancer cells, results in the inhibition of breast CSC expansion in hypoxia. We identify the mTORC1 axis as a critical pathway downstream of CAIX in the regulation of cancer stem cell function. CAIX is also required for expression of EMT markers and regulators, as well as drivers of 'stemness', such as Notch1 and Jagged1 in isolated CSCs. In addition, treatment of mice bearing orthotopic breast tumors with CAIX-specific small-molecule inhibitors results in significant depletion of CSCs within these tumors. Furthermore, combination treatment with paclitaxel results in enhanced tumor growth delay and eradication of lung metastases. These data demonstrate that CAIX is a critical mediator of the expansion of breast CSCs in hypoxic niches by sustaining the mesenchymal and 'stemness' phenotypes of these cells, making CAIX an important therapeutic target for selectively depleting breast CSCs.

BackgroundAcute myeloid leukemia (AML) is a highly heterogeneous disease characterized by complex genetic and molecular features that contribute to poor prognosis and low cure rates. Therefore, identifying novel therapeutic targets is crucial for improving treatment efficacy and patient survival. This study investigated the potential role of cyclin-dependent kinase 9 (CDK9), a known regulator of gene expression, in AML pathogenesis and prognosis. MethodsThis study employed multiple bioinformatics approaches, including analysis of CDK9 expression across various cancers using the Tumor Immune Estimation Resource (TIMER2.0) database and further investigation of its expression and prognostic significance in AML using data from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases. Survival analysis and Cox regression analysis were used to assess the association between CDK9 expression and patient prognosis. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) were performed to elucidate the pathways and biological processes influenced by CDK9. Furthermore, the relationship between CDK9 expression and tumor immune infiltration was evaluated, and a protein–protein interaction (PPI) network was constructed. In vitro experiments, including Western blotting, CCK-8 assays, and flow cytometry, were conducted to validate the bioinformatics findings. ResultsBioinformatics analysis revealed significantly elevated CDK9 expression in AML samples, which correlated with poor patient prognosis. Functional enrichment analysis indicated that CDK9 is involved in key pathways related to cell proliferation, differentiation, and the tumor microenvironment. Moreover, the study observed a strong correlation between CDK9 expression and altered immune cell infiltration, suggesting a potential role in immune evasion. In vitro experiments confirmed that CDK9 overexpression promoted AML cell proliferation and inhibited apoptosis. Additionally, CDK9 showed a strong correlation with epithelial-mesenchymal transition (EMT)-related proteins, suggesting a potential role in AML progression and the EMT process. ConclusionsThis study demonstrates that CDK9 is a potential prognostic biomarker and therapeutic target in AML. Its involvement in multiple key pathways during AML development and its influence on the tumor immune microenvironment support further exploration of CDK9-targeted therapies to improve AML treatment outcomes.

The Role of EMT in Pancreatic Cancer Progression