Stem Cell-like Properties Research Articles

Distinct Cell-Intrinsic Functional Differences Between Iliac Crest and Distal Radius Autografts

Autologous bone grafts demonstrate osteoconductive, osteoinductive, and osteogenic properties. Hand surgeons commonly augment surgical fixation with autografts to promote fracture healing. This study compared the intrinsic stem cell-like properties of 2 commonly used autograft sources in hand surgery: the iliac crest and distal radius. A total of 9 subjects who received an iliac crest bone graft and distal radius bonegraft harvest as a part of the standard care of distal radius malunion or nonunion correction or scaphoid nonunion open reduction and internal fixation were enrolled in the study. Cells were isolated by serial collagenase digestion and subjected to fibroblast colony-forming units, osteogenesis, and adipogenesis assays. The expression levels of genes involved in osteogenesis and adipogenesis were confirmed using quantitative polymerase chain reaction. The cells isolated from the iliac crest bone graft compared with those isolated from the distal radius bone graft demonstrated significantly higher mean fibroblast colony-forming unit efficiency; increased osteogenesis, as measured using alizarin red quantification; increased adipogenesis, as measured using oil red O quantification; and higher expression levels of genes involved in osteogenesis and adipogenesis under the respective differentiation conditions. The cells isolated from the iliac crest bone graft demonstrated a higher fibroblast colony-forming unit capacity and an increased capability to undergo both osteogenesis and adipogenesis. Limited evidence exists comparing the intrinsic stem cell-like properties of the iliac crest and distal radius despite the widespread use of each source in hand and wrist surgery. The information from this investigation may assist hand and wrist surgeons with the selection of a source of autograft.

Concise review: Cancer cell reprogramming and therapeutic implications

The cancer stem cell (CSC) hypothesis postulates that cancer originates from the malignant transformation of stem cells and is considered to apply to a variety of cancers. Additionally, cancer cells alter metabolic processes to sustain their characteristic uncontrolled growth and proliferation. Further, microRNAs (miRNAs) are found to be involved in acquisition of stem cell-like properties, regulation and reprogramming of cancer cells during cancer progression through its post-transcriptional-regulatory activity. In this concise review, we aim to integrate the current knowledge and recent advances to elucidate the mechanisms involved in the regulation of cell reprogramming and highlights the potential therapeutic implications for the future.

Striking Circulating Tumour Cells during Sleep

Circulating tumour cells (CTCs) with stem cell-like properties and epithelial-mesenchymal transition phenotype are precursor cells responsible for dissemination and metastatic spread of cancer [...]

Uncovering the Heterogeneity and Clinical Relevance of Circulating Tumor-Initiating Cells in Hepatocellular Carcinoma Using an Integrated Immunomagnetic-Microfluidic Platform.

Circulating tumor-initiating cells (CTICs) with stem cell-like properties play pivotal roles in tumor metastasis and recurrence. However, little is known about the biology and clinical relevance of CTICs in hepatocellular carcinoma (HCC). Here, we investigated the molecular heterogeneity and clinical relevance of CTICs in HCC using a novel integrated immunomagnetic-microfluidic platform (iMAC). We constructed the iMAC and evaluated its ability to detect CTICs using a series of spiked cell experiments. A four-channel microfluidic chip was applied to investigate the composition of CTICs in patients with primary and recurrent HCC utilizing microbeads labeled with one of four stem-related markers: epithelial cell adhesion molecule (EpCAM), CD133, CD90, and CD24. The dynamic changes of these four CTIC subsets were serially monitored during treatment courses. Finally, single-cell RNA profiling was used to reveal the molecular characteristics of the four CTIC subsets. The iMAC platform detected significantly more EpCAM+ CTICs in the blood samples from 33 HCC patients than the FDA-approved CellSearch system (0.92 ± 0.94 vs 0.23 ± 0.36, P < 0.001). The number of EpCAM+ CTICs (≥0.75/mL) detected by iMAC was a predictor of early recurrence (P = 0.007). The distinct stem-related markers' expression of CTICs could distinguish primary HCC, recurrent HCC, and TACE-resistant HCC. Single-cell transcriptional profiling proved the heterogeneity among individual CTICs and separated the four CTIC subsets into distinct phenotypes. Dissecting the heterogeneity of CTICs using the iMAC represents a novel and informative method for accurate CTIC detection and characterization. This innovative technology will enable more indepth cancer biology research and clinical cancer management than is currently available.

Loss of exosomal miR-34c-5p in cancer-associated fibroblast for the maintenance of stem-like phenotypes of laryngeal cancer cells.

Cancer-associated fibroblasts (CAFs) reconstitute cancer stemness. This study aims to investigate whether the loss of CAF-derived exosomal miR-34c-5p contributes to the maintenance of stem-like properties of laryngeal squamous cell carcinoma (LSCC). Exosomes from primarily cultured CAFs and paired normal fibroblasts (NFs) were collected and identified. The differential expression of exosomal miR-34c-5p between CAFs and NFs was detected by next-generation sequencing. In vitro and in vivo assays were performed to examine the effects of miR-34c-5p on the maintenance of stem-like properties. MiR-34c-5p expression is significantly reduced in CAF-derived exosomes. In vitro and in vivo assays revealed that exosomal miR-34c-5p can regulate the stem-like properties of LSCC cells, such as proliferation, invasion, sphere and plate colony formation, chemoresistance, tumorigenicity in nude mice, as well as the expression of cancer stem cell genes. Loss of miR-34c-5p in CAF-derived exosomes contributes to the maintenance of stem-like phenotypes of LSCC.

AKT inhibition sensitizes EVI1 expressing colon cancer cells to irinotecan therapy by regulating the Akt/mTOR axis.

Ecotropic viral integration site 1 (EVI1) is an oncogenic transcription factor that has been attributed to chemotherapy resistance in different cancers. As yet, however, its role in colon cancer drug resistance is not completely understood. Here, we set out to investigate the functional and therapeutic relevance of EVI1 in colon cancer drug resistance. The EVI1 gene was knocked down in colon cancer cells that were subsequently tested for susceptibility to irinotecan using in vitro assays and in vivo subcutaneous mouse colon cancer models. The effect of EVI1 knockdown on the AKT-mTOR signaling pathway was assessed using cell line models, immunohistochemistry and bioinformatics tools. The anti-proliferative activity of AKT inhibitor GSK690693 and its combination with irinotecan was tested in colon cancer cell line models (2D and 3D). Finally, the therapeutic efficacy of GSK690693 and its combination with irinotecan was evaluated in xenografted EVI1 expressing colon cancer mouse models. We found that EVI1 knockdown decreased cancer stem cell-like properties and improved irinotecan responses in both cell line and subcutaneous mouse models. In addition, we found that EVI1 downregulation resulted in inhibition of AKT/mTOR signaling and RICTOR expression. Knocking down RICTOR expression increased the cytotoxic effects of irinotecan in EVI1 downregulated colon cancer cells. Co-treatment with irinotecan and ATP-competitive AKT inhibitor GSK690693 significantly reduced colon cancer cell survival and tumor progression rates. Inhibition of the AKT signaling cascade by GSK690693 may serve as an alternative to improve the irinotecan response in EVI1-expressing colon cancer cells.

Antitumor Effect of Si-Jun-Zi Decoction on SGC7901 Gastric Cancer Cells by CMTM2 Activation.

The Si-Jun-Zi decoction (SJZ), a traditional Chinese medicine (TCM) formula, is used clinically against multiple malignancies, including gastric cancer (GC). In previous study, we have shown that SJZ plays an anticancer role in SGC7901 cell xenograft mice models. However, the underlying mechanisms are unclear. The objective of this study was to evaluate the effect and mechanism of SJZ on the proliferation, migration, invasion, and cancer stem cell-like properties of GC cells. High-throughput mRNA sequencing analysis was performed to investigate the global alterations in gene expression in xenograft tumors, and 56 significantly differentially expressed genes (43 upregulated and 13 downregulated genes) were identified between the SJZ group and the Model group totally. We focused on CMTM2, which was significantly increased after SJZ intervention, as a candidate target gene of SJZ. The results indicated that CMTM2 expression was elevated in SJZ-treated SGC7901 cells and knocking-down CMTM2 expression partially hampered the inhibitory effects of SJZ on the proliferation, migration, and invasion of GC cells. Moreover, SJZ treatment repressed the spheroid and colony-forming capacity in GC cells, accompanied by downregulation of stem cell markers including SOX2, NANOG, and CD44. CMTM2 knockdown antagonized the effects of SJZ on the cancer stem cell-like properties of SGC7901 cells. Thus, SJZ effectively suppressed the proliferation, migration, invasion, and cancer stem cell-like properties of GC cells in vitro by upregulating CMTM2 expression.

MiR-26a-5p Suppresses Wnt/β-Catenin Signaling Pathway by Inhibiting DNMT3A-Mediated SFRP1 Methylation and Inhibits Cancer Stem Cell-Like Properties of NSCLC

Background Lung cancer is a malignant cancer which results in the most cancer incidence and mortality worldwide. There is increasing evidence that the pattern of DNA methylation affects tumorigenesis and progression. However, the molecules and mechanisms regulating DNA methylation remain unclear. Methods The expression of miR-26a-5p in NSCLC cell lines was detected by qPCR and verified in NSCLC tissues from TCGA using Limma R package. CCK-8 assay, plate clone formation assay, flow cytometry, and sphere formation assay were used to detect the cell proliferation, colony formation, cell cycle, and cancer stem cell- (CSC-) like property in NSCLC cell lines. The immunoblotting was used to detect the protein levels of DNMT3A, SFRP1, and Ki67. Global DNA methylation levels and DNA methylation levels of SFRP1 promoter were examined using ELISA and MSP-PCR assay, respectively. The distribution of β-catenin was examined using immunofluorescence (IF). Besides, xenograft mouse model was used to investigate the antitumor effects of miR-26a-5p in vivo. The pathology and protein levels were, respectively, detected by hematoxylin and eosin (H&E) and immunocytochemistry (IHC). Results The expression of miR-26a-5p was downregulated in the tumor tissues comparted to adjacent normal tissues as well as NSCLC cell lines compared to normal lung epithelial cell (BEAS2B). The overexpression of miR-26a-5p inhibited cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase. DNMT3A was a target of miR-26a-5p and upregulated DNA methylation on SFRP1 promoter. Mechanistically, miR-26a-5p repressed cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase by binding DNMT3A to reduce DNA methylation levels of SFRP1 then upregulated SFRP1 expression. Moreover, miR-26a-5p exerted antitumor effects in vivo. Conclusion Our results revealed that miR-26a-5p acted as a tumor suppressor through targeting DNMT3A to upregulate SFRP1 via reducing DNMT3A-dependent DNA methylation.

Activation of MYO1G by lncRNA MNX1-AS1 Drives the Progression in Lung Cancer.

Lung cancer represents the most prevalent cancer worldwide and causes the death of many patients. Cancer stem cells (CSCs), a subpopulation of cancer cells, have the capacities of self-renewal, unlimited proliferation, and multiple differentiation potential. The purpose of this study was to explore the potential role of long noncoding RNA (lncRNA) MNX1-AS1 on maintaining the stemness of CSC in lung cancer. CSCs were firstly sorted by flow cytometry. After the determination of the target of the present study using Gene Expression Omnibus dataset, MNX1-AS1was found to be highly expressed in lung cancer tissues and cells. Deletion of MNX1-AS1 inhibited proliferation, migration, invasion and sphere-forming abilities of CSC. Furthermore, subcellular fractionation, fluorescence in situ hybridization, RNA immunoprecipitation, and dual-luciferase experiments demonstrated that MNX1-AS1 recruited the transcription factor POU domain class 2 transcription factor 2 (POU2F2) to the nucleus and activated the myosin IG (MYO1G) expression. MYO1G overexpression partially reversed the si-MNX1-AS1-decreased stemness of CSCs. Finally, MNX1-AS1 suppression significantly repressed the growth of xenografts in vivo. Our study highlights the importance of the MNX1-AS1/POU2F2/MYO1G axis in stem cell-like properties of lung cancer cells.

JIB-04, a Pan-Inhibitor of Histone Demethylases, Targets Histone-Lysine-Demethylase-Dependent AKT Pathway, Leading to Cell Cycle Arrest and Inhibition of Cancer Stem-Like Cell Properties in Hepatocellular Carcinoma Cells.

JIB-04, a pan-histone lysine demethylase (KDM) inhibitor, targets drug-resistant cells, along with colorectal cancer stem cells (CSCs), which are crucial for cancer recurrence and metastasis. Despite the advances in CSC biology, the effect of JIB-04 on liver CSCs (LCSCs) and the malignancy of hepatocellular carcinoma (HCC) has not been elucidated yet. Here, we showed that JIB-04 targeted KDMs, leading to the growth inhibition and cell cycle arrest of HCC, and abolished the viability of LCSCs. JIB-04 significantly attenuated CSC tumorsphere formation, growth, relapse, migration, and invasion in vitro. Among KDMs, the deficiency of KDM4B, KDM4D, and KDM6B reduced the viability of the tumorspheres, suggesting their roles in the function of LCSCs. RNA sequencing revealed that JIB-04 affected various cancer-related pathways, especially the PI3K/AKT pathway, which is crucial for HCC malignancy and the maintenance of LCSCs. Our results revealed KDM6B-dependent AKT2 expression and the downregulation of E2F-regulated genes via JIB-04-induced inhibition of the AKT2/FOXO3a/p21/RB axis. A ChIP assay demonstrated JIB-04-induced reduction in H3K27me3 at the AKT2 promoter and the enrichment of KDM6B within this promoter. Overall, our results strongly suggest that the inhibitory effect of JIB-04 on HCC malignancy and the maintenance of LCSCs is mediated via targeting the KDM6B-AKT2 pathway, indicating the therapeutic potential of JIB-04.

The Road Less Taken: Less Appreciated Pathways for Manipulating CD8+ T Cell Exhaustion.

Exhausted CD8+ T (Tex) cells are a distinct cell population that arise during persistent antigen exposure in the context of chronic infections and cancers. Although characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression and distinct transcriptional and epigenetic programs, Tex cells are heterogeneous. Among these, a self-renewing TCF-1+ Tex population, having unique characteristics and the ability to respond to immune-checkpoint blockade, gives rise to TCF-1- terminally Tex cells. These TCF-1+ cells have stem cell-like properties similar to memory T cell populations, but the signals that regulate the developmental pathways and relationships among exhausted cell populations are still unclear. Here, we review our current understanding of Tex cell biology, and discuss some less appreciated molecules and pathways affecting T cell exhaustion. We highlight two co-stimulatory receptors, CD226 and CD137, and their role in inducing or restraining T cell exhaustion, as well as signaling pathways that may be amenable to pharmacological inhibition with a focus on Phosphoinositide-3 Kinase and IL-2 partial agonists. Finally, we discuss novel methods that may increase TCF-1+ populations and therefore improve immunotherapy responsiveness. Understanding features of and pathways to exhaustion has important implications for the success of immunotherapy, including checkpoint blockade and adoptive T-cell transfer therapies.

Cytokine‐ and chemokine‐induced inflammatory colorectal tumor microenvironment: Emerging avenue for targeted therapy

Colorectal cancer (CRC) is a predominant life‐threatening cancer, with liver and peritoneal metastases as the primary causes of death. Intestinal inflammation, a known CRC risk factor, nurtures a local inflammatory environment enriched with tumor cells, endothelial cells, immune cells, cancer‐associated fibroblasts, immunosuppressive cells, and secretory growth factors. The complex interactions of aberrantly expressed cytokines, chemokines, growth factors, and matrix‐remodeling enzymes promote CRC pathogenesis and evoke systemic responses that affect disease outcomes. Mounting evidence suggests that these cytokines and chemokines play a role in the progression of CRC through immunosuppression and modulation of the tumor microenvironment, which is partly achieved by the recruitment of immunosuppressive cells. These cells impart features such as cancer stem cell‐like properties, drug resistance, invasion, and formation of the premetastatic niche in distant organs, promoting metastasis and aggressive CRC growth. A deeper understanding of the cytokine‐ and chemokine‐mediated signaling networks that link tumor progression and metastasis will provide insights into the mechanistic details of disease aggressiveness and facilitate the development of novel therapeutics for CRC. Here, we summarized the current knowledge of cytokine‐ and chemokine‐mediated crosstalk in the inflammatory tumor microenvironment, which drives immunosuppression, resistance to therapeutics, and metastasis during CRC progression. We also outlined the potential of this crosstalk as a novel therapeutic target for CRC. The major cytokine/chemokine pathways involved in cancer immunotherapy are also discussed in this review.

Tumor necrosis factor-α-inducing protein of Helicobacter pylori promotes epithelial-mesenchymal transition and cancer stem-like cells properties via activation of Wnt/β-catenin signaling pathway in gastric cancer cells.

Tumor necrosis factor-α-inducing protein (Tipα) is a newly identified toxin that promotes the inflammation and carcinogenesis caused by Helicobacter pylori. However, its mechanism of pathogenesis is still unclear. To investigate the carcinogenic mechanisms of Tipα, SGC7901 cells and SGC7901-derived cancer stem-like cells (CSCs) were stimulated by recombinant Tipα with or without Wnt/β-catenin signaling inhibitor XAV939. qRT-PCR and Western blotting were employed to detect expression of epithelial-mesenchymal transition (EMT), CSCs markers and downstream target genes of this signaling pathway. The cell migration ability was measured by wound healing assay and transwell assay. Our results indicated that Tipα promoted CSC properties of SGC7901 spheroids, including increased expression of CSC specific surface markers CD44, Oct4 and Nanog and an increased capacity for self-renewal. Tipα activated Wnt/β-catenin signaling in both SGC7901 cells or CSCs. Furthermore, Tipα induced the EMT and increased the expressions of downstream target genes of this signaling, including c-myc, cyclin D1 and CD44. However, XAV939 pretreatment inhibited Tipα-induced EMT and CSC properties in SGC7901 cells or CSCs. These results suggest that Tipα promotes EMT and CSC-like properties in gastric cancer cells through activation of Wnt/β-catenin signaling pathway, thereby accelerating the progression of gastric cancer.

WNT/β-Catenin-Mediated Resistance to Glucose Deprivation in Glioblastoma Stem-like Cells.

Simple SummaryThe malignant growth and therapy resistance of isocitrate dehydrogenase (IDH)-wildtype glioblastoma is thought to be driven by a subpopulation of tumor cells with cancer stem-like cell (CSC) properties. Employing a high-throughput in vitro drug screen, we identified LGK974 and berberine as drugs that impair wingless (WNT) signaling and can thereby sensitize glioblastoma stem-like cells (GSCs) to glucose starvation-induced cell death. The main goal of this study was to characterize the role of the WNT pathway in mediating the survival and metabolic plasticity of GSCs under nutrient-restricted growth conditions. Gas chromatography mass spectrometry (GC-MS) was used to determine WNT-specific alterations of intracellular metabolites in GSCs grown under nutrient restriction, i.e., glucose depletion, or under standard conditions. Metabolic fingerprints hold the promise to complement classic biomarkers, thus potentially aiding the prediction of tumor behavior and patient prognosis.Isocitrate dehydrogenase (IDH)-wildtype glioblastoma is the most common primary malignant brain tumor. It is associated with a particularly poor prognosis, as reflected by an overall median survival of only 15 months in patients who undergo a supramarginal surgical reduction of the tumor mass followed by combined chemoradiotherapy. The highly malignant nature of IDH-wildtype glioblastoma is thought to be driven by glioblastoma stem-like cells (GSCs) that harbor the ability of self-renewal, survival, and adaptability to challenging environmental conditions. The wingless (WNT) signaling pathway is a phylogenetically highly conserved stemness pathway, which promotes metabolic plasticity and adaptation to a nutrient-limited tumor microenvironment. To unravel the reciprocal regulation of the WNT pathway and the nutrient-limited microenvironment, glioblastoma cancer stem-like cells were cultured in a medium with either standard or reduced glucose concentrations for various time points (24, 48, and 72 h). Glucose depletion reduced cell viability and facilitated the survival of a small population of starvation-resistant tumor cells. The surviving cells demonstrated increased clonogenic and invasive properties as well as enhanced chemosensitivity to pharmacological inhibitors of the WNT pathway (LGK974, berberine). Glucose depletion partially led to the upregulation of WNT target genes such as CTNNB1, ZEB1, and AXIN2 at the mRNA and corresponding protein levels. LGK974 treatment alone or in combination with glucose depletion also altered the metabolite concentration in intracellular compartments, suggesting WNT-mediated metabolic regulation. Taken together, our findings suggest that WNT-mediated metabolic plasticity modulates the survival of GSCs under nutrient-restricted environmental conditions.

Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

Simple SummaryCertain tumor aggressiveness-associated mediators from cancer-associated fibroblasts (CAFs) in tumor microenvironment have been reported. Using gene expression analysis, we identified that CAFs overexpress Chemokine ligand 11 (CCL11), which is associated with tumor migration and invasion, increased expression of cancer stem cell properties, and induction of the epithelial-to-mesenchymal transition. Neutralization of CAF-induced CCL11 reversed the aggressive phenotype of cancer cells. Based on the immunohistochemical staining of clinical samples, we found that increased co-expression of CCL11 and its receptor, C-C Motif Chemokine Receptor 3 (CCR3), was associated with poor overall survival. Our results suggest that targeting CCL11-CCR3 signaling is a potential therapeutic strategy for patients with aggressive head and neck cancer. The tumor microenvironment (TME) plays a crucial role in tumor progression. One of its key stromal components, cancer-associated fibroblasts (CAFs), may crosstalk with cancer cells by secreting certain cytokines or chemokines. However, which important mediator(s) are released by CAFs, and the underlying molecular mechanism, remain largely unknown. In the present study, we isolated patient-derived CAFs and normal fibroblasts (NFs). Using microarray analysis, we detected chemokine ligand 11 (CCL11) overexpression in CAFs compared to NFs. CCL11 administration promoted the migration and invasion of head and neck cancer (HNC) cells with enhanced cancer stem cell-like properties and induction of epithelial-to-mesenchymal transition. Furthermore, neutralization of CCL11 activity reversed the aggressive phenotype of CAF-induced cancer cells. Confocal microscopy showed colocalization of CCL11 and CC chemokine receptor 3 (CCR3) on HNC cells. Moreover, immunohistochemical analysis of clinical samples from 104 patients with HNC showed that expression of CCL11 and CCR3 were significantly correlated with poor overall survival (p = 0.003 and 0.044, respectively). Collectively, CCL11 expressed on CAFs promotes HNC invasiveness, and neutralization of CCL11 reverses this effect. We propose that the CCL11/CCR3 signaling circuit is a potential target for optimizing therapeutic strategies against HNC.

Activation of NOTCH signaling via DLL1 is mediated by APE1-redox-dependent NF-κB activation in oesophageal adenocarcinoma

ObjectiveOesophageal adenocarcinoma (EAC) arises in the setting of Barrett’s oesophagus, an intestinal metaplastic precursor lesion that can develop in patients with chronic GERD. Here, we investigated the role of acidic...

In Vitro Characterization of the Human Skeletal Stem Cell-like Properties of Primary Bone-Derived Mesenchymal Stem/Stromal Cells in Patients with Late and Early Hip Osteoarthritis.

Human skeletal stem cells (hSSCs) were recently identified as podoplanin (PDPN)/CD73/CD164-positive and CD146-negative cells that decline with age, and play a role in the pathogenesis of osteoarthritis (OA). The aim of this study was to identify the hSSC-like properties of bone-derived mesenchymal stem/stromal cells (MSCs) of patients with late and early OA. Methods: First, we performed gene expression profiling for the hSSC markers in 32 patients with late and early OA, and donors without OA. Having identified the low expression of hSSC markers in late OA patients, we further performed trilineage differentiation and immunophenotyping for hSSC makers in the selected subsets from each donor group. Results: Our results show no differences in osteogenesis, chondrogenesis, and adipogenesis between the MSCs from the three groups. However, the immunophenotyping shows lower CD164 in MSCs from early OA patients in comparison with late and no OA subjects (p = 0.002 and p = 0.017). Conclusions: Our study shows that the in vitro hSSC-like properties of bone-derived MSCs are similar in patients with early and late OA, and in donors without OA. However, the lower percentage of CD164-positive MSCs in early OA patients indicates the potential of CD164 as a marker of the onset of OA.

Abstract 896: An antifungal, Itraconazole, induces cell death by targeting HER2 signaling and stem-like properties in trastuzumab-resistant HER2-positive breast cancer

Abstract Trastuzumab resistance in HER2-positive breast cancer leads to a poorer prognosis and shorter overall survival. Identifying existing drugs for another purpose of use may be considered as a part of new approaches to overcome resistance in cancer biology. Itraconazole is a FDA-approved drug, which is one of anti-fungal drugs. This drug has been reported to exploit as a cancer therapeutic agent in several cancer types. We sought to investigate the anti-cancer effects of itraconazole on cell proliferation, apoptosis, autophagy and breast cancer stem cell-like properties in terms of challenging trastuzumab resistance in HER2-positive breast cancer. The effect of itraconazole on trastuzumab-resistant cell line, JIMT-1, in vitro was examined in aspects of cell viability, apoptosis, autophagy, and impact on cancer stem cells. As in vivo experimental model, trastuzumab-resistant JIMT-1 cells were implanted to generate xenografts to study anti-tumor efficacy of itraconazole. Treatment of itraconazole significantly suppressed the growth of JIMT-1 cells with marked induction of apoptosis. Itraconazole downregulated p185HER2 and truncated-p95HER2, and their phosphorylation levels in JIMT-1 cells. In addition, the outcome of the decrease in the levels of Beclin-1 and p62 and the increase of LC3 I/II after the exposure to itraconazole supported that itraconazole induced autophagy as well. Importantly, itraconazole not only killed proliferating tumor cells but also effectively eradicated cancer stem-like populations. To elucidate eradication of cancer stem-like population by itraconazole, ALDH1 activity assay and FACS analysis of CD44+/CD24- stem-like phenotype in JIMT-1 cells were carried out. As a result, stem-cell like populations were impaired as evidenced by a significant decrease in ALDH1 activity and CD44+/CD24- stem-like populations. Through in vivo mouse model, the physiological relevance of in vitro observations was confirmed. Evident inhibition of tumor burden and growth in trastuzumab-resistant xenografts was shown in consequence of itraconazole administration, accompanying by substantial downregulation of HER2, ALDH1 and microvessel density as well as dramatic decrease of p62 in vivo. No injury to liver or kidneys by itraconazol was found based on no statistically significant difference in serum levels of ALT, AST and BUN between vehicle- and itraconazole-administrated groups. We have demonstrated that itraconazole, which is FDA-approved drug as an anti-fungal drug, exerts anti-tumor activity in trastuzumab-resistant HER2-positive breast cancer by targeting cancer stem-like properties, suppression of the HER2 signaling as well as induction of autophagy. These findings support the notion that itraconazole might be a new strategic approach as a treatment for trastuzumab-resistant HER2-positive breast cancers. Citation Format: Jung Min Park, Soeun Park, Minsu Park, Seongjae Kim, Juyeon Seo, Dongmi Ko, Kee Dal Nam, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. An antifungal, Itraconazole, induces cell death by targeting HER2 signaling and stem-like properties in trastuzumab-resistant HER2-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 896.

Abstract 777: Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties

Abstract Mantle cell lymphoma (MCL) is a mature B cell neoplasm with two distinct biological subtypes regarding clinical, molecular and pathological features. Conventional MCL (cMCL) subtype is characterized by high aggressiveness and poor outcome with frequent relapses after initial response to treatment, suggesting the presence of a population of cancer stem cells (CSC) able to self-renew and responsible for drug resistance. SOX11 embryonic transcription factor is aberrantly overexpressed mostly in cMCL and has an oncogenic role in MCL. MCL-CSC have been isolated from MCL cases by different groups. However, its relationship to SOX11 expression and contribution to MCL clinical features and aggressive behavior remains unknown. We hypothesized that SOX11 may promote stem cell-like properties through the activation of stem cell-related genes, leading to an aggressive and incurable tumor in MCL patients. Here, we integrated the differential gene expression profile (GEP) between SOX11+ and SOX11- MCL primary cases with stem cell-related genes, and specific SOX11 ChIP-chip and epigenetic data in MCL cell lines and primary cases, to identify potential mediators of CSC directly regulated by SOX11 in MCL. We observed an enrichment of hematopoietic (HSC) and leukemic stem cells (LSC) gene signatures in SOX11+ compared to SOX11- MCL primary cases. MSI2, an RNA binding protein that maintains self-renewal and prevents differentiation in HSC, emerged as one of the most significant upregulated CSC-related gene in SOX11+ compared to SOX11- MCLs, positively correlating with SOX11 expression in MCL primary cases. Our in vitro experiments showed that SOX11 binds to MSI2 regulatory regions increasing its expression in MCL cells. MSI2 enhancers located in intronic regions were activated in SOX11+ but not in SOX11- MCL cell lines and primary cases. MSI2 upregulation significantly associated with poor overall survival, independently of common risk factors (SOX11 expression, high copy number alterations, 17p/TP53, 11q/ATM and 9p/CDKN2A alterations), in MCL patients. MSI2 knockdown MCL cell lines had a gene expression profile enriched in proapoptotic-related genes; whereas HSC and LSC gene signatures were downregulated compared to its control cells. MSI2 knockdown or MSI2 inhibition with Ro 08-2750 treatment decreased clonogenic growth, tumor survival and chemoresistance in MCL cells. SOX11+/MSI2 high MCL primary leukemic samples showed higher percentage of ALDH+ cells compared to SOX11-/MSI2 low samples, which decreased after Ro 08-2750 treatment. Besides, MSI2 silencing delayed tumor growth in vivo in MCL xenograft mice models. Overall, our results suggest that SOX11+ MCL cases acquire stemness features through the upregulation of MSI2 expression, which promotes chemoresistance, self-renewal and tumor survival. MSI2 represents a novel biomarker for MCL-CSC and therapeutic target for relapsed MCL. Citation Format: Marta Sureda-Gómez, Patricia Balsas, Marta Leonor Rodríguez, Ferran Nadeu, Anna De Bolòs, Álvaro Eguileor, Marta Kulis, Giancarlo Castellano, José Ignacio Martin-Subero, Santiago Demajo, Pedro Jares, Eva Giné, Elias Campo, Virginia Amador. Deciphering the role of MSI2 as a regulator of mantle cell lymphoma stem-like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 777.

Androgen receptor-mediated CD8+ T cell stemness programs drive sex differences in antitumor immunity

The incidence and mortality rates of many non-reproductive human cancers are generally higher in males than in females. However, the immunological mechanism underlying sexual differences in cancers remains elusive. Here, we demonstrated that sex-related differences in tumor burden depended on adaptive immunity. Male CD8+ Tcells exhibited impaired effector and stem cell-like properties compared with female CD8+ Tcells. Mechanistically, androgen receptor inhibited the activity and stemness of male tumor-infiltrating CD8+ Tcells by regulating epigenetic and transcriptional differentiation programs. Castration combined with anti-PD-L1 treatment synergistically restricted tumor growth in male mice. In humans, fewer male CD8+ Tcells maintained a stem cell-like memory state compared with female counterparts. Moreover, AR expression correlated with tumor-infiltrating CD8+ Tcell exhaustion in cancer patients. Our findings reveal sex-biased CD8+ Tcell stemness programs in cancer progression and in the responses to cancer immunotherapy, providing insights into the development of sex-based immunotherapeutic strategies for cancer treatment.