Cancer Stem Cells In Malignancies Research Articles

Photothermal-Triggered Sulfur Oxide Gas Therapy Augments Type I Photodynamic Therapy for Potentiating Cancer Stem Cell Ablation and Inhibiting Radioresistant Tumor Recurrence.

Despite advances in cancer therapy, the existence of self-renewing cancer stem cells (CSC) can lead to tumor recurrence and radiation resistance, resulting in treatment failure and high mortality in patients. To address this issue, a near-infrared (NIR) laser-induced synergistic therapeutic platform has been developed by incorporating aggregation-induced emission (AIE)-active phototheranostic agents and sulfur dioxide (SO2 ) prodrug into a biocompatible hydrogel, namely TBH, to suppress malignant CSC growth. Outstanding hydroxyl radical (·OH) generation and photothermal effect of the AIE phototheranostic agent actualizes Type I photodynamic therapy (PDT) and photothermal therapy through 660nm NIR laser irradiation. Meanwhile, a large amount of SO2 is released from the SO2 prodrug in thermo-sensitive TBH gel, which depletes upregulated glutathione in CSC and consequentially promotes ·OH generation for PDT enhancement. Thus, the resulting TBH hydrogel can diminish CSC under 660nm laser irradiation and finally restrain tumor recurrence after radiotherapy (RT). In comparison, the tumor in the mice that were only treated with RT relapsed rapidly. These findings reveal a double-boosting ·OH generation protocol, and the synergistic combination of AIE-mediated PDT and gas therapy provides a novel strategy for inhibiting CSC growth and cancer recurrence after RT, which presents great potential for clinical treatment.

Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs.

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple-negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC , respectively) from the MDA-MB-231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL-6/IL-8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α-SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.

NRF2 and Mitochondrial Function in Cancer and Cancer Stem Cells.

The NRF2–KEAP1 system is a fundamental component of the cellular response that controls a great variety of transcriptional targets that are mainly involved in the regulation of redox homeostasis and multiple cytoprotective mechanisms that confer adaptation to the stress conditions. The pleiotropic response orchestrated by NRF2 is particularly relevant in the context of oncogenic activation, wherein this transcription factor acts as a key driver of tumor progression and cancer cells’ resistance to treatment. For this reason, NRF2 has emerged as a promising therapeutic target in cancer cells, stimulating extensive research aimed at the identification of natural, as well as chemical, NRF2 inhibitors. Excitingly, the influence of NRF2 on cancer cells’ biology extends far beyond its mere antioxidant function and rather encompasses a functional crosstalk with the mitochondrial network that can influence crucial aspects of mitochondrial homeostasis, including biogenesis, oxidative phosphorylation, metabolic reprogramming, and mitophagy. In the present review, we summarize the current knowledge of the reciprocal interrelation between NRF2 and mitochondria, with a focus on malignant tumors and cancer stem cells.

Abstract 6218: Oncolytic Seneca Valley virus (SVV-001) overcomes checkpoint inhibitor resistance and demonstrates a systemic anti-tumor immune response in a syngeneic pancreatic cancer murine model

Abstract Here we evaluated the efficacy of Seneca Valley Virus (SVV), an oncolytic virus in the Picornaviridae family, in combination with checkpoint inhibitors (CPI) using the CPI-resistant murine syngeneic pancreatic tumor model Pan02. SVV, serendipitously identified in 2001, is an extremely tumor selective and potent oncolytic virus for a variety of solid human cancers, primarily due to the expression of the receptor of SVV, TEM8, being selectively expressed on the surface of malignant cancer cells and cancer stem cells within a variety of solid tumors. SVV has been tested in three clinical trials of patients having neuroendocrine neoplasms as a single intravenous dose monotherapy. Despite this, the results were encouraging with patients having evidence of clinical benefit and only one DLT was reported in the 76 patients treated. Recent data have demonstrated that some oncolytic viruses can enhance efficacy in preclinical models and in clinical trials when the oncolytic virus was injected intratumorally in combination with systemic administration of a CPI(s). The mechanism is primarily believed to be due to the oncolytic virus enhancing T cell infiltration and fostering an anti-tumor immune response. SVV was injected intra-tumorally in Pan02 tumors along with aPD1and/or aCTLA4. SVV reversed any resistance to CPIs and enhanced efficacy over CPI(s) alone resulting in complete cures in >83% of mice with primary and abscopal tumors. SVV plus aPD1+aCTLA4 resulted in 5 of 6 mice cured of their primary tumor. The animals were challenged on their contralateral flank with Pan02 to evaluate systemic and abscopal immune effects. All mice (5/5) that cleared the primary Pan02 tumor also eradicated the abscopal secondary tumors. Control treated Pan02 tumor-bearing mice were all sacrificed due to tumor burden by day 70 (median survival <50 days). Control animals treated with aPD1+aCTLA4 showed transient tumor regressions but these animals all grew large tumors, requiring sacrifice. In contrast, the SVV+aPD1+aCTLA4 primary tumors were eliminated within 44 days and these animals remain tumor-free out to >110+ days. Contralateral tumors were eradicated in all 5 animals that rejected the primary tumor, demonstrating potent systemic immunity. In the control aPD1+aCTLA4 group, only one animal with contralateral tumor showed prolonged tumor regression. SVV treatment caused increases in T-cell infiltration; the combination of SVV+aPD1+aCTLA4 showing the highest infiltration. Phenotypic and gene expression analyses of infiltrating cells will be presented. These studies serve as a foundation for translating SVV oncolytic virotherapy combined with anti-PD-1 and anti-CTLA4 antibodies in patients with neuroendocrine neoplasms with a clinical trial anticipated to begin in H1, 2021. Citation Format: Paul L. Hallenbeck, Sunil Chada. Oncolytic Seneca Valley virus (SVV-001) overcomes checkpoint inhibitor resistance and demonstrates a systemic anti-tumor immune response in a syngeneic pancreatic cancer murine model [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 6218.

Long Noncoding RNA MALAT1 Promotes Laryngocarcinoma Development by Targeting miR-708-5p/BRD4 Axis to Regulate YAP1-Mediated Epithelial-Mesenchymal Transition.

Objective The objective of this study was to investigate whether long noncoding RNA Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) contributes to laryngocarcinoma development via regulating the Yes-associated protein 1- (YAP1-) mediated epithelial-mesenchymal transition (EMT) and the underlying mechanism. Methods The effects of MALAT1 suppression and BET inhibitor JQ1 on the malignant phenotypes and cancer stem cell- (CSC-) like properties of laryngocarcinoma cells as well as the expression of bromodomain-containing protein 4 (BRD4), YAP1, and EMT markers were investigated. Moreover, the relationships between MALAT1 and miR-708-5p as well as between miR-708-5p and BRD4 were explored. Furthermore, whether MALAT1 regulated the malignant phenotypes of laryngocarcinoma cells via sponging miR-708-5p to target BRD4 was revealed by both in vitro and in vivo experiments. Results MALAT1 suppression inhibited the malignant phenotypes of laryngocarcinoma cells, such as decreased proliferation, promoted apoptosis, suppressed migration, and inhibited the CSC properties. Suppression of MALAT1 increased miR-708-5p expression and decreased the expression of BRD4 and YAP1 and inhibited EMT. Moreover, there were target relationships between MALAT1 and miR-708-5p as well as between miR-708-5p and BRD4. miR-708-5p overexpression and MALAT1 suppression had synergistic inhibitory effects on the malignant phenotypes of laryngocarcinoma cells and the expression of BRD4, YAP1, and EMT. Furthermore, in vivo experiments confirmed that MALAT1/miR-708-5p regulated tumorigenicity by regulating BRD4 and YAP1-mediated EMT. Conclusions Our results indicate that suppression of MALAT1 may inhibit laryngocarcinoma development by sponging miR-708-5p/BRD4 to regulate YAP1-mediated EMT. Targeting MALAT1/miR-708-5p/BRD4 axis may provide a promising therapeutic strategy for laryngocarcinoma.

IMMUNOHISTOCHEMICAL EXPRESSION OF CD44 AND CD56 IN MALIGNANT SALIVARY GLAND TUMORS

Introduction: Salivary gland cancers impose a huge part in oral and maxillofacial pathologies. Mucoepidermoid (MEC) and adenoid cystic carcinomas (ACC) are salivary malignancies, where (MEC) being the most common cancer. CD44, is a family of transmembrane non-kinase cell surface glycoproteins, that was broadly used as a marker for cancer stem cells (CSCs) in various malignancies. CD44 was found to be overexpressed in various types of cells including cancer stem cells (CSCs) and it has been postulated that CD44 has a part in carcinogenesis. Natural killer (NK) cells are one of the components of the innate immune system, which express the phenotypic marker CD56 in the absence of CD3, and has a vital role in tumor-cell surveillance. This study aimed to assess the expression of CD44 and CD56 in different grades of mucoepidermoid carcinomas and different patterns of adenoid cystic carcinoma, and correlate between the expressions of the markers (CD44 and CD56) in different grades and patterns of the tumors. Material &Methods: Thirty-three paraffin embedded blocks of salivary gland tumors (18 mucoepidermoid carcinoma and 15 adenoid cystic carcinoma) were selected. Those tumorswere classified into four groups: intermediate grade (IG) MEC, high grade (HG) MEC, ACC (tub-crib) and ACC (solid) patterns.Immunohistochemical staining for CD44 and CD56 was performed. Data were analyzed using SPSS (24), the independent-T test and Anova to analyze the statistical significance. Results: for CD44, inMEC group, we found no significant difference between both grades for CD44 expression, however, the difference was found to be significant between both patterns of ACC. While for CD56, the difference was statistically significant between both grades of MEC, and both patterns of ACC. Conclusion:CD44 could be promising goal for cancer therapy, mostly for CD44 expressing cancers. NK cells have a crucial role in the anti-tumor immune mechanism. Moreover, more studies are significantly demanded to assess the promising role and function of CD56 in diagnosis and prognosis of salivary gland cancers, and the tumor-cell surveillance capacity of the NK cells.

Stem Cell Marker Aldehyde Dehydrogenase 1A1 Expression in Triple-negative Breast Carcinoma

Background: Increasing evidence has proposed that tumor contains tumor initiating cells or cancer stem cells (CSCs) are responsible for its progression and relapse. Aldehyde dehydrogenase 1A1 (ALDH1A1) has recently been identified as a marker for cancer stem cells in some human malignancies including breast cancer.Triple negative breast carcinomas (TNBCs) are group of primary breast tumors with aggressive clinical behavior that have no targeted therapy at present.
 Aim: The assessment of immunohistochemical expression of ALDH1A1 in triple negative breast carcinoma and its correlation with the clinicopathological features of TNBC.
 Material and Methods: This study consisted of 60 cases of TNBC. Immunohistochemical reactions were carried out by using ALDH1A1 monoclonal antibody. A total score of ALDH1A1 expression is obtained by multiplying the score of staining intensity & percentage of stained cells to obtain score ranging from 0 up to 300. Due to the relatively large number of positive cases, a statistical analysis was performed with a negative (score ≤ 10) and positive (score > 10) cutoff [1].
 Results: Evaluation of the results of immunostaining for ALDH1A1 showed 88.3% of totalcases (53 cases) having a positive cytoplasmic reactivity. Statistical analysis for a possible correlation between ALDH1A1 expression and prognostic clinicopathological parameters; age, size, tumor grade, histologic subtypes, lymphovascular invasion, intraductal components, tumor infiltrating lymphocytes and TNM stage grouping revealed a non-significant correlation.
 Conclusion: ALDH1A1 couldn’t be used solely as a diagnostic or prognostic marker In TNBCs. Further research combining with other biomarkers and with a greater number of patients is necessary to confirm the role of ALDH1A1 in TNBC.
 Key words: Cancer stem cells (CSCs), Aldehyde dehydrogenase 1A1 (ALDH1A1), Triple Negative Breast cancer (TNBC).

Advances in glioma-associated oncogene (GLI) inhibitors for cancer therapy.

The Hedgehog/Glioma-associated oncogene homolog (HH/GLI) signaling pathway regulates self-renewal of rare and highly malignant cancer stem cells, which have been shown to account for the initiation and maintenance of tumor growth as well as for drug resistance, metastatic spread and relapse. As an important component of the Hh signaling pathway, glioma-associated oncogene (GLI) acts as a key signal transmission hub for various signaling pathways in many tumors. Here, we review direct and indirect inhibitors of GLI; summarize the abundant active structurally diverse natural GLI inhibitors; and discuss how to better develop and utilize GLI inhibitors to solve the problem of drug resistance in tumors of interest. In summary, GLI inhibitors will be promising candidates for various cancer treatments.

NR2F2 controls malignant squamous cell carcinoma state by promoting stemness and invasion and repressing differentiation.

The nongenetic mechanisms required to sustain malignant tumor state are poorly understood. During the transition from benign tumors to malignant carcinoma, tumor cells need to repress differentiation and acquire invasive features. Using transcriptional profiling of cancer stem cells from benign tumors and malignant skin squamous cell carcinoma (SCC), we identified the nuclear receptor NR2F2 as uniquely expressed in malignant SCC. Using genetic gain of function and loss of function in vivo, we show that NR2F2 is essential for promoting the malignant tumor state by controlling tumor stemness and maintenance in mouse and human SCC. We demonstrate that NR2F2 promotes tumor cell proliferation, epithelial-mesenchymal transition and invasive features, while repressing tumor differentiation and immune cell infiltration by regulating a common transcriptional program in mouse and human SCCs. Altogether, we identify NR2F2 as a key regulator of malignant cancer stem cell functions that promotes tumor renewal and restricts differentiation to sustain a malignant tumor state.

Nanoparticle-mediated specific elimination of soft cancer stem cells by targeting low cell stiffness

As the driving force of tumor progression, cancer stem cells (CSCs) hold much lower cellular stiffness than bulk tumor cells across many cancer types. However, it remains unclear whether low cell stiffness can be harnessed in nanoparticle-based therapeutics for CSC targeting. We report that breast CSCs exhibit much lower stiffness but considerably higher uptake of nitrogen-doped graphene quantum dots (N-GQDs) than bulk tumor cells. Softening/stiffening cells enhances/suppresses nanoparticle uptake through activating/inhibiting clathrin- and caveolae-mediated endocytosis, suggesting that low cell stiffness mediates the elevated uptake in soft CSCs that may lead to the specific elimination. Further, soft CSCs enhance drug release, cellular retention, and nuclear accumulation of drug-loaded N-GQDs by reducing intracellular pH and exocytosis. Remarkably, drug-loaded N-GQDs specifically eliminate soft CSCs both in vitro and in vivo, inhibit tumor but not animal growth, and reduce the tumorigenicity of xenograft cells. Our findings unveil a new mechanism by which low cellular stiffness can be harnessed in nanoparticle-based strategies for specific CSC elimination, opening a new paradigm of cancer mechanomedicine. Statement of significanceLow cell stiffness is associated with high malignancy of tumor cells and thus serves as a mechanical hallmark of CSCs. However, it remains unclear whether cellular stiffness can be exploited for specific targeting of soft CSCs. This work reports that soft CSCs exhibit high N-GQD uptake compared to stiff tumor cells, which is regulated by cellular stiffness. Further, soft CSCs have enhanced drug release, cellular retention, and nuclear accumulation of drug-loaded N-GQDs, which enable the specific elimination of malignant CSCs both in vitro and in vivo with minimal side effect. In summary, our study demonstrates that CSC's low stiffness can be harnessed as a mechanical target for specific eradication, which provides a new paradigm of cancer mechanomedicine.

Proposed Models of Functional Heterogeneity in Cancer and the Effects of Microenvironmental Factors on Cancer Stem Cells: A Literature Review

Introduction: Functional heterogeneity in cancer may result in the metastasis of various types of tumour cells throughout the body. Attempting to explain functional heterogeneity in cancer cells has given rise to two models. The Cancer Stem Cell model proposes that a subset of tumour cells self-replicate and that heterogeneity is a progeny of various cancer stem cells (CSCs). The Clonal Evolution Model proposes heterogeneity as a product of mutations across tumour cells that accumulate and metastasize linearly or branching. Methods: Research was conducted through open-access journals and information was compiled surrounding CSC models using the Google Scholar and McMaster Library database search engines. Inclusions were sources that detailed the relationship between both models of functional heterogeneity and microenvironments and treatments. Literature that did not center around tumour microenvironments was not included in this literature review. Results: The two main models of tumour proliferation were explored and related to hypoxic tumour microenvironments. Various markers, etiologic agents and toxins were identified that contribute to tumour progression. Cell signalling and pathways that contribute to major cellular functions were identified, along with possible disruptions and epigenetic changes that lead to tumour and CSC proliferation. Discussion: This study reveals that the tumour microenvironment plays a large role in the proliferation of CSCs. Although the therapies targeting microenvironments are in early stages of development, focusing on these CSC targeted- therapies may lead to better treatments for cancer or more effective combination therapies. Strengths of the paper include the compilation of major contributing areas to CSC proliferation, whereas limitations encompass the high variability of tumour cells that are not all covered in this review. Conclusion: While no definitively eradicating treatment for CSCs currently exist, the recent developments in cancer research indicate promising new techniques for its management. Implications: By further studying malignant CSCs, highly effective cancer treatments may result, leading to the advancement of CSC recognition and combination therapy.

Targeting cancer stem cells by disulfiram and copper sensitizes radioresistant chondrosarcoma to radiation.

Overcoming the radiosensitivity of chondrosarcoma (CS), the second most common primary bone tumor, is needed. Radioresistance is attributed to cancer stem cells (CSCs) in many malignancies. Disulfiram (DSF), an FDA-approved anti-alcoholism drug, complexed with Cu (DSF/Cu) can radiosensitize epithelial CSCs. This prompted us to investigate the radiosensitizing effect of DSF/Cu on CS CSCs (CCSCs). The radiosensitizing effects of DSF/Cu on CCSCs were investigated in vitro using cell lines SW1353 and CS-1. Stemness was identified independently by flow cytometry for CCSCs (ALDH+CD133+), sphere-forming ability, and Western blot analysis of stemness gene protein expression. The radiosensitizing effect of DSF/Cu was studied in an orthotopic CS xenograft mouse model by analyzing xenograft growth and residual xenografts for stemness. CCSCs were found to be resistant to single-dose (IR) and fractionated irradiation (FIR). IR and FIR increased CS stemness. Combined with DSF/Cu in vitro and in vivo, IR and FIR eliminated CS stemness. RT + DSF/Cu was safer and more effective than either RT ± DSF in inhibiting growth of orthotopic CS xenografts. In conclusion, DSF/Cu radiosensitizes CCSCs. These results can be translated into clinical trials for CS patients requiring RT for improved outcomes.

The small molecule drug CBL0137 increases the level of DNA damage and the efficacy of radiotherapy for glioblastoma.

Glioblastoma (GBM) is an incurable brain tumor with inevitable recurrence. This is in part due to a highly malignant cancer stem cell (CSC) subpopulation of tumor cells that is particularly resistant to conventional treatments, including radiotherapy. Here we show that CBL0137, a small molecule anti-cancer agent, sensitizes GBM CSCs to radiotherapy. CBL0137 sequesters the FACT (facilitates chromatin transcription) complex to chromatin, resulting in cytotoxicity preferentially within tumor cells. We show that when combined with radiotherapy, CBL0137 inhibited GBM CSC growth and resulted in more DNA damage in the CSCs compared to irradiation or drug alone. Using an in vivo subcutaneous model, we showed that the frequency of GBM CSCs was reduced when tumors were pretreated with CBL0137 and then exposed to irradiation. Survival studies with orthotopic GBM models resulted in significantly extended survival for mice treated with combinatorial therapy. As GBM CSCs contribute to the inevitable recurrence in patients, targeting them is imperative. This work establishes a new treatment paradigm for GBM that sensitizes CSCs to irradiation and may ultimately reduce tumor recurrence.

Abstract 1746: The BET inhibitor JQ-1 enhances radio-sensitivity in pediatric cancer cell lines by sustained p-gH2AX expression and enhanced differentiation

Abstract Purpose: Pediatric high grade gliomas (HGG), high risk medulloblastomas (MB) and neuroblastomas (NB) are malignant neurological tumors with poor prognosis despite major advancements in current therapeutic interventions. The standard of care therapy for such high risk tumors includes surgical resection, when possible, and radio-therapy with or without chemo-therapy. Therapeutic failure may be due to radio- and/or chemo-resistance and the maintenance of an aggressive stem-like sub-population of cells within the bulk tumors. Epigenetic deregulation is implicated in pediatric neurological cancer malignancies where the bromo- and extra-terminal domain (BET) protein activity may play a role in histone H3K27 acetylation leading to tumorigenesis and radio-resistance. Methods and Results: We used the BET inhibitor JQ-1 to determine the radio-sensitizing effect on a panel of human pediatric neurological cancer cell lines including: NB (IMR-32 and SKNSH), MB (D556 and D283) and HGG (GBM01 and DIPG07). Cells were seeded 24 hours (h) before treatment with 1 µM dose of JQ-1 for an additional 24 h before irradiation (IR) with 6Gy (JL Shepherd Mark 1 137Cs Irradiator). IncuCyte (Essen Biosciences, Ann Arbor, MI) live cell imaging recorded cellular proliferation (confluency) over 6 days post IR, revealing that while single treatment with JQ-1 or 6Gy irradiation alone significantly reduced cell proliferation in all the tested cell lines, the combination of JQ-1 pre-treatment followed by 6Gy IR differentially impaired proliferation capacity in all cell lines except DIPG07 by further reducing cell proliferation. To determine the mechanism of radio-sensitization induced by JQ-1, Western blot and Immunofluorescence analysis revealed increased p-γH2AX foci at 24 h post IR in JQ-1 pre-treated cells. Furthermore, the stem cell maintenance and tumorigenic proteins OCT-4, MYCN, HSP90, L1-CAM, RAB5C and FABP5 were differentially expressed after JQ-1 treatment. Next we assessed the effect of JQ-1 on cellular migration and stem-cell maintenance using the ‘wound-healing' scratch and tumor-sphere self-renewal assays, respectively. Cell migration was significantly (P < 0.05) inhibited with JQ-1 in both human NB (IMR-32 and SKNSH) and MB (D556 and D283) cell lines. Tumor-sphere formation efficiency was significantly (P < 0.05) reduced in IMR-32, SKNSH, D556, D283 and DIPG cells after treatment with JQ-1. Morphological cellular differentiation, evidenced by long neurite extensions, was induced after JQ-1 treatment in the D556, IMR-32 and SKNSH cells. Conclusions: Our data show potent anti-stem cell activity as well as decreased proliferation, migration and enhanced radiosensitivity following JQ-1 treatment. These effects may be due to delayed DNA repair mechanisms, differential expression of stem cell maintenance and tumorigenic proteins and induced differentiation of the malignant cancer stem cells. Citation Format: Tamara Mary Abou-Antoun, Jad Abdallah, Amanda R. Khater, Sarra El-Soussi, Aysegul Balyimez, Shinjini Ganguly, Anthony Sandler, Omar Mian. The BET inhibitor JQ-1 enhances radio-sensitivity in pediatric cancer cell lines by sustained p-gH2AX expression and enhanced differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1746.

Targeting Aldehyde Dehydrogenases to Eliminate Cancer Stem Cells in Gynecologic Malignancies.

Gynecologic cancers cause over 600,000 deaths annually in women worldwide. The development of chemoresistance after initial rounds of chemotherapy contributes to tumor relapse and death due to gynecologic malignancies. In this regard, cancer stem cells (CSCs), a subpopulation of stem cells with the ability to undergo self-renewal and clonal evolution, play a key role in tumor progression and drug resistance. Aldehyde dehydrogenases (ALDH) are a group of enzymes shown to be robust CSC markers in gynecologic and other malignancies. These enzymes also play functional roles in CSCs, including detoxification of aldehydes, scavenging of reactive oxygen species (ROS), and retinoic acid (RA) signaling, making ALDH an attractive therapeutic target in various clinical scenarios. In this review, we discuss the critical roles of the ALDH in driving stemness in different gynecologic malignancies. We review inhibitors of ALDH, both general and isoform-specific, which have been used to target CSCs in gynecologic cancers. Many of these inhibitors have been shown to be effective in preclinical models of gynecologic malignancies, supporting further development in the clinic. Furthermore, ALDH inhibitors, including 673A and CM037, synergize with chemotherapy to reduce tumor growth. Thus, ALDH-targeted therapies hold promise for improving patient outcomes in gynecologic malignancies.

All-trans retinoic acid therapy induces asymmetric division of glioma stem cells from the U87MG cell line.

The poor therapeutic effect of the current treatments for malignant glioma may be attributed to glioma stem cells (GSCs), which have been demonstrated to divide symmetrically. All-trans retinoic acid (ATRA)-induced differentiation is considered to target GSCs and has been reported to have the capability of eradicating cancer stem cells in specific malignancies. The aim of the present study was to investigate the effects of ATRA on the division mode of GSCs isolated from the U87MG glioblastoma cell line of unknown origin. The expressions of the GSC markers CD133 and nestin were detected using immunocytochemistry to identify GSCs. In addition, the differentiation potency of these GSCs was observed by detecting the expression of glial fibrillary acidic protein, β-tubulin III and galactosylceramidase using immunofluorescent staining. The Numb protein distribution was analyzed in two daughter cells following a GSC division. The results of the present study demonstrated that Numb protein is symmetrically segregated into two daughter cells during GSC division. Furthermore, the present study demonstrated that treatment with ATRA increased the asymmetric cell division of GSCs. In conclusion, these results suggest a therapeutic effect from ATRA-induced asymmetric division of GSCs from the U87MG cell line.

Abstract 165: CUCDC2 regulates malignant phenotype of triple-negative breast cancer through interacting with â-catenin

Abstract Triple-negative breast cancer (TNBC) has the worst prognosis among all subtypes of breast cancer. CUE domain-containing protein-2 (CUEDC2) protein has reported to confer endocrine resistance in breast cancer by down-regulation of estrogen receptor α (ERα) expression. However, the function of CUEDC2 in TNBC subtype is unclear. Here, we showed CUEDC2 is expressed in triple-negative breast cancer and associated with its malignant phenotype. Knockdown of CUEDC2 not only inhibits TNBC cell proliferation, clone formation, migration, and invasion, but also suppresses TNBC stem cell mammosphere formation ability and cancer stem cell markers expression. While over-expression of CUEDC2 enhances its cell malignant phenotype. Furthermore, we determined that the ARM domain of β-catenin interacts with CUE domain of CUEDC2 through immunoprecipitation and pull-down assay. In the 34 TNBC samples, the expression levels of CUEDC2 and β-catenin also showed a positive correlation. Taken together, our study revealed CUEDC2 is associated with malignant phenotype and breast cancer stem cell properties of TNBC by interacting with β-catenin, and suggested that CUEDC2 may be a potential promising prognosis and therapy target for TNBC. Citation Format: Shuyan Han, Hai-Bo Han, Yan-Na Jiao, Pang-Kuo Lo, Yuan Yao, Qun Zhou, Ping-Ping Li. CUCDC2 regulates malignant phenotype of triple-negative breast cancer through interacting with â-catenin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 165.

Next-Generation Hedgehog/GLI Pathway Inhibitors for Cancer Therapy.

The Hedgehog/Glioma-associated oncogene homolog (HH/GLI) signaling pathway regulates self-renewal of rare and highly malignant cancer stem cells (CSC), which have been shown to account for the initiation and maintenance of tumor growth as well as for drug resistance, metastatic spread and relapse. Efficacious therapeutic approaches targeting CSC pathways, such as HH/GLI signaling in combination with chemo, radiation or immunotherapy are, therefore, of high medical need. Pharmacological inhibition of HH/GLI pathway activity represents a promising approach to eliminate malignant CSC. Clinically approved HH/GLI pathway inhibitors target the essential pathway effector Smoothened (SMO) with striking therapeutic efficacy in skin and brain cancer patients. However, multiple genetic and molecular mechanisms resulting in de novo and acquired resistance to SMO inhibitors pose major limitations to anti-HH/GLI therapies and, thus, the eradication of CSC. In this review, we summarize reasons for clinical failure of SMO inhibitors, including mechanisms caused by genetic alterations in HH pathway effectors or triggered by additional oncogenic signals activating GLI transcription factors in a noncanonical manner. We then discuss emerging novel and rationale-based approaches to overcome SMO-inhibitor resistance, focusing on pharmacological perturbations of enzymatic modifiers of GLI activity and on compounds either directly targeting oncogenic GLI factors or interfering with synergistic crosstalk signals known to boost the oncogenicity of HH/GLI signaling.

Do Different Stemness Markers Identify Different Pools of Cancer Stem Cells in Malignancies: A Study on ER+ and ER-Breast Cancer Cell Lines.

In view of popularity of cancer stem cell (CSC) model all events in evolution of cancer are being explained in that context. Breast cancer is first solid tumor in which CSCs were identified. We aimed to compare stemness profile of two major subtypes [Estrogen receptor positive (ER+) and negative (ER-)] breast cancer using different sets of markers. Expression of CD44/CD24, CK/Vimentin, E-Cadherin/Fibronectin and percentage of side population (SP) was studied in ER+ (T47D) and ER- (MDA-MB-231) cell lines by flow cytometry. Breast CSCs (BCSCs) were sorted using CD44+/CD24-/low expressionand SP analysis and cultured. BCSCs were then compared with Non-CSCs (NCSCs) for response to drugs (Paclitaxel and Cisplatin), Ki67 and ER expression. Results showed higher expression of stemness markers (CD44+/CD24-/low, CK+/Vimentin+ and E-Cadherin-/FibrinectinF+) in MDA-MB-231 cells. Percentage SP representing BCSCs was found to be significantly more in later (3.20 ± 0.002 cf. T47D 1.25% ± 0.0007). BCSCs were found to be more resistant to drugs as compared to NCSCs in both cell lines. ER expression was weak in BCSCs sorted from T47D as compared to NCSCs. Ki67 was expressed in both BCSCs and NCSCs. Differences in expression of stemness markers help to explain aggressive behavior, higher recurrence rate and metastatic potential of MDA-MB-231 cells. However, no correlation amongst different markers used suggests that they may be identifying varied populations of cells in tumor hierarchy. A weak ER expression in BCSCs may be strategy used by BCSCs to escape effect of hormone therapy in ER+ breast cancers.

Abstract LB-297: HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1

Abstract HER2 represents an ideal target for cancer immunotherapy. HER2-targeted antibody drug conjugate (ADC) was tested in immunocompromised (SCID) host, which doesn't allow the evaluation of the ADC-induced immune responses. In addition, no potential impact of ADC on cancer stem cells (CSCs) has been reported. Furthermore, tumor cells with PD-L1 expression may escape immune attack by converting PD-1 positive immune effector cells into anergy. We hypothesized that HER2-targeted ADC could modulate host immune response, target HER2high CSCs, and the efficacy of ADC could be enhanced by anti-PD-L1. To test this hypothesis, we used two immunocompetent murine models: mouse breast tumor D2/F2 and TNBC 4T1 with enforced expression of HER2, D2F2/E2 and HER2-4T1 respectively. Both D2F2/E2 and HER2-4T1 could generate tumor in immunocompetent Balb/c mice, which allowed us to test the efficacy and specificity of HER2-targeted ADC, and to evaluate host immune responses. In D2F2/E2 mouse model, we found that HER2-targeted ADC significantly inhibited D2F2/E2 tumor growth, but not the parental D2F2 tumors, in a dose dependent manner. The specificity was confirmed in HER2-4T1 model because we observed significant suppression of HER2-targted ADC on HER2-4T1 tumor growth, but not on the parental 4T1 tumor. In parallel, we observed that HER2-targeted ADC treatment reduced the number of HER2 positive cells, and ALDHhigh cells. ALDHhigh cells have been identified as cancer stem cells in multiple malignancies. We have characterized the stemness of ALDHhigh 4T1 cells. In the present study, we confirmed that the ALDHhigh sub-population of D2F2/E2 cells are highly tumorigenic compared to ALDHlow cells. Next, we tested and observed that PD-L1 expression increased in D2F2/E2 and HER2-4T1 fresh tumor cells than in cell lines. Anti-PD-L1 mAb significantly augmented the therapeutic efficacy of HER2-targeted ADC in both D2F2/E2 and HER2-4T1 tumor models. In addition, anti-PD-L1 plus HER2-targeted ADC further reduced the number of HER2 positive cells, and ALDHhigh cells, but increased the number of CD3+ and CD19+ tumor infiltrating lymphocytes (TILs). Expanded CD3+ TILs from the tumor subjected to HER2-targeted ADC plus anti-PD-L1 treatment killed tumor cells significantly more than mono-treatment. Expanded CD19+ TILs from tumor subjected to HER2-targeted ADC plus anti-PD-L1 produced more IgG than all the controls. Importantly, the IgG could specifically bind to tumor cells, resulting in the highest cytotoxicity of the tumor cells via ADCC. Together, our data indicate that HER2-targeted ADC could induce significant host immune responses, which was evidenced by the detection of anti-tumor CD3+ and CD19+ TILs. We first show that HER2-targeted ADC could target ALDHhigh cancer stem cells, and such effect was significantly enhanced by anti-PD-L1 administration. Citation Format: Leiming Xia, Lu Wen, Ming Lin, Comer Frank, Hinrichs Mary Jane, Oberst Michael, Coats Steven, Yangyi Bao, Alfred E. Chang, Max Wicha, Qiao Li. HER2-targeted antibody drug conjugates induce host immunity and target cancer stem cells and the efficacy can be enhanced by anti-PD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-297.