Elimination Of Cancer Stem Cells Research Articles

Mitophagy and cancer: role of BNIP3/BNIP3L as energetic drivers of stemness features, ATP production, proliferation, and cell migration.

Mitophagy is a selective form of autophagy which permits the removal of dysfunctional or excess mitochondria. This occurs as an adaptative response to physiological stressors, such as hypoxia, nutrient deprivation, or DNA damage. Mitophagy is promoted by specific mitochondrial outer membrane receptors, among which are BNIP3 and BNIP3L. The role of mitophagy in cancer is being widely studied, and more specifically in the maintenance of cancer stem cell (CSC) properties, such as self-renewal. Given that CSCs are responsible for treatment failure and metastatic capacity, targeting mitophagy could be an interesting approach for CSC elimination. Herein, we describe a new model system to enrich sub-populations of cancer cells with high basal levels of mitophagy, based on the functional transcriptional activity of BNIP3 and BNIP3L. Briefly, we employed a BNIP3(L)-promoter-eGFP-reporter system to isolate cancer cells with high BNIP3/BNIP3L transcriptional activity by flow cytometry (FACS). The model was validated by using complementary lysosomal and mitophagy-specific probes, as well as the mitochondrially-targeted red fluorescent protein (RFP), namely mt-Keima. High BNIP3/BNIP3L transcriptional activity was accompanied by increases in i) BNIP3/BNIP3L protein levels, ii) lysosomal mass, and iii) basal mitophagy activity. Furthermore, cancer cells with increased BNIP3/BNIP3L transcriptional activity exhibited CSC features, such as greater mammosphere-forming ability and high CD44 levels. To further explore the model, we also analysed other stemness characteristics in MCF7 and MDA-MB-231 breast cancer cell lines, directly demonstrating that BNIP3(L)-high cells were more metabolically active, proliferative, migratory, and drug-resistant, with elevated anti-oxidant capacity. Therefore, high levels of basal mitophagy appear to enhance CSC features.

Targeted Therapy of Tumors and Cancer Stem Cells based on Oxidant-Regulated Redox Pathway and its Mechanism.

A malignant tumor is a frequent and common disease that severely threatens human health. Many mechanisms, such as cell signaling pathway, anti-apoptosis mechanism, cell stemness, metabolism, and cell phenotype, have been studied to explain the reasons for chemotherapy, radioresistance, and tumor recurrences in antitumor treatment. Cancer stem cells (CSCs) are important tumor cell subclasses that can potentially organize and regulate stem cell properties. Growing evidence suggests that CSCs can initiate tumors and constitute a significant factor in metastasis, recurrence, and treatment resistance. The inability to completely target and remove CSCs is a considerable obstacle in tumor treatment. Therefore, drugs and therapeutic strategies that can effectively intervene with CSCs are essential for the treatment of different tumor types. However, the current strategies and efficacy of targeted elimination of CSCs are very limited. Oxidative stress has been recognized to play a crucial role in cancer pathophysiology. Moreover, reactive oxygen species (ROS) production and imbalance of the built-in cellular antioxidant defense system are hallmarks of tumor and cancer etiology. The current paper will focus on the regulation and mechanism behind oxidative stress in tumors and cancer stem cells and its tumor therapy applications. Additionally, the article discusses the role of CSCs in causing tumor treatment resistance and recurrence based on a redox perspective. The study also emphasizes that targeted modulation of oxidative stress in CSCs has great potential in tumor therapy, providing novel prospects for tumor therapy.

Hierarchical self-recognition and response in CSC and non-CSC micro-niches for cancer therapy

Cancer stem cells (CSCs) characterized by self-renewal, invasiveness, tumorigenicity and resistance to treatment are regarded as the thorniest issues in refractory tumors. We develop a targeted and hierarchical controlled release nano-therapeutic platform (SEED-NPs) that self-identifies and responds to CSC and non-CSC micro-niches of tumors. In non-CSC micro-niche, reactive oxygen species (ROS) trigger the burst release of the chemotherapeutic drug and photosensitizer to kill tumor cells and reduce tumor volume by combining chemotherapy and photodynamic therapy (PDT). In CSC micro-niche, the preferentially released differentiation drug induces CSC differentiation and transforms CSCs into chemotherapy-sensitive cells. SEED-NPs exhibit an extraordinary capacity for downregulating the stemness of CD44+/CD24- SP (side population) cell population both in vitro and in vivo, and reveal a 4-fold increase of tumor-targeted accumulation. Also, PDT-generated ROS promote the formation of tunneling nanotubes and facilitate the divergent network transport of drugs in deep tumors. Moreover, ROS in turn promotes CSC differentiation and drug release. This positive-feedback-loop strategy enhances the elimination of refractory CSCs. As a result, SEED-NPs achieve excellent therapeutic effects in both 4T1 SP tumor-bearing mice and regular 4T1 tumor-bearing mice without obvious toxicities and eradicate half of mice tumors. SEED-NPs integrate differentiation, chemotherapy and PDT, which proved feasible and valuable, indicating that active targeting and hierarchical release are necessary to enhance antitumor efficacy. These findings provide promising prospects for overcoming barriers in the treatment of CSCs.

Bimetallic Gold/Silver and Bioactive Camptothecin Hybrid Nanoparticles for Eradication of Cancer Stem Cells in a Combination Manner.

The defeat of cancer is still a challenge due to the existence of cancer stem cells (CSCs) because they resist conventional chemotherapy via multifactor regulated mechanisms. Consequently, one-dimensional action toward CSCs cannot work. Herein, we used rationally designed hybrid nanoparticles as a combined cancer therapy, hoping to form a multidimensional control network. In this paper, gold/silver alloy nanoparticle decorated camptothecin nanocrystals were formulated according to complementary anti-CSC mechanisms from gold, silver, and organic drug. This smart drug formulation could combine chemotherapy and thermotherapy, target different tumor sites, and demonstrate versatile toxicity profiles from each component. Major results indicated that this nanosystem demonstrated indiscriminately effective cytotoxic/proapoptotic/necrotic activity against bulk MCF-7 cells and their CSC subpopulation, in particular under laser ablation. Moreover, this nanosystem displayed enhanced antineoplastic activity against CSC spheroids, resulting in a significant reduction in their number and size, that is, their self-renewal capacity. All the results indicated that CSCs upon treatment of these new hybrid nanoparticles underwent reduced stemness and conversion from the original quiescent state and recovered their sensitivity toward chemotherapy. The relevant anticancer mechanism was ascribed to NIR-pH dual responsive drug release, synergistic/combined thermo-chemotherapy of organic drug and inorganic alloy nanoparticles, enhanced cellular uptake mediated by alloy nanoparticles, and Ag+-induced biomembrane damage. This thermo-chemotherapy platform provides a new combinatorial strategy for inorganic and organic agents in the complete elimination of CSCs.

Glutaminolysis inhibition boosts photodynamic therapy to eliminate cancer stem cells

High reactive oxygen species (ROS) levels provide a therapeutic opportunity to eradicate cancer stem cells (CSCs), a population of cells responsible for tumorigenesis, progression, metastasis, and recurrence. However, enhanced antioxidant systems in this population of cells attenuate ROS-inducing therapies. Here, we developed a nanoparticle-assisted combination therapy to eliminate CSCs by employing photodynamic therapy (PDT) to yield ROS while disrupting ROS defense with glutaminolysis inhibition. Specifically, we leveraged an oleic acid-hemicyanine conjugate (CyOA) as photosensitizer, a new entity molecule HYL001 as glutaminolysis inhibitor, and a biocompatible folic acid-hydroxyethyl starch conjugate (FA-HES) as amphiphilic surfactant to construct cellular and mitochondrial hierarchical targeting nanomedicine (COHF NPs). COHF NPs inhibited glutaminolysis to reduce intracellular ROS scavengers, including glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADPH), and to blunt oxidative phosphorylation (OXPHOS) for oxygen-conserved PDT. Compared to COLF NPs without glutaminolysis inhibitor, COHF NPs exhibited higher phototoxicity to breast cancer stem cells (BCSCs) both in vitro and in vivo. More importantly, we corroborated that marketed glutaminolysis inhibitors, such as CB839 and V9302, augment the clinically used photosensitizer (Hiporfin) for BCSCs elimination. This study develops a potent CSCs targeting strategy by combining glutaminolysis inhibition with PDT and provides significant implications for cancer therapy.

Platelet Membrane Biomimetic Manganese Carbonate Nanoparticles Promote Breast Cancer Stem Cell Clearance for Sensitized Radiotherapy.

The presence of cancer stem cells (CSCs) significantly limits the therapeutic efficacy of radiotherapy (RT). Efficient elimination of potential CSCs is crucial for enhancing the effectiveness of RT. In this study, we developed a biomimetic hybrid nano-system (PMC) composed of MnCO3 as the inner core and platelet membrane (PM) as the outer shell. By exploiting the specific recognition properties of membrane surface proteins, PMC enables precise targeting of CSCs. Sonodynamic therapy (SDT) was employed using manganese carbonate nanoparticles (MnCO3 NPs), which generate abundant reactive oxygen species (ROS) upon ultrasound (US) irradiation, thereby impairing CSC self-renewal capacity and eradicating CSCs. Subsequent RT effectively eliminates common tumor cells. Both in vitro cell experiments and in vivo animal studies demonstrate that SDT mediated by PMC synergistically enhances RT to selectively combat CSCs while inhibiting tumor growth without noticeable side effects. Our findings offer novel insights for enhancing the efficacy and safety profiles of RT.

Hyperbaric Oxygen Boosts Antitumor Efficacy of Copper-Diethyldithiocarbamate Nanoparticles against Pancreatic Ductal Adenocarcinoma by Regulating Cancer Stem Cell Metabolism.

Cuproptosis-based cancer nanomedicine has received widespread attention recently. However, cuproptosis nanomedicine against pancreatic ductal adenocarcinoma (PDAC) is severely limited by cancer stem cells (CSCs), which reside in the hypoxic stroma and adopt glycolysis metabolism accordingly to resist cuproptosis-induced mitochondria damage. Here, we leverage hyperbaric oxygen (HBO) to regulate CSC metabolism by overcoming tumor hypoxia and to augment CSC elimination efficacy of polydopamine and hydroxyethyl starch stabilized copper-diethyldithiocarbamate nanoparticles (CuET@PH NPs). Mechanistically, while HBO and CuET@PH NPs inhibit glycolysis and oxidative phosphorylation, respectively, the combination of HBO and CuET@PH NPs potently suppresses energy metabolism of CSCs, thereby achieving robust tumor inhibition of PDAC and elongating mice survival importantly. This study reveals novel insights into the effects of cuproptosis nanomedicine on PDAC CSC metabolism and suggests that the combination of HBO with cuproptosis nanomedicine holds significant clinical translation potential for PDAC patients.

Recent Advances in the Targeting of Head and Neck Cancer Stem Cells

Head and neck squamous cell carcinoma (HNSCC) is a very heterogeneous cancer with a poor overall response to therapy. One of the reasons for this therapy resistance could be cancer stem cells (CSCs), a small population of cancer cells with self-renewal and tumor-initiating abilities. Tumor cell heterogeneity represents hurdles for therapeutic elimination of CSCs. Different signaling pathway activations, such as Wnt, Notch, and Sonic-Hedgehog (SHh) pathways, lead to the expression of several cancer stem factors that enable the maintenance of CSC features. Identification and isolation of CSCs are based either on markers (CD133, CD44, and aldehyde dehydrogenase (ALDH)), side populations, or their sphere-forming ability. A key challenge in cancer therapy targeting CSCs is overcoming chemotherapy and radiotherapy resistance. However, in novel therapies, various approaches are being employed to address this hurdle such as targeting cell surface markers, other stem cell markers, and different signaling or metabolic pathways, but also, introducing checkpoint inhibitors and natural compounds into the therapy can be beneficial.

ID1 expressing macrophages support cancer cell stemness and limit CD8+ T cell infiltration in colorectal cancer

Elimination of cancer stem cells (CSCs) and reinvigoration of antitumor immunity remain unmet challenges for cancer therapy. Tumor-associated macrophages (TAMs) constitute the prominant population of immune cells in tumor tissues, contributing to the formation of CSC niches and a suppressive immune microenvironment. Here, we report that high expression of inhibitor of differentiation 1 (ID1) in TAMs correlates with poor outcome in patients with colorectal cancer (CRC). ID1 expressing macrophages maintain cancer stemness and impede CD8+ T cell infiltration. Mechanistically, ID1 interacts with STAT1 to induce its cytoplasmic distribution and inhibits STAT1-mediated SerpinB2 and CCL4 transcription, two secretory factors responsible for cancer stemness inhibition and CD8+ T cell recruitment. Reducing ID1 expression ameliorates CRC progression and enhances tumor sensitivity to immunotherapy and chemotherapy. Collectively, our study highlights the pivotal role of ID1 in controlling the protumor phenotype of TAMs and paves the way for therapeutic targeting of ID1 in CRC.

Piperlongumine based nanomedicine impairs glycolytic metabolism in triple negative breast cancer stem cells through modulation of GAPDH & FBP1

BackgroundTriple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and exhibits high rate of chemoresistance, metastasis, and relapse. This can be attributed to the failure of conventional therapeutics to target a sub-population of slow cycling or quiescent cells called as cancer stem cells (CSCs). Therefore, elimination of CSCs is essential for effective TNBC treatment. PurposeResearch suggests that breast CSCs exhibit elevated glycolytic metabolism which directly contributes in maintenance of stemness, self-renewability and chemoresistance as well as in tumor progression. Therefore, this study aimed to target rewired metabolism which can serve as Achilles heel for CSCs population and have far reaching effect in TNBC treatment. MethodsWe used two preclinical models, zebrafish and nude mice to evaluate the fate of nanoparticles as well as the therapeutic efficacy of both piperlongumine (PL) and its nanomedicine (PL-NPs). ResultsIn this context, we explored a phytochemical piperlongumine (PL) which has potent anti-cancer properties but poor pharmacokinetics impedes its clinical translation. So, we developed PLGA based nanomedicine for PL (PL-NPs), and demonstrated that it overcomes the pharmacokinetic limitations of PL, along with imparting advantages of selective tumor targeting through Enhanced Permeability and Retention (EPR) effect in zebrafish xenograft model. Further, we demonstrated that PL-NPs efficiently inhibit glycolysis in CSCs through inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by modulating glutathione S-transferase pi 1 (GSTP1) and upregulation of fructose-1,6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis. We also illustrated that inhibition of glycolysis results in overall tumor regression in two preclinical models. ConclusionThis study discusses novel mechanism of action by which PL acts on CSCSs. Taken together our study provides insight into development of PL based nanomedicine which could be exploited in clinics to achieve complete eradication of TNBC by targeting CSCs.

Boron-Doped Carbon Nanodots as a Theranostic Agent for Colon Cancer Stem Cells.

Carbon nanodots have drawn a great deal of attention due to their green and expedient opportunities in biological and chemical sciences. Their high fluorescence capabilities and low toxicity for living cells and tissues make them excellent imaging agents. In addition, they have a fluorimetric response against inorganic and organic species. Boron-doped carbon nanodots (B-CDs) with high fluorescence yield were produced from phenylboronic acid and glutamine as boron and carbon sources, respectively, by a hydrothermal method. First, the effects of the temperature on their fluorescence yield and the structural characteristics of B-CDs were investigated. Second, their cytotoxicity and cell death and proliferation behaviors were examined. The cytotoxicity was evaluated by the MTT assay. The cellular properties were evaluated with the distribution of caspase 3, Ki67, lamin B1, P16, and cytochrome c after the indirect immunoperoxidase technique. After the MTT assay, 1:1 dilution of all applicants for 24 h was used in the study. After immunohistochemical analyses, the application of B-CDs synthesized at 230 °C did not change control cell (Vero) proliferation, and also apoptosis was not triggered. Colo 320 CD133+ and CD133- cell-triggered apoptosis and cellular senescence were found to be synthesis temperature dependent. In addition, Colo 320 CD133- cells were affected relatively more than CD133+ cells from B-CDs. While B-CDs did not affect the control cells, the colon cancer stem cells (Colo 320 CD133+) were affected in a time-dependent manner. Therefore, the use of the synthesized B-CD product may be an alternative method for controlling or eliminating cancer stem cells in the tumor tissue.

Deregulation of All-Trans Retinoic Acid Signaling and Development in Cancer.

Cancer stem cells are the root cause of cancer, which, in essence, is a developmental disorder. All-trans retinoic acid (ATRA) signaling via ligand-activation of the retinoic acid receptors (RARs) plays a crucial role in tissue patterning and development during mammalian embryogenesis. In adults, active RARγ maintains the pool of hematopoietic stem cells, whereas active RARα drives myeloid cell differentiation. Various findings have revealed that ATRA signaling is deregulated in many cancers. The enzymes for ATRA synthesis are downregulated in colorectal, gastric, lung, and oropharyngeal cancers. ATRA levels within breast, ovarian, pancreatic, prostate, and renal cancer cells were lower than within their normal counterpart cells. The importance is that 0.24 nM ATRA activates RARγ (for stem cell stemness), whereas 100 times more is required to activate RARα (for differentiation). Moreover, RARγ is an oncogene regarding overexpression within colorectal, cholangiocarcinoma, hepatocellular, ovarian, pancreatic, and renal cancer cells. The microRNA (miR) 30a-5p downregulates expression of RARγ, and miR-30a/miR-30a-5p is a tumor suppressor for breast, colorectal, gastric, hepatocellular, lung, oropharyngeal, ovarian, pancreatic, prostate, and renal cancer. These complementary findings support the view that perturbations to ATRA signaling play a role in driving the abnormal behavior of cancer stem cells. Targeting ATRA synthesis and RARγ has provided promising approaches to eliminating cancer stem cells because such agents have been shown to drive cell death.

A traditional gynecological medicine inhibits ovarian cancer progression and eliminates cancer stem cells via the LRPPRC–OXPHOS axis

BackgroundOvarian cancer (OC) is the most lethal malignant gynecological tumor type for which limited therapeutic targets and drugs are available. Enhanced mitochondrial oxidative phosphorylation (OXPHOS), which enables cell growth, migration, and cancer stem cell maintenance, is a critical driver of disease progression and a potential intervention target of OC. However, the current OXPHOS intervention strategy mainly suppresses the activity of the electron transport chain directly and cannot effectively distinguish normal tissues from cancer tissues, resulting in serious side effects and limited efficacy.MethodsWe screened natural product libraries to investigate potential anti-OC drugs that target OXPHOS. Additionally, LC-MS, qRT-PCR, western-blot, clonogenic assay, Immunohistochemistry, wound scratch assay, and xenograft model was applied to evaluate the anti-tumor mechanism of small molecules obtained by screening in OC.ResultsGossypol acetic acid (GAA), a widely used gynecological medicine, was screened out from the drug library with the function of suppressing OXPHOS and OC progression by targeting the leucine-rich pentatricopeptide repeat containing (LRPPRC) protein. Mechanically, LRPPRC promotes the synthesis of OXPHOS subunits by binding to RNAs encoded by mitochondrial DNA. GAA binds to LRPPRC directly and induces LRPPRC rapid degradation in a ubiquitin-independent manner. LRPPRC was overexpressed in OC, which is highly correlated with the poor outcomes of OC and could promote the malignant phenotype of OC cells in vitro and in vivo. GAA management inhibits cell growth, clonal formation, and cancer stem cell maintenance in vitro, and suppresses subcutaneous graft tumor growth in vivo.ConclusionsOur study identified a therapeutic target and provided a corresponding inhibitor for OXPHOS-based OC therapy. GAA inhibits OC progression by suppressing OXPHOS complex synthesis via targeting LRPPRC protein, supporting its potential utility as a natural therapeutic agent for ovarian cancer.

Potent salinomycin C20-O-alkyl oxime derivative SAL-98 efficiently inhibits tumor growth and metastasis by affecting Wnt/β-catenin signal pathway

The dysregulation of Wnt/β-catenin signaling pathway is closely related to tumorigenesis, metastasis and cancer stem cell maintenance. Salinomycin is a polyether ionophore antibiotic that selectively eliminates cancer stem cells by inhibiting the Wnt/β-catenin signal pathway. Salinomycin selectively target cancer stem cells, but the toxicity limits its further use. In this study, we explore the anti-tumor mechanism of one most active salinomycin C20-O-alkyl oximederivative SAL-98 and found that SAL-98 exerts 10 times higher anti-tumor and anti-CSCs activities compared with salinomycin, which induces cell cycle arrest, ER stress and mitochondria dysfunction and inhibits Wnt/β-catenin signal pathway in vitro with high efficacy. Moreover, SAL-98 shows good anti-metastasis effect in vivo. In addition, SAL-98 demonstrates same anti-tumor activities as salinomycin with less 5 times concentration in vivo, the ER stress, autophagy and anti-CSCs effects were also confirmed in vivo. Mechanistically, SAL-98 inhibits the Wnt/β-catenin signaling pathway associated with CHOP expression induced by ER stress, the induced CHOP disrupts the β-catenin/TCF4 complex and represses the Wnt targeted genes. This study provides an alternative strategy for rational drug development to target Wnt/β-catenin signaling pathway.

Near-infrared-activated anticancer platinum(IV) complexes directly photooxidize biomolecules in an oxygen-independent manner.

Conventional light-driven cancer therapeutics require oxygen and visible light to indirectly damage biomolecules, limiting their efficacy in deep, hypoxic tumours. Here we report the use of near-infrared-activated small-molecule Pt(IV) photooxidants to directly oxidize intracellular biomolecules in an oxygen-independent manner, achieving controllable and effective elimination of cancer stem cells. These Pt(IV) complexes accumulate in the endoplasmic reticulum and show low toxicity in the dark. Upon irradiation, the resultant metal-enhanced photooxidation effect causes them to robustly photooxidize survival-related biomolecules, induce intense oxidative stress, disrupt intracellular pH (pHi) homeostasis and initiate nonclassical necrosis. In vivo experiments confirm that the lead photooxidant can effectively inhibit tumour growth, suppress metastasis and activate the immune system. Our study validates the concept of metal-enhanced photooxidation and the subsequent chemotherapeutic applications, supporting the development of such localized photooxidants to directly damage intracellular biomolecules and decrease pHi as a strategy for effective metal-based drugs.

Effects of DNA-binding ligands from dimeric bisbenzimidazoles series DBA(n) and DBPA( n) in combination with γ-radiation on epithelial-mesenchymal transition and pool size of MCF-7 breast cancer stem cells

Radiation therapy is one of the main treatments for malignant neoplasms, including breast cancer. However, it is known, that radiation therapy can lead to an increase in the number of cancer stem cells, which are resistant to traditional antitumor effects, and are believed to be responsible for the development of recurrences and metastases. Therefore, the development of cancer stem cells elimination means is of considerable interest, especially in combination with ionizing radiation. The effects of single and combined exposure to new series of minor-groove DNA ligands - dimeric bisbenzimidazoles - DBA(n) and DBPA(n) (where n is the number of methylene groups between two bisbenzimidazole blocks) and Y-radiation on human Breast cancer cells were studied in our work for MCF-7 line in vitro. Compounds with maximum cytotoxic effect and cell binding were selected, and then the effects of the latter on the CD44+CD24-/low cancer stem cells population and radiation-induced epithelial-mesenchymal transition were studied by the criterion of vimentin expression. An increase in the expression level of this protein and simultaneously in the relative number of cancer stem cells after a single exposure of Y-radiation at a dose of 4 Gy were shown. DBPA(1,4) in combination with irradiation blocked the radiation-induced vimentin expression and decreased the relative number of cancer stem cells by 1.7 and 4.1 times compared with irradiation (p = 0.041 and p = 0.005), respectively. At the same time, the absolute number of cancer stem cells decreased by 2.8 and 12.0 times compared to irradiation (p = 0.029 and p = 0.004), respectively. In contrast, DBA(5,7) by itself or in combination with Y-irradiation increased the level of vimentin expression and the same compounds when combined with irradiation increased the relative number of cancer stem cells by 3.1 and 3.6 times (p = 0.006 and p = 0.005), respectively, compared with irradiation. The absolute number of cancer stem cells increased by 2.2 and 1.5 times (p = 0.017 and p = 0.032), respectively. The data obtained show a close relationship between epithelial-mesenchymal transition and formation of the cancer stem cells pool after radiation exposure, and also indicate the prospects for further study of DBPA(1,4) as a means of cancer stem cells elimination under in vivo conditions.

Pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor increases the efficacy of cabazitaxel by inducing apoptosis and decreasing cancer stem cells.

Cancer stem cells (CSCs) are associated with metastasis and recurrence in prostate cancer as well as other cancers. We aimed to enhance the sensitivity of cabazitaxel in prostate cancer cell therapy by targeting CSCs with a Wnt inhibitor and salinomycin pretreatment. PC3, DU-145, and LNCaP human prostate cancer cells were exposed to Wnt/β-catenin pathway inhibitor CCT036477 (iWnt) with salinomycin for 48h, followed by cabazitaxel treatment for 48h. Cell viability, mRNA, and protein expression changes were evaluated by MTT, RT-qPCR, and Western blot assays, respectively. Apoptosis was determined by image-based cytometry, and cell migration was assessed by wound healing assay. Three-dimensional culture was established to assess the malignant phenotype and stemness potential of transformed or cancer cells. CD44 + CSCs were isolated using magnetic-activated cell sorting system. Pretreatment of PC3, DU-145, and LNCaP cells with salinomycin iWnt significantly sensitized the cells to cabazitaxel therapy. Spheroid culture confirmed that the treatment modality was more effective than a single administration of chemotherapy. The pretreatment of PC3 cells increased the rate of apoptosis compared to single administration of cabazitaxel, which downregulated Bcl-2 and upregulated caspase 3, caspase 8 expressions. The pretreatment suppressed cell migration, downregulated the expression of Sox2 and Nanog, and significantly reduced CD44 + CSC numbers. Notably, the treatment modality reduced pAKT, p-P38 MAPK, and pERK1/2. The data suggest that pretreatment of prostate cancer cells with salinomycin and Wnt inhibitor may increase the efficacy of cabazitaxel therapy by inhibiting cell proliferation and migration, and eliminating cancer stem cells.

Evaluation of the Effectiveness of Various Autophagy Inhibitors in A549 Cancer Stem Cells.

Numerous studies have already established that autophagy plays a central role in the survival of all cells, including malignant ones. Autophagy is a central cog in the general mechanism that provides the intracellular proteostasis determining cellular physiological and phenotypic characteristics. The accumulated data show that autophagy largely contributes to cancer cell stemness. Thus, autophagy modulation is considered one of the promising pharmacological targets in therapy aimed at cancer stem cell elimination. However, autophagy is a multi-stage intracellular process that involves numerous protein participants. In addition, the process can be activated simultaneously by various signaling modules. Therefore, it is no small feat to select an effective pharmacological drug against autophagy. What's more, the search for potential chemotherapeutic agents that could eliminate cancer stem cells through pharmacological inhibition of autophagy is still under way. In the present work, we selected a panel of autophagy inhibitors (Autophinib, SBI-0206965, Siramesine, MRT68921, and IITZ-01), some of whom have been recently identified as effective autophagy inhibitors in cancer cells. Using A549 cancer cells, which express the core stem factors Oct4 and Sox2, we evaluated the effect of these drugs on the survival and preservation of the original properties of cancer stem cells. Among the agents selected, only Autophinib demonstrated a significant toxic effect on cancer stem cells. The obtained results demonstrate that autophagy inhibition by Autophinib downregulates the expression of the Sox2 protein in A549 cells, and that this downregulation correlates with a pronounced induction of apoptosis. Moreover, Autophinib-treated A549 cells are unable to form spheroids, which indicates a reduction in stemness. Thus, among the drugs studied, only Autophinib can be considered a potential agent against cancer stem cells.

Hyperbaric Oxygen Activates Enzyme-Driven Cascade Reactions for Cooperative Cancer Therapy and Cancer Stem Cells Elimination.

Tumor starvation induced by intratumor glucose depletion emerges as a promising strategy for anticancer therapy. However, its antitumor potencies are severely compromised by intrinsic tumor hypoxia, low delivery efficiencies, and undesired off-target toxicity. Herein, a multifunctional cascade bioreactor (HCG), based on the self-assembly of pH-responsive hydroxyethyl starch prodrugs, copper ions, and glucose oxidase (GOD), is engineered, empowered by hyperbaric oxygen (HBO) for efficient cooperative therapy against aggressive breast cancers. Once internalized by tumor cells, HCG undergoes disassembly and releases cargoes in response to acidic tumor microenvironment. Subsequently, HBO activates GOD-catalyzed oxidation of glucose to H2 O2 and gluconic acid by ameliorating tumor hypoxia, fueling copper-catalyzed •OH generation and pH-responsive drug release. Meanwhile, HBO degrades dense tumor extracellular matrix, promoting tumor accumulation and penetration of HCG. Moreover, along with the consumption of glucose and the redox reaction of copper ions, the antioxidant capacity of tumor cells is markedly reduced, collectively boosting oxidative stress. As a result, the combination of HCG and HBO can not only remarkably suppress the growth of orthotopic breast tumors but also restrain pulmonary metastases by inhibiting cancer stem cells. Considering the clinical accessibility of HBO, this combined strategy holds significant translational potentials for GOD-based therapies.

Abstract 459: Targeting the non-junctional tumorigenic function of connexin43 in cancer cell plasticity

Abstract Tumors are highly heterogeneous and dynamic, with both cancer cells and cancer stem cells (CSCs) displaying a high degree of plasticity contributing to metastasis and tumor recurrence following treatment. Epithelial-mesenchymal transition (EMT) enables cancer cells to acquire migratory and invasive characteristics, access the circulatory system, and metastasize. Moreover, EMT is associated with therapeutic resistance and CSC generation. Our previous research demonstrated a redistribution of the gap junction protein connexin43 (Cx43) from the plasma membrane to the cytosol during EMT. Using super-resolution STochastic Optical Reconstruction Microscopy (STORM), we have recently identified increased interaction of such intracellular Cx43 with microtubules during EMT and in primary glioblastoma (GBM) CSCs (GSCs). We further report a tumorigenic function for cytosolic Cx43/microtubule complexing in cancer cell and CSC proliferation, migration, and survival. Cx43 localization and activity is regulated by multiple sites for protein-protein interaction within the Cx43 carboxy-terminus (CT), including a tubulin binding domain. We employed a Cx43 mimetic peptide named JM2 (juxtamembrane 2) encompassing the microtubule binding sequence of the Cx43 CT. Following administration, JM2 efficiently and specifically disrupts the interaction of Cx43 with microtubules, inhibits GSC-derived neurosphere formation in vitro, and patient GBM-derived organoid growth ex vivo. Using an orthotopic GBM patient derived xenograft mouse model, we demonstrate in vivo that JM2 significantly decreases the number of GSCs within brain tumors, inhibits the formation of highly invasive GBM tumors, and increases mouse survival in combination with the chemotherapy alkylating agent temozolomide. Further examining the role of Cx43/microtubule complexing in the context of metastatic disease, we observed JM2 also decreases mesenchymal marker expression and inhibits cell migration and invasion in EMT models. Using an in vivo orthotopic breast cancer mouse model we find that intratumoral administration of JM2 prevents metastasis, significantly decreasing cancer cell lung colonization. Our findings validate targeting Cx43 complexing with microtubules, with JM2, as a novel therapeutic approach for cancer treatment through inhibition of EMT-associated increases in invasion and metastasis, and elimination of CSCs. Modulation of non-junctional Cx43 functions therefore represents a viable therapeutic approach to target cellular plasticity with broad therapeutic implications for many cancers. Citation Format: Samy Lamouille, Christina E. Wheeler, Stacie E. Deaver, Michael J. Zeitz, Zhi Sheng, Robert G. Gourdie, James W. Smyth. Targeting the non-junctional tumorigenic function of connexin43 in cancer cell plasticity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 459.