Cancer Stem-like Cells Population Research Articles

Development of a novel N14-substituted antitumor evodiamine derivative with inhibiting heat shock protein 70 in non-small cell lung cancer

Notwithstanding the latest advancements in anticancer therapy, non-small cell lung cancer (NSCLC) remains a prominent contributor to cancer-associated mortality worldwide. Therefore, effective anti-cancer agents are required for the treatment of NSCLC. We previously demonstrated that the natural alkaloid evodiamine efficiently suppressed lung cancer cells and lung cancer stem-like cell populations by suppressing heat shock protein 70 (Hsp70). This finding inspired us to formulate evodiamine-based anti-cancer compounds against NSCLC. In this study, we synthesized a series of evodiamine derivatives with substitutions at the N14 position. EV206 was chosen for further study because it was the most effective among the 22 evodiamine derivatives at stopping H1299 cell growth. EV206 treatment efficiently suppressed cell viability and colony formation in both attached cells and in soft agar, even in those carrying drug resistance, by inducing apoptosis. The effectiveness of EV206 is approximately ten times greater than that of evodiamine. Normal cell viability was marginally affected by EV206 treatment. Additionally, EV206 efficiently decreased the cancer stem cell (CSC) population in the NSCLC cells. EV206 reduced the growth of H460 xenograft tumors without exhibiting toxic effects. These data implied that EV206 has the potential to be an effective Hsp70-targeting anticancer drug with low toxicity.

RNA binding protein ZCCHC24 promotes tumorigenicity in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expression of hormone and HER2 receptors and is highly malignant with no effective therapeutic targets. In TNBC, the cancer stem-like cell (CSC) population is considered to be the main cause of resistance to treatment. Thus, the therapeutic targeting of this population could substantially improve patient survival. Here, we identify the RNA-binding protein ZCCHC24 as enriched in the mesenchymal-like TNBC population. ZCCHC24 promotes the expression of a set of genes related to tumorigenicity and treatment resistance by directly binding to the cis-element "UGUWHWWA" in their mRNAs, thereby stabilizing them. One of the ZCCHC24 targets, ZEB1, is a transcription factor that promotes the expression of cancer stemness genes and reciprocally induces ZCCHC24 expression. ZCCHC24 knockdown by siRNAs shows a therapeutic effect and reduces the mesenchymal-like cell population in TNBC patient-derived xenografts. ZCCHC24 knockdown also has additive effects with the BET inhibitor JQ1 in suppressing tumor growth in TNBC patient-derived xenografts.

A live single-cell reporter system reveals drug-induced plasticity of a cancer stem cell-like population in cholangiocarcinoma.

Cancer stem cells (CSC) play an important role in carcinogenesis and are acknowledged to be responsible for chemoresistance in cholangiocarcinoma (CCA). Studying CCA CSC has been challenging, due to lack of consensus CSC markers, and to their plastic nature. Since dual expression of the core pluripotent factors SOX2/OCT4 has been shown to correlate with poor outcome in CCA patients, we selected the SOX2/OCT4 activating short half-life GFP-based live reporter (SORE6-dsCopGFP) to study CSC dynamics at the single-cell level. Transduction of five human CCA cell lines resulted in the expression of 1.8-13.1% GFP-positive (SORE6POS) cells. By live imaging, we found that SORE6POS CCA cells possess self-renewal capacity and that they can be induced to differentiate. Significantly, the SORE6POS cells were highly tumorigenic, both in vitro and in vivo, thus implicating the characteristics of primary CSCs. When we then analyzed for selected CSC-related markers, we found that the majority of both CD133+/CD44+, and CD133+/LGR5+ CCA cells were SORE6POS cells. Exposing transduced cells to standard CCA chemotherapy revealed higher growth rate inhibition at 50% (GR50s) for SORE6POS cells compared to GFP-negative (SORE6NEG) ones indicating that these CSC-like cells were more resistant to the treatment. Moreover, the chemotherapy induced SORE6POS from SORE6NEG cells, while retaining the existing SORE6POS population. Finally, treatment of transduced cells with CDK4/6 inhibitors in vitro for 3 days resulted in a lowered CSC number in the culture. Thus, applying a live reporter system allowed us to elucidate the stem cell diversity and drug-induced plasticity of CCA CSCs. These findings have clear implications for future management of such patients.

Abstract 3584: Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer

Abstract Exposure to progesterone is a recognized risk factor for breast cancer, and PGR polymorphisms are associated with various malignancies. The two progesterone receptor (PR) isoforms, full length PR-B and truncated PR-A, are expressed from the PGR gene in breast tissue and play crucial roles in normal physiology. An imbalance in the expression ratio of these isoforms, favoring increased levels of PR-A, is common in breast cancer and is associated with resistance to tamoxifen in Luminal A-type tumors. Notably, PRs have recently been implicated in promoting endocrine resistance and driving the expansion of cancer stem-like cell (CSC) populations implicated in early dissemination and maintenance of metastatic populations. Because prior gene expression studies were primarily conducted in 2D culture conditions, the isoform-specific molecular and epigenetic mechanisms underlying PR actions in advanced ER+ breast cancer remain elusive. Our goal was to define progesterone-driven gene expression in CSC cells by employing transcriptomic profiling of T47D cells cultivated in spheroid-culture conditions (3D) expressing exclusively PR-A or PR-B. Utilizing CUT&RUN, we have mapped the genomic binding sites unique to each PR isoform and identified shared sites. This genomic data has been integrated with phenotypic studies in cells expressing a single PR isoform. Our findings indicate that PR-A acts as a regulator of the cell cycle and senescence, while PR-B plays a pivotal role in cell metabolism and intracellular signaling. We find little overlap between PR-regulated gene sets derived from the same hormone-treated cells harvested from 2D relative to 3D conditions. Our genomic profiling of PRs in this 3D model system has unveiled novel strategies to combat CSC outgrowth and late recurrence in luminal breast cancer patients. This work has shifted our prior understanding of the role of PRs in gene regulation within the CSC population, and offers potential insights for therapeutic interventions in ER+ breast cancer. Citation Format: Noelle E. Gillis, Thu H. Truong, Jessica Finlay-Schultz, Carol A. Sartorius, Carol A. Lange. Context matters: Genomic profiling of PR isoform-specific actions in breast cancer stem cells reveals novel therapeutic insights for ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3584.

Tumor-derived apoptotic extracellular vesicle-mediated intercellular communication promotes metastasis and stemness of lung adenocarcinoma

Apoptosis has long been recognized as a significant mechanism for inhibiting tumor formation, and a plethora of stimuli can induce apoptosis during the progression and treatment of tumors. Moreover, tumor-derived apoptotic extracellular vesicles (apoEVs) are inevitably phagocytosed by live tumor cells, promoting tumor heterogeneity. Understanding the mechanism by which apoEVs regulate tumor cells is imperative for enhancing our knowledge of tumor metastasis and recurrence. Herein, we conducted a series of in vivo and in vitro experiments, and we report that tumor-derived apoEVs promoted lung adenocarcinoma (LUAD) metastasis, self-renewal and chemoresistance. Mechanistically, we demonstrated that apoEVs facilitated tumor metastasis and stemness by initiating the epithelial-mesenchymal transition program and upregulating the transcription of the stem cell factor SOX2. In addition, we found that ALDH1A1, which was transported by apoEVs, activated the NF-κB signaling pathway by increasing aldehyde dehydrogenase enzyme activity in recipient tumor cells. Furthermore, targeting apoEVs-ALDH1A1 significantly abrogated these effects. Collectively, our findings elucidate a novel mechanism of apoEV-dependent intercellular communication between apoptotic tumor cells and live tumor cells that promotes the formation of cancer stem cell-like populations, and these findings reveal that apoEVs-ALDH1A1 may be a potential therapeutic target and biomarker for LUAD metastasis and recurrence.

Up-regulation of RAN by MYBL2 maintains osteosarcoma cancer stem-like cells population during heterogeneous tumor generation

Intratumor heterogeneity is one of the major features of cancers, leading to aggressive disease and treatment failure. Cancer stem-like cells (CSCs) are believed to give rise to the heterogeneous cell types within tumors. Hence, understanding the regulatory mechanism underlying the recurrence process of heterogeneous tumor by CSCs could facilitate the development of CSC-targeted therapies. Here, utilizing single-cell transcriptomics, we present the molecular profile of osteosarcoma CSCs-derived heterogeneous tumors consisting of CSC clusters, osteoprogenitor and differentiated cell types, such as pre-osteoblasts, osteoblasts and chondroblasts. Furthermore, by constructing the comprehensive map of modulated genes during CSCs self-renewal and differentiation, we identify RAN exhibiting specific peak expression in osteosarcoma CSCs clusters which is transcriptionally up-regulated by MYBL2. Functionality, MYBL2-RAN pathway promotes the CSCs self-renewal by enhancing the nuclear accumulation of MYC protein, which in turn boosts the overexpression of RAN as a positive feedback. Importantly, blockage of MYBL2-RAN pathway sensitizes CSCs to cisplatin treatment and synergistically enhanced the cisplatin-induced cytotoxicity. Both MYBL2 and RAN are highly expressed in clinical osteosarcoma tissues which indicate poor prognosis. Collectively, our study provides advanced insights into the regeneration process of heterogeneous tumor originating from CSCs and highlights the MYBL2-RAN pathway as a promising target for CSC-based therapy in osteosarcoma.

Abstract PR01: Identification and pharmacological targeting of treatment-resistant, stem-like breast cancer cells for combination therapy

Abstract Marker-based isolation of Cancer Stem-Like Cells (CSLCs), which can display preferential resistance to standard-of-care chemotherapy, has proven challenging, due to their very small frequency in breast cancer tissue, thus impeding their characterization, as well as identification and targeting of mechanistic dependencies. To address this challenge, we leveraged a systems biology framework for the protein-activity based characterization of CSLC biology at the single cell level and for the identification of drugs that could reprogram them to a differentiated, chemotherapy-sensitive state. To enrich for CSLCs we performed flow cytometry-based sorting of cells dissociated from 7 metastatic breast cancer patients, both TNBC and HR+, using the EpCAM and CD49f markers, followed by RNA-seq profiling. Protein activity-based clustering, using the VIPER algorithm (Alvarez et al. Nat Genet 2017), identified complementary cell states associated with high activity of either established CSLC markers (BMI1, NOTCH1, etc.) or markers of differentiated cells (GATA3, KRT18, etc.). VIPER-inferred Master Regulator (MR) proteins, identified by comparing highest versus lowest stemness score cells in each patient, were virtually identical across patients and independent of hormone receptor status. Following MR validation by pooled, CRISPR/Cas9-mediated KO followed by single cell RNA-seq profiling (CROP-seq) in cell lines comprising both CLSC and differentiated BRCA cells, we used the OncoTreat algorithm (Alvarez et al. Nat Genet 2018) to identify small molecule compounds capable of inverting their activity (MR-inverter drugs), thus potentially inducing differentiation or cell death. For this purpose, we leveraged gene expression profiles of BT-20 cells—which significantly recapitulate the CSLC MR signature—following treatment with a repertoire of 91 clinically relevant drugs. Albendazole, the top drug identified by OncoTreat, depleted CSLC population (p = 2.0 × 10−4, by Fisher’s exact test, w.r.t. CSLCs to differentiated cells ratio) without noticeable cytotoxicity in the experiment of single cell-based assays in a TNBC PDX model, at 14 days after treatment. In contrast, paclitaxel, a commonly used chemotherapeutic, significantly increase the ratio of CSLC to differentiated cells (p = 2.6 × 10−4, by Fisher’s exact test), suggesting that alternating treatment with the two drugs may abrogate the tumor-initiating potential of paclitaxel-resistant cells, while also preventing uncontrolled tumor growth. This rationale of combination therapy was confirmed by preclinical study, where treatment with multiple cycles of albendazole and paclitaxel displayed significant synergy (p = 0.00869), compared to the corresponding monotherapies. In conclusion, our systems biology framework is a proof-of-concept that drugs can be effectively repurposed to enhance the therapeutic activity of anti-tumor agents used in conventional chemotherapy, and this method can be generalizable to address the challenge of other cancers with heterogeneity. Citation Format: Jeremy Worley, Heeju Noh, Daoqi You, Mikko M Turunen, Hongxu Ding, Evan Paull, Aaron T Griffin, Adina Grunn, Kristina Guillan, Erin C Bush, Samantha J Brosius, Prabhjot S Mundi, Peter Sims, Piero Dalerba, Filemon S Dela Cruz, Andrew L Kung, Andrea Califano. Identification and pharmacological targeting of treatment-resistant, stem-like breast cancer cells for combination therapy [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 PR01.

Deciphering head and neck cancer microenvironment: Single-cell and spatial transcriptomics reveals human papillomavirus-associated differences.

Human papillomavirus (HPV) is a major causative factor of head and neck squamous cell carcinoma (HNSCC), and the incidence of HPV- associated HNSCC is increasing. The role of tumor microenvironment in viral infection and metastasis needs to be explored further. We studied the molecular characteristics of primary tumors (PTs) and lymph node metastatic tumors (LNMTs) by stratifying them based on their HPV status. Eight samples for single-cell RNA profiling and six samples for spatial transcriptomics (ST), composed of matched primary tumors (PT) and lymph node metastases (LNMT), were collected from both HPV- negative (HPV- ) and HPV-positive (HPV+ ) patients. Using the 10x Genomics Visium platform, integrative analyses with single-cell RNA sequencing were performed. Intracellular and intercellular alterations were analyzed, and the findings were confirmed using experimental validation and publicly available data set. The HPV+ tissues were composed of a substantial amount of lymphoid cells regardless of the presence or absence of metastasis, whereas the HPV- tissue exhibited remarkable changes in the number of macrophages and plasma cells, particularly in the LNMT. From both single-cell RNA and ST data set, we discovered a central gene, pyruvate kinase muscle isoform 1/2 (PKM2), which is closely associated with the stemness of cancer stem cell-like populations in LNMT of HPV- tissue. The consistent expression was observed in HPV- HNSCC cell line and the knockdown of PKM2 weakened spheroid formation ability. Furthermore, we found an ectopic lymphoid structure morphology and clinical effects of the structure in ST slide of the HPV+ patients and verified their presence in tumor tissue using immunohistochemistry. Finally, the ephrin-A (EPHA2) pathway was detected as important signals in angiogenesis for HPV- patients from single-cell RNA and ST profiles, and knockdown of EPHA2 declined the cell migration. Our study described the distinct cellular composition and molecular alterations in primary and metastatic sites in HNSCC patients based on their HPV status. These results provide insights into HNSCC biology in the context of HPV infection and its potential clinical implications.

THU570 Isoform Specific Gene Regulation By Progesterone Receptors Drives Expansion Of Breast Cancer Stem Cell Expansion In 3D Cell Culture Models

Abstract Disclosure: N.E. Gillis: None. T.H. Truong: None. C.A. Lange: None. Exposure to progesterone is a risk factor for breast cancer, and numerous PGR polymorphisms are linked to breast, endometrial, and ovarian cancer. Two progesterone receptor (PR) isoforms are expressed from PGR in breast tissues: full-length PR-B and truncated PR-A. In normal breast epithelia, PR isoforms are equally expressed and mostly function as A:B heterodimers. An imbalance of this ratio, and thus a predominance of homodimers, is frequent in ER+ breast cancer. Luminal A-type estrogen receptor positive (ER+) tumors often exhibit an elevated PR-A:PR-B ratio that predicts resistance to tamoxifen. It has recently been shown that PRs promote endocrine resistance and drive expansion of breast cancer stem-like cell (CSC) populations. However, the isoform-specific molecular mechanisms of PR action in ER+ breast cancers remain undefined. The goal of our current work is to map the progesterone-driven gene expression in CSC cells through transcriptomic profiling of T47D cells grown in spheroid-culture conditions that express exclusively PR-A or PR-B. This transcriptomic data has been integrated with phenotypic studies of T47D and MCF7 cells that express a single isoform of PR. Our data shows PR-A is a regulator of the cell cycle and of senescence, while PR-B mediates cell metabolism and intracellular signaling. We further integrated our cell line data with transcriptomes from patient tumors that have been classified based on their PR-A:PR-B ratio and their response to treatment with anti-progestins. Our results are a first step towards re-defining the role of PRs in gene regulation within the CSC population. Presentation Date: Thursday, June 15, 2023

Orai3 Calcium Channel Contributes to Oral/Oropharyngeal Cancer Stemness through the Elevation of ID1 Expression.

Emerging evidence indicates that intracellular calcium (Ca2+) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca2+ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation.

TRβ Agonism Induces Tumor Suppression and Enhances Drug Efficacy in Anaplastic Thyroid Cancer in Female Mice.

Thyroid hormone receptor beta (TRβ) is a recognized tumor suppressor in numerous solid cancers. The molecular signaling of TRβ has been elucidated in several cancer types through re-expression models. Remarkably, the potential impact of selective activation of endogenous TRβ on tumor progression remains largely unexplored. We used cell-based and in vivo assays to evaluate the effects of the TRβ agonist sobetirome (GC-1) on a particularly aggressive and dedifferentiated cancer, anaplastic thyroid cancer (ATC). Here we report that GC-1 reduced the tumorigenic phenotype, decreased cancer stem-like cell populations, and induced redifferentiation of the ATC cell lines with different mutational backgrounds. Of note, this selective activation of TRβ amplified the effects of therapeutic agents in blunting the aggressive cell phenotype and stem cell growth. In xenograft assays, GC-1 alone inhibited tumor growth and was as effective as the kinase inhibitor, sorafenib. These results indicate that selective activation of TRβ not only induces a tumor suppression program de novo but enhances the effectiveness of anticancer agents, revealing potential novel combination therapies for ATC and other aggressive solid tumors.

Kruppel-like factor 8 regulates triple negative breast cancer stem cell-like activity.

Breast tumor development is regulated by a sub-population of breast cancer cells, termed cancer stem-like cells (CSC), which are capable of self-renewing and differentiating, and are involved in promoting breast cancer invasion, metastasis, drug resistance and relapse. CSCs are highly adaptable, capable of reprogramming their own metabolism and signaling activity in response to stimuli within the tumor microenvironment. Recently, the nutrient sensor O-GlcNAc transferase (OGT) and O-GlcNAcylation was shown to be enriched in CSC populations, where it promotes the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. This enrichment was associated with upregulation of the transcription factor Kruppel-like-factor 8 (KLF8) suggesting a potential role of KLF8 in regulating CSCs properties. Triple-negative breast cancer cells were genetically modified to generate KLF8 overexpressing or KLF8 knock-down cells. Cancer cells, control or with altered KLF8 expression were analyzed to assess mammosphere formation efficiency, CSCs frequency and expression of CSCs factors. Tumor growth in vivo of control or KLF8 knock-down cells was assessed by fat-pad injection of these cell in immunocompromised mice. Here, we show that KLF8 is required and sufficient for regulating CSC phenotypes and regulating transcription factors SOX2, NANOG, OCT4 and c-MYC. KLF8 levels are associated with chemoresistance in triple negative breast cancer patients and overexpression in breast cancer cells increased paclitaxel resistance. KLF8 and OGT co-regulate each other to form a feed-forward loop to promote CSCs phenotype and mammosphere formation of breast cancer cells. These results suggest a critical role of KLF8 and OGT in promoting CSCs and cancer progression, that may serve as potential targets for developing strategy to target CSCs specifically.

Abstract 2442: KLF8 and OGT/O-GlcNAcylation regulate stem-like cell properties in breast cancer

Abstract Breast cancer is the most common cancer and the second leading cause of cancer death among women overall. One of the main challenges in fighting breast cancer is the intratumor heterogeneity as they are composed of different subpopulations of cancer cells. This heterogeneity of breast cancer is promoted and maintained by a subpopulation of cancer stem-like cells (CSC). CSC share properties of adult stem cells, capable of self-renewing, differentiating & repopulating a tumor. In addition to CSC role in promoting tumor growth, CSC are involved in metastasis, drug resistance and relapse of breast cancer. Therefore, understanding the mechanisms that drive and maintain CSC will help identify novel therapeutic targets in treating breast cancer. Functional activities of CSC are heavily influenced by modulations in the tumor microenvironment, such as alteration in nutrient status. One key component connecting signaling pathway and cancer cell metabolism is the enzyme O-GlcNAc Transferase (OGT), which utilize UDP-GlcNAc to modify nuclear and cytoplasmic proteins in a process termed O-GlcNAcylation. OGT and O-GlcNAc are upregulated in many cancers. Recently, we showed a direct connection between OGT/O-GlcNAc and CSC in breast cancer, in which OGT/O-GlcNAc is required and sufficient to promote the stemness of breast cancer cells in vitro, as well as tumorigenesis in vivo. Global transcriptomic analysis of CSCs-enriched mammospheres with OGT overexpression revealed Kruppel-like-factor 8 (KLF8) as a downstream target of OGT in CSC. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE), reduced CSC population, and tumor growth in vivo. In contrast, KLF8 overexpression increased MFE and CSC population. Importantly, KLF8 knockdown blocked the effect of elevated OGT and O-GlcNAcylation in promoting MFE, while KLF8 overexpression rescued OGT inhibition in regulating in breast cancer cells. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level, while KLF8 overexpression increased expression of these CSC factors. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O-GlcNAcylation, suggesting a possible feed-forward loop between OGT and KLF8 in promoting CSC. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple-negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem-like cells population. Citation Format: Giang Le Minh, Emily Esquea, Tejsi Dhamelia, Neha Akella, Mauricio J. Reginato. KLF8 and OGT/O-GlcNAcylation regulate stem-like cell properties in breast cancer [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 2442.

An A-ring substituted evodiamine derivative with potent anticancer activity against human non-small cell lung cancer cells by targeting heat shock protein 70

The heat shock protein (HSP) system is essential for the conformational stability and function of several proteins. Therefore, the development of efficacious HSP-targeting anticancer agents with minimal toxicity is required. We previously demonstrated that evodiamine is an anticancer agent that targets HSP70 in non-small cell lung cancer (NSCLC) cells. In this study, we synthesized a series of evodiamine derivatives with improved efficacy and limited toxicity. Among the 14 evodiamine derivatives, EV408 (10-hydroxy-14-methyl-8,13,13b,14-tetrahydroindolo[2′,3′:3,4]pyrido[2,1-b]quinazolin-5(7H)-one) exhibited the most potent inhibitory effects on viability and colony formation under anchorage-dependent and -independent culture conditions in various human NSCLC cells, including those that are chemoresistant, by inducing apoptosis. In addition, EV408 suppressed the cancer stem-like cell (CSC) population of NSCLC cells and the expression of stemness-associated markers. Mechanistically, EV408 inhibited HSP70 function by directly binding and destabilizing the HSP70 protein. Furthermore, EV408 significantly inhibited the growth of NSCLC cell line tumor xenografts without overt toxicity. Additionally, EV408 had a negligible effect on the viability of normal cells. These results suggest the potential of EV408 as an efficacious HSP70-targeting evodiamine derivative with limited toxicity that inhibits both non-CSC and CSC populations in NSCLC.

Docetaxel in combination with metformin enhances antitumour efficacy in metastatic breast carcinoma models: a promising cancer targeting based on PEGylated liposomes.

Metformin has been shown to kill cancer stem-like cells in genetically various types of breast carcinoma. With the aim to simultaneously eradicate the bulk population of tumour cells and the rare population of cancer stem-like cells in breast cancer tissues, we used the combination chemotherapy of docetaxel (DTX) with metformin (MET). Furthermore, we introduce an active loading method based on ammonium sulphate 250 mM (SA) for encapsulating docetaxel into liposomes. Docetaxel and metformin encapsulated into PEGylated liposomes with two different methods based on remote or passive loading methods, respectively. The size and surface charge of the liposomes were characterized. DTX content in the nanoliposomes was measured by the high-performance liquid chromatography method. The drug release profiles were evaluated in phosphate-buffered dextrose 5% with the pH of 6.5 and 7.4. We examined the antitumour activity of Taxotere (TAX), and liposomal formulation of DTX and MET as a monotherapy or combination therapy. The biodistribution of liposomes was also investigated using 99mTc hexamethyl propylene amine oxime method in BALB/c mice bearing 4T1 breast carcinoma tumours. The final formulations were prepared according to the best physicochemical characteristics which were HSPC/mPEG2000-DSPE/Chol (DTX liposomes) and HSPC/DPPG/mPEG2000-DSPE/Chol (MET liposomes), at molar ratios of 85/5/10 and (55/5/5/35), respectively. In vivo experiments showed that when free or liposomal metformin used in combination with liposomal docetaxel, they prolonged median survival time (MST) from 31 in the control group to 46 days, which demonstrates their promising effects on the survival of the 4T1 breast carcinoma mice models. Moreover, combination therapies could significantly increase life span in comparison with phosphate-buffered saline (PBS) and Taxotere groups at the same dose. Furthermore, in the combination therapy study, treatment with DTX liposomes prepared by ammonium sulphate 250 mM buffer alone resulted in similar therapeutic efficacy to combination therapy. The biodistribution study exhibited significant accumulation of DTX liposomes in the tumours due to the Enhanced Permeability and Retention effect. This study also showed that metformin-based combinatorial chemotherapies have superior efficacy versus their corresponding monotherapy counterparts at same doses. The findings confirm that liposomes based on ammonium sulphate 250 mM could be as a promising formulation for efficient DTX delivering and cancer targeting and therefore merit further investigations.

Unifying Different Cancer Theories in a Unique Tumour Model: Chronic Inflammation and Deaminases as Meeting Points.

The increase in cancer incidences shows that there is a need to better understand tumour heterogeneity to achieve efficient treatments. Interestingly, there are several common features among almost all types of cancers, with chronic inflammation induction and deaminase dysfunctions singled out. Deaminases are a family of enzymes with nucleotide-editing capacity, which are classified into two main groups: DNA-based and RNA-based. Remarkably, a close relationship between inflammation and the dysregulation of these molecules has been widely documented, which may explain the characteristic intratumor heterogeneity, both at DNA and transcriptional levels. Indeed, heterogeneity in cancer makes it difficult to establish a unique tumour progression model. Currently, there are three main cancer models—stochastic, hierarchic, and dynamic—although there is no consensus on which one better resembles cancer biology because they are usually overly simplified. Here, to accurately explain tumour progression, we propose interactions among chronic inflammation, deaminases dysregulation, intratumor genetic heterogeneity, cancer phenotypic plasticity, and even the previously proposed appearance of cancer stem-like cell populations in the edges of advanced solid tumour masses (instead of being the cells of origin of primary malignancies). The new tumour development model proposed in this study does not contradict previously accepted models and it may open up a window to interesting therapeutic approaches.

Trabectedin suppresses escape from therapy-induced senescence in tumor cells by interfering with glutamine metabolism

Conventional and targeted cancer therapies may induce a cellular senescence program termed therapy-induced senescence. However, unlike normal cells, cancer cells are able to evade the senescence cell cycle arrest and to resume proliferation, driving tumor recurrence after treatments. Cells that escape from therapy-induced senescence are characterized by a plastic, cancer stem cell-like phenotype, and recent studies are beginning to define their unique metabolic features, such as glutamine dependence.Here, we show that the antineoplastic drug trabectedin suppresses escape from therapy-induced senescence in all cell lines studied, and reduces breast cancer stem-like cells, at concentrations that do not affect the viability of senescent tumor cells. We demonstrate that trabectedin downregulates both the glutamine transporter SLC1A5 and glutamine synthetase, thereby interfering with glutamine metabolism. On the whole, our results indicate that trabectedin targets a glutamine-dependent cancer stem-like cell population involved in evasion from therapy-induced senescence and suggest a therapeutic potential for trabectedin combined with pro-senescence chemotherapy in tumor treatment.

Abstract 901: Sox2 and Oct4 activity identify CSC populations in ovarian cancer

Abstract High Grade Serous Ovarian Cancer is the most lethal gynecological cancer, mainly due to late-stage diagnosis and prevalence of chemotherapy-resistant disease. Tumors are comprised of heterogenous cancer cells, which makes targeted therapies challenging. Studies have identified a subset of cancer stem-like cells (CSCs) that, unlike bulk tumor cells, can evade chemotherapy and induce relapse. Cell surface markers such as CD133, CD44, CD117, or ALDH enzyme activity are typically used to identify and isolate CSCs; however, these markers are inconsistent across cell lines and patient samples making them unreliable for isolating CSCs. Consistent indicators of CSCs may be activity of stem cell transcription factors, such as SOX2, OCT4, and NANOG, which are known to promote long-term self-renewal and asymmetric division, processes required for tumor repopulation. We hypothesize that a reporter responding to SOX2 and OCT4 activity will represent a functionally relevant and reliable marker for identifying CSCs capable of enabling relapse. We have stably transduced a reporter (termed SORE6), which detects SOX2 and OCT4 activity, into OV90 and ACI23 ovarian cancer cells. SORE6+ cells, isolated using fluorescence assisted cell sorting (FACS), have 2 to 15-fold increases in SOX2/OCT4/NANOG transcripts, relative to SORE6- cells. Comparison of CSC markers in chemotherapy versus vehicle conditions suggests that SORE6 more consistently identifies the CSC population. Specifically, flow cytometry analysis of ACI23 cells showed a 2-fold increase in SORE6+ cells, 7-fold increase in CD117+ cells, and a 1.2-fold increase in CD133 cells in carboplatin treated cells relative to vehicle. For OV90, SORE6+ increases again by 2-fold, while CD117+ increases by 5-fold and CD133 increases by 1.5-fold. SORE6+ cells also showed significantly enhanced spheroid formation efficiency in low serum conditions and resistance to carboplatin and paclitaxel chemotherapies, relative to SORE6- cells. Finally, in vivo limiting dilution studies in mice showed a significant difference in tumorigenicity of SORE6+ cells at low dilutions in ACI23 cells, but not for OV90 cells. Flow cytometry analysis of disassociated tumors showed that tumors created using pure SORE6- cells remained SORE6-, while tumors created using pure SORE6+ cells maintained only 10% SORE6+, indicating the ability of SORE6+ cells to asymmetrically divide to create a heterogeneous tumor. In conclusion, these data suggest that the SORE6 reporter identifies a CSC population within ovarian cancer cell lines and could be a useful tool for consistent isolation of CSCs. Use of this reporter will enhance our understanding of mechanisms, such as SOX2 and OCT4 activity, that support chemoresistance and tumor repopulation. More reliable identification of CSCs will enable the design of therapies to overcome chemotherapy resistance and disease recurrence. Citation Format: Luisjesus S. Cruz, Mikella Robinson, Samuel F. Gilbert, Omar Lujano-Olazaba, Logan A. Alexander, Alex Horkowitz, Carrie D. House. Sox2 and Oct4 activity identify CSC populations in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 901.

Identification of two cancer stem cell-like populations in triple-negative breast cancer xenografts.

ABSTRACTGene expression analysis at the single-cell level by next-generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell–cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-sequencing datasets confirmed that the two CSC-like populations existed in TNBC xenograft models and in TNBC patients. The diversity of these multiple CSC-like populations could cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

KLF8 and OGT/O‐GlcNAcylation regulate breast cancer stem‐like cells

One of the main challenges in treating breast cancer is the intra‐tumor heterogeneity, which, partly, is maintained and promoted by a sub‐population of breast cancer cells called breast cancer stem‐like cells (BCSCs). BCSCs shares traits of mammary stem cells, capable of self‐renewing and differentiating, while promotes invasion, metastasis, drug resistance and relapse. BCSCs are highly adaptive, capable of reprogramming metabolism and signaling activity in response to tumor microenvironment. One key component of cell nutrient sensing mechanism, linking alteration in metabolism to cell signaling, is O‐GlcNAc transferase (OGT). OGT is responsible to adding O‐GlcNAc moieties to target nuclear, cytoplasmic and mitochondrial proteins from UDP‐GlcNAc – final product of the hexosamine biosynthetic pathway. This modification is termed O‐GlcNAcylation and both OGT and O‐GlcNAcylation are upregulated in many cancers. Recently, we showed that OGT/O‐GlcNAcylation is enriched in BCSCs, promoting the stemness and tumorigenesis of breast cancer cells in vitro and in vivo. RNA‐seq analysis of BCSC‐enriched mammospheres with OGT overexpression revealed Kruppel‐like‐factor 8 (KLF8) as a downstream target of OGT in promoting BCSCs. Here, we show that KLF8 expression in breast cancer cells is regulated by OGT at the mRNA and protein level. Knockdown of KLF8 reduced mammosphere forming efficiency (MFE) and NANOG expression and ALDH activity in breast cancer cells, while overexpressing KLF8 increased MFE, NANOG+ and ALDH+BCSC population. Knockdown of KLF8 also reduced breast tumor growth in vivo. Importantly, KLF8 knockdown abolished the effect of elevated OGT and O‐GlcNAcylation in breast cancer cell MFE and BCSC population, while KLF8 overexpression rescued OGT inhibition in MFE and BCSC population. suggesting that KLF8 is required in promoting BCSCs. Consistently, KLF8 knockdown in breast cancer cells significantly reduced expression of stem cells markers at both mRNA and protein level. Interestingly, KLF8 knockdown in breast cancer cell reduced OGT expression, while KLF8 overexpression increased OGT and O‐GlcNAcylation, suggesting a possible feed‐forward loop between OGT and KLF8 in promoting BCSCs. Supporting the idea that KLF8 and OGT are critical for breast cancer progression, high expression of KLF8 and OGT is correlated with poor breast cancer patient outcome. Furthermore, overexpression of KLF8 showed increase in resistance to paclitaxel in triple‐negative breast cancer cells in vitro, suggesting a possible role of KLF8 in promoting chemotherapy resistance in breast cancer. Together, our results suggested that KLF8 and OGT may work in concert in promoting breast cancer stem‐like cells population.