Chemoresistant Cancer Stem Cells Research Articles

Abstract IA23: "NO Target for Cancer Stem Cells"

Abstract Developing novel therapies that target cancer metastasis, chemoresistant cancer stem cells, and augment the efficacy of systemic chemo/radiotherapies is crucial to reducing cancer-associated mortality. A protein within the tumor microenvironment that has gained interest as a promising therapeutic target is nitric oxide synthase (NOS). Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in triple negative breast cancer (TNBC) patients. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway may be one of the most common oncogenic aberrations in certain TNBC subsets, in particular metaplastic breast cancer (MpBC). We will summarize the role of NO in PI3K-Akt pathway activation in human TNBC, as well as data from The Cancer Genome Atlas of the role of iNOS and PI3K-Akt interaction. This review discusses the current knowledge of the roles of nitric oxide (NO) in tumor progression and cancer metastasis, and promising preclinical studies that evaluated NOS inhibitors as an anti-cancer therapy. Lastly, we will offer a perspective on utilizing NOS inhibitors in the clinical setting, highlighting the prospects and outstanding challenges. Citation Format: Jenny C. Chang. "NO Target for Cancer Stem Cells" [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA23.

Histone demethylase KDM6A coordinating with KMT2B regulates self-renewal and chemoresistance of non-small cell lung cancer stem cells

Background and aimsWnt signaling is essential for the maintenance of cancer stem cells (CSCs), but mutations in the β-catenin and APC genes are less common in non-small cell lung carcinoma (NSCLC). Thus, the mechanism underlying the constitutive activation of Wnt signaling in lung CSCs is still unknown. Materials and methodsGene set enrichment analysis and immunohistochemistry were performed to establish the correlation between KDM6A/KM2B and CSC stemness. Human NSCLC cell lines were genetically manipulated for functional studies. Sphere formation assay and stemness gene expression profiling were examined to investigate the role of KDM6A/KMT2B in lung CSCs. Tumor xenograft assay were used to identify the function of KDM6A/KMT2B on tumorigenicity and tumor recurrence in vivo. Western blot analysis, coimmunoprecipitation and chromatin immunoprecipitation were performed to understand KDM6A/KMT2B mediated epigenetic regulation of Histone 3 lysine 4 methylation (H3K4me) on Wnt signaling pathway. ResultsWe discovered that the expression of Histone demethylase KDM6A and methyltransferase KMT2B correlate with the stemness of CSCs in NSCLC. KDM6A coordinates with KMT2B to activate the Wnt/β-catenin signaling pathway by regulating the H3K4me3 level and promotes the tumorigenicity and maintenance of CSC stemness. Furthermore, KDM6A/ KMT2B overexpression promotes the CSC chemoresistance and tumor recurrence both in vitro and in vivo. Inhibition of KDM6A and KMT2B potently suppress tumor initiation and recurrence in xenografted animal models. ConclusionOur findings suggest that KDM6A and KMT2B mediate the constitutive activation of Wnt/β-catenin signaling in lung CSCs, potentially providing a therapeutic target for NSCLC.

Colorectal cancer and therapy response: a focus on the main mechanisms involved.

The latest GLOBOCAN 2021 reports that colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. Most CRC cases are sporadic and associated with several risk factors, including lifestyle habits, gut dysbiosis, chronic inflammation, and oxidative stress. To summarize the biology of CRC and discuss current therapeutic interventions designed to counteract CRC development and to overcome chemoresistance. Literature searches were conducted using PubMed and focusing the attention on the keywords such as "Current treatment of CRC" or "chemoresistance and CRC" or "oxidative stress and CRC" or "novel drug delivery approaches in cancer" or "immunotherapy in CRC" or "gut microbiota in CRC" or "systematic review and meta-analysis of randomized controlled trials" or "CSCs and CRC". The citations included in the search ranged from September 1988 to December 2022. An additional search was carried out using the clinical trial database. Rounds of adjuvant therapies, including radiotherapy, chemotherapy, and immunotherapy are commonly planned to reduce cancer recurrence after surgery (stage II and stage III CRC patients) and to improve overall survival (stage IV). 5-fluorouracil-based chemotherapy in combination with other cytotoxic drugs, is the mainstay to treat CRC. However, the onset of the inherent or acquired resistance and the presence of chemoresistant cancer stem cells drastically reduce the efficacy. On the other hand, the genetic-molecular heterogeneity of CRC often precludes also the efficacy of new therapeutic approaches such as immunotherapies. Therefore, the CRC complexity made of natural or acquired multidrug resistance has made it necessary the search for new druggable targets and new delivery systems. Further knowledge of the underlying CRC mechanisms and a comprehensive overview of current therapeutic opportunities can provide the basis for identifying pharmacological and biological barriers that render therapies ineffective and for identifying new potential biomarkers and therapeutic targets for advanced and aggressive CRC.

Therapeutic inhibition of Bmi-1 ablates chemoresistant cancer stem cells in adenoid cystic carcinoma

ObjectivesAdenoid Cystic Carcinomas (ACC) typically show modest responseto cytotoxic therapy. Cancer stem cells (CSC) have been implicated in chemoresistance and tumor relapse. However, their role in ACC remains unknown. The purpose of this work was to evaluate the impact of targeting ACC CSCs with Bmi-1 inhibitors on resistance to cytotoxic therapy and tumor relapse. Materials and methodsTherapeutic efficacy of a small molecule inhibitor of Bmi-1 (PTC596; Unesbulin) and/or Cisplatin on ACC stemness was evaluated in immunodeficient mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in human ACC cell-lines (UM-HACC-2A,-14) or low passage primary human ACC cells (UM-HACC-6). The effect of therapy on stemness was examined by salisphere assays, flow cytometry for ALDH activity and CD44 expression, and Western blots for Bmi-1 (self-renewal marker) and Oct4 (embryonic stem cell marker) expression. ResultsPlatinum-based agents (Cisplatin, Carboplatin) induced Bmi-1 and Oct4 expression, increased salisphere formation and the CSC fraction in vitro and in vivo. In contrast, PTC596 inhibited expression of Bmi-1, Oct4 and pro-survival proteins Mcl-1 and Claspin; decreased the number of salispheres, and the fraction of ACC CSCs in vitro. Silencing Claspin decreased salisphere formation and CSC fraction. Both, single agent PTC596 and PTC596/Cisplatin combination decreased the CSC fraction in PDX ACC tumors. Notably, short-term combination therapy (2 weeks) with PTC596/Cisplatin prevented tumor relapse for 150 days in a preclinical trial in mice. ConclusionTherapeutic inhibition of Bmi-1 ablates chemoresistant CSCs and prevents ACC tumor relapse. Collectively, these results suggest that ACC patients might benefit from Bmi-1-targeted therapies.

SETD4 Confers Cancer Stem Cell Chemoresistance in Nonsmall Cell Lung Cancer Patients via the Epigenetic Regulation of Cellular Quiescence.

Increasing evidence indicates that quiescent cancer stem cells (CSCs) are a root cause of chemoresistance. SET domain-containing protein 4 (SETD4) epigenetically regulates cell quiescence in breast cancer stem cells (BCSCs), and SETD4-positive BCSCs are chemoradioresistant. However, the role of SETD4 in chemoresistance, tumor progression, and prognosis in nonsmall cell lung cancer (NSCLC) patients is unclear. Here, SETD4-positive cells were identified as quiescent lung cancer stem cells (qLCSCs) since they expressed high levels of ALDH1 and CD133 and low levels of Ki67. SETD4 expression was significantly higher in advanced-stage NSCLC tissues than in early-stage NSCLC tissues and significantly higher in samples from the chemoresistant group than in those from the chemosensitive group. Patients with high SETD4 expression had shorter progression-free survival (PFS) times than those with low SETD4 expression. SETD4 facilitated heterochromatin formation via H4K20me3, thereby leading to cell quiescence. RNA-seq analysis showed upregulation of genes involved in cell proliferation, glucose metabolism, and PI3K-AKT signaling in activated qLCSCs (A-qLCSCs) compared with qLCSCs. In addition, SETD4 overexpression facilitated PTEN-mediated inhibition of the PI3K-mTOR pathway. In summary, SETD4 confers chemoresistance, tumor progression, and a poor prognosis by regulating CSCs in NSCLC patients.

A Tumor Microenvironment-Driven Network Regulated by STAT3 and p65 Negatively Controls the Enrichment of Cancer Stem Cells in Human HR+/HER2− Breast Cancer

Hormone receptor-positive and HER2-negative (HR+/HER2-; luminal A) tumors are prevalent in breast cancer. Our past studies demonstrated that "TME Stimulation" (estrogen + TNFα + EGF, representing three arms of the tumor microenvironment, TME) has enriched metastasis-forming cancer stem cells (CSCs) in HR+/HER2- human breast cancer cells. Here, following information obtained by RNAseq analyses of TME-stimulated CSCs and Non-CSCs, we found that TME Stimulation has induced the activation of S727-STAT3, Y705-STAT3, STAT1 and p65. Upon TME Stimulation, stattic (STAT3 inhibitor) usage demonstrated that Y705-STAT3 activation negatively controlled CSC enrichment and epithelial-to-mesenchymal transition (EMT) traits, while inducing CXCL8 (IL-8) and PD-L1 expression. However, STAT3 knock-down (siSTAT3) had no effect on these functions; in terms of CSC enrichment, p65 had down-regulatory roles that compensated for the loss of an entire STAT3 protein. Y705-STAT3 and p65 acted additively in reducing CSC enrichment, and Y705A-STAT3 variant + sip65 has enriched chemo-resistant CSCs. Clinical data analyses revealed an inverse correlation between Y705-STAT3 + p65 phosphorylation and CSC signature in luminal A patients, and connection to improved disease course. Overall, we find regulatory roles for Y705-STAT3 and p65 in TME-stimulated HR+/HER2- tumors, with the ability to limit CSC enrichment. These findings raise concerns about using inhibitors of STAT3 and p65 as therapeutic strategies in the clinic.

Bench to Bedside: New Therapeutic Approaches with Extracellular Vesicles and Engineered Biomaterials for Targeting Therapeutic Resistance of Cancer Stem Cells.

Cancer has recently been the second leading cause of death worldwide, trailing only cardiovascular disease. Cancer stem cells (CSCs), represented as tumor-initiating cells (TICs), are mainly liable for chemoresistance and disease relapse due to their self-renewal capability and differentiating capacity into different types of tumor cells. The intricate molecular mechanism is necessary to elucidate CSC's chemoresistance properties and cancer recurrence. Establishing efficient strategies for CSC maintenance and enrichment is essential to elucidate the mechanisms and properties of CSCs and CSC-related therapeutic measures. Current approaches are insufficient to mimic the in vivo chemical and physical conditions for the maintenance and growth of CSC and yield unreliable research results. Biomaterials are now widely used for simulating the bone marrow microenvironment. Biomaterial-based three-dimensional (3D) approaches for the enrichment of CSC provide an excellent promise for future drug discovery and elucidation of molecular mechanisms. In the future, the biomaterial-based model will contribute to a more operative and predictive CSC model for cancer therapy. Design strategies for materials, physicochemical cues, and morphology will offer a new direction for future modification and new methods for studying the CSC microenvironment and its chemoresistance property. This review highlights the critical roles of the microenvironmental cues that regulate CSC function and endow them with drug resistance properties. This review also explores the latest advancement and challenges in biomaterial-based scaffold structure for therapeutic approaches against CSC chemoresistance. Since the recent entry of extracellular vesicles (EVs), cell-derived nanostructures, have opened new avenues of investigation into this field, which, together with other more conventionally studied signaling pathways, play an important role in cell-to-cell communication. Thus, this review further explores the subject of EVs in-depth. This review also discusses possible future biomaterial and biomaterial-EV-based models that could be used to study the tumor microenvironment (TME) and will provide possible therapeutic approaches. Finally, this review concludes with potential perspectives and conclusions in this area.

Chemoresistance-Related Stem Cell Signaling in Osteosarcoma and Its Plausible Contribution to Poor Therapeutic Response: A Discussion That Still Matters.

Osteosarcoma is amongst the most prevalent bone sarcomas and majorly afflicts children and adolescents. Therapeutic regimens based on the triad of doxorubicin, cisplatin and methotrexate have been used as the state-of-the-art approach to clinical treatment and management, with no significant improvements in the general outcomes since their inception in the early 1970s. This fact raises the following problematic questions: Why do some patients still relapse despite an initial good response to therapy? Why do nearly 30% of patients not respond to neoadjuvant therapies? Does residual persistent disease contribute to relapses and possible metastatic dissemination? Accumulating evidence suggests that chemoresistant cancer stem cells may be the major culprits contributing to those challenging clinical outcomes. Herein, we revisit the maneuvers that cancer stem cells devise for eluding cell killing by the classic cytotoxic therapies used in osteosarcoma, highlighting studies that demonstrate the complex crosstalk of signaling pathways that cancer stem cells can recruit to become chemoresistant.

High AUF1 level in stromal fibroblasts promotes carcinogenesis and chemoresistance and predicts unfavorable prognosis among locally advanced breast cancer patients

BackgroundLocally advanced breast cancer (LABC), the most aggressive form of the disease, is a serious threat for women's health worldwide. The AU-rich RNA-binding factor 1 (AUF1) promotes the formation of chemo-resistant breast cancer stem cells. Thereby, we investigated the power of AUF1 expression, in both cancer cells and their stromal fibroblasts, as predictive biomarker for LABC patients’ clinical outcome following neoadjuvant treatment.MethodsWe have used immunohistochemistry to assess the level of AUF1 on formalin-fixed paraffin-embedded tissues. Immunoblotting was utilized to show the effect of AUF1 ectopic expression in breast stromal fibroblasts on the expression of various genes both in vitro and in orthotopic tumor xenografts. Cytotoxicity was evaluated using the WST1 assay, while a label-free real-time setting using the xCELLigence RTCA technology was utilized to assess the proliferative, migratory and invasive abilities of cells.ResultsWe have shown that high AUF1 immunostaining (≥ 10%) in both cancer cells and their adjacent cancer-associated fibroblasts (CAFs) was significantly associated with higher tumor grade. Kaplan–Meier univariate analysis revealed a strong correlation between high AUF1 level in CAFs and poor patient’s survival. This correlation was highly significant in patients with triple negative breast cancer, who showed poor disease-free survival (DFS) and overall survival (OS). High expression of AUF1 in CAFs was also associated with poor OS of ER+/Her2− patients. Similarly, AUF1-positive malignant cells tended to be associated with shorter DFS and OS of ER+/Her2+ patients. Interestingly, neoadjuvant therapy downregulated AUF1 to a level lower than 10% in malignant cells in a significant number of patients, which improved both DFS and OS. In addition, ectopic expression of AUF1 in breast fibroblasts activated these cells and enhanced their capacity to promote, in an IL-6-dependent manner, the epithelial-to-mesenchymal transition and stemness processes. Furthermore, these AUF1-expressing cells enhanced the chemoresistance of breast cancer cells and their growth in orthotopic tumor xenografts.ConclusionsThe present findings show that the CAF-activating factor AUF1 has prognostic/predictive value for breast cancer patients and could represent a great therapeutic target in order to improve the precision of cancer treatment.

Nestin as a Prognostic Biomarker in High-grade Epithelial Ovarian Cancer Treated by Neoadjuvant Chemotherapy.

High-grade epithelial ovarian cancer (HGEOC) is a heterogeneous disease and among the deadliest types of cancer. It often acquires resistance to conventional chemotherapy and its prognosis remains highly poor. The tissue protein nestin, implicated in the assembly and disassembly of intermediate filaments, has been reported to be an unfavourable prognostic factor in several cancer types. We hypothesized that HGEOC progression is regulated by the proliferation of chemoresistant cancer stem cells, in which nestin might be implicated. This preliminary study aimed to evaluate nestin as a prognostic biomarker in HGEOC treated by neoadjuvant chemotherapy (NACT) followed by cytoreductive surgery. A retrospective study (2009-2019) was conducted on 92 patients with primary ovarian, fallopian tube or peritoneal HGEOC who underwent NACT followed by cytoreductive surgery. Nestin expression in tissue samples was semi-quantitatively evaluated defining nestin positivity for those with histochemical score ≥30. We then evaluated the prognostic value of nestin expression. The median progression-free survival was similar between nestin-positive (22 months) and nestin-negative (19 months) groups (p=0.57). Interestingly, the median overall survival was shorter for the nestin-positive group (48 vs. 67 months, respectively), however the difference did not reach statistical significance (p=0.43). Tissue nestin expression does not appear to be a relevant prognostic biomarker in HGEOC treated by NACT. However, we believe that prospective studies in larger cohorts should be conducted and evaluation of nestin in pre-NACT HGEOC samples needs to be explored.

Cancer stem cell assay for identification of effective treatments for platinum-resistant recurrent ovarian cancer.

e17584 Background: Disease recurrence and progression of ovarian cancer is a common event, which is accompanied by the development of platinum-resistant or refractory disease. The presence of chemo-resistant cancer stem cells (CSCs) contributes to tumor propagation, maintenance, and treatment resistance of this difficult to treat disease. The choice of appropriate chemotherapy is critical to the management of advanced epithelial ovarian cancer. We developed a CSCs assay (ChemoID) that identifies the most effective chemotherapy for individual patients from a panel of FDA-approved chemotherapies. Methods: Ascites or interventional radiology biopsies were collected under physician order from 85 consecutive patients affected by 3rd relapsed ovarian cancer. Test results from the assay were used to treat patients with the highest cell kill drugs, taking into consideration their health status and using dose reductions, if needed. Our study assessed the correlation of the ChemoID test results to treatment outcomes independently of other biomarkers. A retrospective chart analysis and review of CT and PET scans were performed to determine patients’ outcomes for tumor response, progression-free survival (PFS), and overall survival (OS). Results: We observed that recurrent ovarian cancer patients treated with high-cell kill chemotherapy agents guided by the ChemoID drug response assay had an improvement in their median PFS and OS when compared to historical median PFS and OS and/or when compared to patients who could not receive high cell kill chemotherapies (PFS low cell kill 3.5 months vs. high cell kill 12.0 months; OS low cell kill 6.0 months vs. high cell kill 15.0 months). Conclusions: The current evidence strongly suggests that the ChemoID assay predicted the effectiveness of specific therapies, as well as improved outcomes for patients treated with assay-sensitive therapies.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Cancer Stem Cells: Innovative Approach for Testable Alternatives against Therapy Resistant Cancer

Chemo-resistant cancer stem cells represent a sub-population of cancer initiating phenotype in primary cancer. These cells evolve in to a metastatic phenotype via activation of multiple cell signaling pathways for cancer cell survival and epithelial-mesenchymal transition. Reliable cancer stem cell models represent a valuable experimental approach for drug discovery platform to identify efficacious testable alternatives against therapy-resistant cancer. Present commentary provides a systematic discussion of relevant conceptual and technical aspects of cancer stem cell biology and its significance for therapeutic alternatives.

A specific inhibitor of ALDH1A3 regulates retinoic acid biosynthesis in glioma stem cells

Elevated aldehyde dehydrogenase (ALDH) activity correlates with poor outcome for many solid tumors as ALDHs may regulate cell proliferation and chemoresistance of cancer stem cells (CSCs). Accordingly, potent, and selective inhibitors of key ALDH enzymes may represent a novel CSC-directed treatment paradigm for ALDH+ cancer types. Of the many ALDH isoforms, we and others have implicated the elevated expression of ALDH1A3 in mesenchymal glioma stem cells (MES GSCs) as a target for the development of novel therapeutics. To this end, our structure of human ALDH1A3 combined with in silico modeling identifies a selective, active-site inhibitor of ALDH1A3. The lead compound, MCI-INI-3, is a selective competitive inhibitor of human ALDH1A3 and shows poor inhibitory effect on the structurally related isoform ALDH1A1. Mass spectrometry-based cellular thermal shift analysis reveals that ALDH1A3 is the primary binding protein for MCI-INI-3 in MES GSC lysates. The inhibitory effect of MCI-INI-3 on retinoic acid biosynthesis is comparable with that of ALDH1A3 knockout, suggesting that effective inhibition of ALDH1A3 is achieved with MCI-INI-3. Further development is warranted to characterize the role of ALDH1A3 and retinoic acid biosynthesis in glioma stem cell growth and differentiation.

Therapeutic potential of thymoquinone in combination therapy against cancer and cancer stem cells

The long-term success of standard anticancer monotherapeutic strategies has been hampered by intolerable side effects, resistance to treatment and cancer relapse. These monotherapeutic strategies shrink the tumor bulk but do not effectively eliminate the population of self-renewing cancer stem cells (CSCs) that are normally present within the tumor. These surviving CSCs develop mechanisms of resistance to treatment and refuel the tumor, thus causing cancer relapse. To ensure durable tumor control, research has moved away from adopting the monotreatment paradigm towards developing and using combination therapy. Combining different therapeutic modalities has demonstrated significant therapeutic outcomes by strengthening the anti-tumor potential of monotreatment against cancer and cancer stem cells, mitigating their toxic adverse effects, and ultimately overcoming resistance. Recently, there has been growing interest in combining natural products from different sources or with clinically used chemotherapeutics to further improve treatment efficacy and tolerability. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, has gained great attention in combination therapy research after demonstrating its low toxicity to normal cells and remarkable anticancer efficacy in extensive preclinical studies in addition to its ability to target chemoresistant CSCs. Here, we provide an overview of the therapeutic responses resulting from combining TQ with conventional therapeutic agents such as alkylating agents, antimetabolites and antimicrotubules as well as with topoisomerase inhibitors and non-coding RNA. We also review data on anticancer effects of TQ when combined with ionizing radiation and several natural products such as vitamin D3, melatonin and other compounds derived from Chinese medicinal plants. The focus of this review is on two outcomes of TQ combination therapy, namely eradicating CSCs and treating various types of cancers. In conclusion, the ability of TQ to potentiate the anticancer activity of many chemotherapeutic agents and sensitize cancer cells to radiotherapy makes it a promising molecule that could be used in combination therapy to overcome resistance to standard chemotherapeutic agents and reduce their associated toxicities.

Messing Up the Cancer Stem Cell Chemoresistance Mechanisms Supported by Tumor Microenvironment.

Despite the recent advances in cancer patient management and in the development of targeted therapies, systemic chemotherapy is currently used as a first-line treatment for many cancer types. After an initial partial response, patients become refractory to standard therapy fostering rapid tumor progression. Compelling evidence highlights that the resistance to chemotherapeutic regimens is a peculiarity of a subpopulation of cancer cells within tumor mass, known as cancer stem cells (CSCs). This cellular compartment is endowed with tumor-initiating and metastasis formation capabilities. CSC chemoresistance is sustained by a plethora of grow factors and cytokines released by neighboring tumor microenvironment (TME), which is mainly composed by adipocytes, cancer-associated fibroblasts (CAFs), immune and endothelial cells. TME strengthens CSC refractoriness to standard and targeted therapies by enhancing survival signaling pathways, DNA repair machinery, expression of drug efflux transporters and anti-apoptotic proteins. In the last years many efforts have been made to understand CSC-TME crosstalk and develop therapeutic strategy halting this interplay. Here, we report the combinatorial approaches, which perturb the interaction network between CSCs and the different component of TME.

Abstract 119: Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells

Abstract In colon cancer, the subset of cells fueling the clonal growth of the tumor have been shown to also possess enhanced chemoresistance abilities. These cells, known as cancer stem cells (CSC), are controlled by molecules and cells within the tumor microenvironment. In the latter reside enteric glial cells (EGC) that heavily regulate intestinal epithelial barrier function in a healthy colon. However, EGC influence on colon carcinogenesis remains poorly understood. Recent work from our lab shows that EGC promote CSC-driven tumorigenesis via the release of prostaglandin E2. Here, we investigate the impact of EGC on CSC chemoresistance. CD24Hi-CD44Hi CSC were FACS-isolated from human colon cancer primary tumors or cell lines and 3D-grown in the presence of murine or human EGC seeded on Transwell filters in vitro in the presence of the chemotherapeutic drug 5-Fluorouracil (5-FU) or injected subcutaneously with(out) EGC in vivo in 5-FU treated-immunodeficient mice. EGC impact was assessed on 5-FU-induced restriction of tumor formation and growth in vitro and in vivo. EGC-conditioned medium (CM) impact on 5-FU-induced apoptosis in CSC was measured by flow cytometry. EGC impact on expression of 48 chemoresistance-related genes in CSC was assessed by high throughput RT-qPCR. Impact of inhibition of ATM activity and ATM activation by the Mre11-Rad50-Nbs1 (MRN) complex in CSC was tested using 0.1µM KU-55933 and 12µM mirin. To understand the impact of 5-FU on EGC, levels of senescence markers p16, p19, p21 and H3K9me3 and β-gal activity were assessed. Proteomic analyses of EGC-CM were conducted by mass spectrometry. EGC promoted growth of CSC-derived tumors in the presence of 5-FU in vivo. EGC increased the number and size of tumor-organoids grown from CSC isolated from cell lines (HT29, HCT116, HCT15) and primary tumors in the presence of 5-FU in vitro. EGC-CM reduced apoptosis in 5-FU-treated CSC. Out of all genes tested, only ATM mRNA was significantly enriched in 5-FU-treated CSC cultured with EGC vs. alone. Co-culture studies using CSC derived from the ATM deficient SW1222 cell line or using the ATM inhibitor KU-55933 reduced EGC pro-chemoresistance effects. Furthermore, inhibition of ATM activation by the upstream DNA damage sensor MRN complex using mirin abolished EGC protective effects on CSC resistance to 5-FU. 5-FU-treated EGC showed increased levels of senescence markers and protein secretion, indicative of a senescence-associated secretory phenotype (SASP). Moreover, CM from 5-FU-treated EGC further reduced 5-FU-induced apoptosis in CSC as compared to CM from untreated EGC. Mass spectrometric analyses revealed that IGFBP7, a putative ATM activator, was >100 times more abundant in the CM of 5-FU-treated EGC vs. untreated EGC. Our data strongly indicate that EGC promote CSC chemoresistance via increased ATM signaling. Future studies will investigate the implication of IGFBP7 and downstream targets of ATM involved in DNA repair. Citation Format: Gregory Bacola, Simon Vales, Alice Prigent, Kelsie A. Dougherty, Deanna M. Peperno, Shaian Lashani, Bradley A. Wieland, Melissa Touvron, Lisa Oliver, François M. Vallette, Michel Neunlist, Laurianne Van Landeghem. Enteric glial cells promote chemoresistance in ATM-expressing cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 119.

Dual Targeting of Sorafenib-Resistant HCC-Derived Cancer Stem Cells.

Sorafenib, an oral multi-tyrosine kinase inhibitor, has been the first-line therapy for the treatment of patients with advanced HCC, providing a survival benefit of only three months in approximately 30% of patients. Cancer stem cells (CSCs) are a rare tumour subpopulation with self-renewal and differentiation capabilities, and have been implicated in tumour growth, recurrence and drug resistance. The process of epithelial-to-mesenchymal transition (EMT) contributes to the generation and maintenance of the CSC population, resulting in immune evasion and therapy resistance in several cancers, including HCC. The aim of this study is to target the chemoresistant CSC population in HCC by assessing the effectiveness of a combination treatment approach with Sorafenib, an EMT inhibitor and an immune checkpoint inhibitor (ICI). A stem-cell-conditioned serum-free medium was utilised to enrich the CSC population from the human HCC cell lines Hep3B, PLC/PRF/5 and HepG2. The anchorage independent spheres were characterised for CSC features. The human HCC-derived spheres were assessed for EMT status and expression of immune checkpoint molecules. The effect of combination treatment with SB431542, an EMT inhibitor, and siRNA-mediated knockdown of programmed cell death protein ligand-1 (PD-L1) or CD73 along with Sorafenib on human HCC-derived CSCs was examined with cell viability and apoptosis assays. The three-dimensional spheres enriched from human HCC cell lines demonstrated CSC-like features. The human HCC-derived CSCs also exhibited the EMT phenotype along with the upregulation of immune checkpoint molecules. The combined treatment with SB431542 and siRNA-mediated PD-L1 or CD73 knockdown effectively enhanced the cytotoxicity of Sorafenib against the CSC population compared to Sorafenib alone, as evidenced by the reduced size and proliferation of spheres. Furthermore, the combination treatment of Sorafenib with SB431542 and PD-L1 or CD73 siRNA resulted in an increased proportion of an apoptotic population, as evidenced by flow cytometry analysis. In conclusion, the combined targeting of EMT and immune checkpoint molecules with Sorafenib can effectively target the CSC tumour subpopulation.

The inhibition of ABCB1/MDR1 or ABCG2/BCRP enables doxorubicin to eliminate liver cancer stem cells

Two ATP-binding cassette transporters, ABCB1/MDR1 and ABCG2/BCRP, are considered the most critical determinants for chemoresistance in hepatocellular carcinoma. However, their roles in the chemoresistance in liver cancer stem cells remain elusive. Here we explored the role of inhibition of MDR1 or ABCG2 in sensitizing liver cancer stem cells to doxorubicin, the most frequently used chemotherapeutic agent in treating liver cancer. We show that the inhibition of MDR1 or ABCG2 in Huh7 and PLC/PRF/5 cells using either pharmacological inhibitors or RNAi resulted in the elevated level of intracellular concentration of doxorubicin and the accompanied increased apoptosis as determined by confocal microscopy, high-performance liquid chromatography, flow cytometry, and annexin V assay. Notably, the inhibition of MDR1 or ABCG2 led to the reversal of the chemoresistance, as evident from the enhanced death of the chemoresistant liver cancer stem cells in tumorsphere-forming assays. Thus, the elevation of effective intracellular concentration of doxorubicin via the inhibition of MDR1 or ABCG2 represents a promising future strategy that transforms doxorubicin from a traditional chemotherapy agent into a robust killer of liver cancer stem cells for patients undergoing transarterial chemoembolization.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis.

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.