Expression Of Cancer Stem Cells Research Articles

Adenovirus-mediated expression of MOAP-1, Bax and RASSF1A antagonizes chemo-drug resistance of human breast cancer cells expressing cancer stem cell markers

Cancer is one of the major leading causes of mortality globally and chemo-drug-resistant cancers pose significant challenges to cancer treatment by reducing patient survival rates and increasing treatment costs. Although the mechanisms of chemoresistance vary among different types of cancer, cancer cells are known to share several hallmarks, such as their resistance to apoptosis as well as the ability of cancer stem cells to produce metastatic daughter cells that are resistant to chemotherapy. To address the issue of chemo-drug resistance in cancer cells, a tetracistronic expression construct, Ad-MBR-GFP, encoding adenovirus-mediated expression of MOAP-1, Bax, RASSSF1A, and GFP, was generated to investigate its potential activity in reducing or inhibiting the chemo-drug resistant activity of the human breast cancer cells, MCF-7-CR and MDA-MB-231. When infected by Ad-MBR-GFP, the cancer cells exhibited round cell morphology and nuclei condensation with positive staining for annexin-V. Furthermore, our results showed that both MCF-7-CR and MDA-MB-231 cells stained positively for CD 44 and negatively for CD 24 (CD44+/CD24-) with high levels of endogenous ALDH activity whereas SNU-1581 breast cancer cells were identified as CD 44-/CD 24- cells with relatively low levels of endogenous ALDH activity and high sensitivity toward chemo-drugs, suggesting that both CD 44 and ALDH activity contribute to chemo-drug resistance. Moreover, both MCF-7-CR and MDA-MB-231 cells showed strong chemo-drug sensitivity to cisplatin when the cells were infected by Ad-MBR-GFP, leading to 9-fold and 2-fold reduction in the IC 50 values when compared to cisplatin treatment alone, respectively. The data were further supported by 3D (soft agar) and spheroid cell models of MCF-7-CR and MDA-MB-231 cells which showed a 2-fold reduction of a number of cell colonies and spheroid size when treated with both Ad-MBR-GFP and cisplatin, and compared to control. Other than chemo-sensitivity, Ad-MBR-GFP-infected cancer cells retarded cell migration. Flow cytometry analysis showed that the mechanism of action of Ad-MBR-GFP involved cell cycle arrest at the G1 phase and inhibition of cellular DNA synthesis. Taken together, our investigation showed that Ad-MBR-GFP mediated chemo-drug sensitization in the infected cancer cells involved the activation of apoptosis signaling, cell cycle arrest, and inhibition of DNA synthesis, suggesting that Ad-MBR-GFP is potentially efficacious for the treatment of chemo-drug resistant cancers.

Anti‐Breast Cancer Stem Cell Properties of Silver(I)‐Non‐steroidal Anti‐Inflammatory Drug Complexes

The synthesis and characterisation of a series of silver(I) complexes (1‐7) containing non‐steroidal anti‐inflammatory drugs (NSAIDs) and triphenylphosphine ligands is reported. The diclofenac‐containing silver(I) complex 1 exhibits micromolar potency towards bulk breast cancer cells and breast cancer stem cells (CSCs) cultured in monolayers. Notably, the silver(I) complex 1 displays up to 9.4‐fold higher potency towards three‐dimensional mammospheres than cisplatin (a clinically used anticancer metallodrug) and salinomycin (a breast‐CSC active small molecule). Mechanistic studies show that the silver(I) complex 1 readily interacts with thiol‐containing biomolecules in solution and elevates intracellular reactive oxygen species levels in breast CSCs. Interestingly the silver(I) complex 1 does not perturb cyclooxygenase‐2 (COX‐2) expression in breast CSCs and its cytotoxicity is unaffected in the presence of prostaglandin E2, implicative of a COX‐2 independent mechanism of action.

TRIM58 downregulation maintains stemness via MYH9-GRK3-YAP axis activation in triple-negative breast cancer stem cells.

TRIM58 is a member of the TRIM protein family, which possess with E3 ubiquitin ligase activities. Studies have revealed that low expression of TRIM58 plays key roles, has been implicated in the tumor progression of tumor formation due to its reduced expression. However, its role in regulating the stemness of breast cancer stem cells (CSCs) remains unexplored. Here, we found that TRIM58 was underexpressed in TNBC tissues and cells compared to adjacent mucosa tissue, and its downregulation was significantly associated with shorter survival. Overexpression of TRIM58 reduced the proportion of CD44 + /CD24- cells, upregulated differentiation genes, and inhibited stemness-related gene expression in TNBC CSCs. In vitro and in vivo experiments revealed that TRIM58 overexpression in CSCs suppressed tumor sphere formation and tumorigenic capacity. Co-IP results indicated direct interaction between TRIM58 and MYH9, with TRIM58 inducing MYH9 degradation via ubiquitination in differentiated cells. Label-free quantitative proteomics identified GRK3 and Hippo-YAP as downstream targets and signaling pathways of MYH9. TIMER database analysis, immunohistochemistry, western blotting, DNA-protein pulldown experiments, and dual luciferase reporter assays demonstrated that MYH9 regulated GRK3 transcriptional activation in CSCs. In conclusion, elevated TRIM58 expression in CSCs downregulates MYH9 protein levels by promoting ubiquitin-mediated degradation, thereby inhibiting downstream GRK3 transcription, inactivating the YAP stemness pathway, and ultimately promoting CSC differentiation.

Decorin suppresses stemness and migration potential of malignant peripheral nerve sheath tumor through inhibiting epidermal growth factor receptor signaling

Cancer stem cells (CSCs) play pivotal roles in the growth, invasion, metastasis, chemo-resistance in malignant peripheral nerve sheath tumor (MPNST). The current characterization of CSCs in MPNST is not complete. Decorin is a critical regulator of microenvironment, but its expression and function in CSCs of MPNST has not been studied. In the current study, Decorin levels and its relationship with lung and liver metastasis were determined in clinical specimens. Decorin expression in CD133-positive or CD44-positive CSCs was analyzed by RT-qPCR on cytospun MPNST cells after flow cytometry-based cell sorting. Decorin-positive cells were separated from Decorin-negative cells in transfected MPNST cell lines using a designed plasmid expressing red fluorescent protein (RFP) under a Decorin promoter. Tumor sphere formation, tumor growth, cell invasion, cell migration, and the resistance to chemotherapy-induced apoptosis were determined on Decorin-positive versus Decorin-negative MPNST cells. In vivo tumor growth was analyzed in mice receiving subcutaneous transplantation of Decorin-positive versus Decorin-negative MPNSTs. We found that Decorin levels were significantly downregulated in MPNST specimens, compared to non-tumorous adjacent tissue. Significantly lower Decorin levels were detected in MPNSTs with lung or liver metastasis compared to those without. Poorer patient survival was detected in Decorin-low MPNST, compared to Decorin-high subjects. More Decorin-negative cells were detected in CD133-positive MPNST cells than CD133-negative MPNST cells, and in CD44-positive MPNST cells than in CD44-negative MPNST cells. Compared to Decorin-positive MPNST cells, Decorin-negative MPNST cells generated significantly more tumor spheres in culture, were more invasive and migratory, and were more resistant to chemotherapy-induced apoptosis, likely due to the inhibition of epidermal growth factor receptor signaling by Decorin. Decorin-negative MPNST cells grew significantly larger tumor in vivo. Thus, depletion of Decorin may occur in CSCs in MPNSTs, serving possibly as a new therapeutic target.

Neil 1 deficiency facilitates chemoresistance through upregulation of RAD18 expression in ovarian cancer stem cells

Over the past decades, cancer stem cells (CSCs) have emerged as a critical subset of tumor cells associated with tumor recurrence and resistance to chemotherapy. Understanding the mechanisms underlying CSC-mediated chemoresistance is imperative for improving cancer therapy outcomes. This study delves into the regulatory role of NEIL1, a DNA glycosylase, in chemoresistance in ovarian CSCs.We first observed a decreased expression of NEIL1 in ovarian CSCs, suggesting its potential involvement in CSC regulation. Using pan-cancer analysis, we confirmed the diminished NEIL1 expression in ovarian tumors compared to normal tissues. Furthermore, NEIL1 downregulation correlated with an increase in stemness markers and enrichment of CSCs, highlighting its role in modulating CSC phenotype.Further mechanistic investigation revealed an inverse correlation between NEIL1 and RAD18 expression in ovarian CSCs. NEIL1 depletion led to heightened RAD18 expression, promoting chemoresistance possibly via enhancing Translesion DNA Synthesis (TLS)-mediated DNA lesion bypass. Moreover, dowregulation of NEIL1 results in reduced DNA damage accumulation and suppressed apoptosis in ovarian cancer.Overall, our findings unveil a novel mechanism involving NEIL1 and RAD18 in regulating chemoresistance in ovarian CSCs. Targeting this NEIL1-RAD18 axis may offer promising therapeutic strategies for combating chemoresistance and improving ovarian cancer treatment outcomes.

Long noncoding RNA DLEU2 and ROR1 pathway induces epithelial-to-mesenchymal transition and cancer stem cells in breast cancer

Breast cancer (BC) patient who receives chemotherapy for an extended length of time may experience profound repercussions in terms of metastases and clinical outcomes due to the involvement of the epithelial-to-mesenchymal transition (EMT) mechanism and enriched cancer stem cells (CSCs). BC cells that express high levels of lncRNA deleted in lymphocytic leukemia-2 (lncRNA DLEU2) and type I tyrosine kinase-like orphan receptor ROR1 (ROR1) may play roles in the enhanced ability of the activation EMT and CSC induction. Here we find that lncRNA DLEU2 and ROR1 are specifically upregulated in tumor tissues compared to their normal counterparts in TCGA, PubMed GEO datasets, and samples from archived breast cancer tumor tissues. Following chemotherapy, lncRNA DLEU2 and ROR1 were enhanced in BC tumor cells, coupled with the expression of CSCs, EMT-related genes, and BMI1. Mechanistically, ROR1 and lncRNA DLEU2 overexpression led to enhanced tumor cell proliferation, inhibition of apoptosis, cell-cycle dysregulation, chemoresistance, as well as BC cell’s abilities to invade, migrate, develop spheroids. These findings imply that the role of lncRNA DLEU2 and ROR1 in BC therapeutic failure is largely attributed to EMT, which is intricately linked to enriched CSCs. In conclusion, our findings indicate that a lncRNA DLEU2 and ROR1-based regulatory loop governs EMT and CSC self-renewal, implying that targeting this regulatory pathway may improve patients’ responses to chemotherapy and survival.

RNF183 Promotes Colon Cancer Cell Stemness through Fatty Acid Oxidation

Colon cancer (COAD) is a prevalent gastrointestinal tumor, composed of a few cancer stem cells (CSCs). High expression of RNF183 drives colorectal cancer metastasis, but its role in COAD cell stemness is still unclear. Bioinformatics analyzed expression and enriched pathway of RNF183 in COAD tissue. IHC analyzed RNF183 protein expression in tumor tissue. CD133 + CD44+ CSCs were sorted by flow cytometry, and RNF183 expression in COAD cells or CSCs was detected by qPCR, western blot and immunofluorescence. CCK-8 assay assessed cell viability, and sphere formation assay tested cell sphere-forming ability. Western blot measured protein expression of stem cell markers. qPCR assayed expression of fatty acid oxidation genes. The ability of fatty acid oxidation was analyzed by detecting fatty acid metabolism. RNF183 was highly expressed in COAD and CD133 + CD44+ CSCs, and was enriched in fatty acid metabolism pathway. RNF183 expression was positively correlated with enzymes involved in fatty acid oxidation. RNF183 could promote COAD stemness and fatty acid oxidation. Rescue experiments showed that Orlistat (a fatty acid oxidation inhibitor) reversed stimulative impact of RNF183 overexpression on COAD stemness. RNF183 promoted COAD stemness by affecting fatty acid oxidation, which may be a new therapeutic target for inhibiting COAD development.

Self-Renewal Inhibition in Breast Cancer Stem Cells: Moonlight Role of PEDF in Breast Cancer.

Breast cancer is the leading cause of death among females in developed countries. Although the implementation of screening tests and the development of new therapies have increased the probability of remission, relapse rates remain high. Numerous studies have indicated the connection between cancer-initiating cells and slow cellular cycle cells, identified by their capacity to retain long labeling (LT+). In this study, we perform new assays showing how stem cell self-renewal modulating proteins, such as PEDF, can modify the properties, percentage of biomarker-expressing cells, and carcinogenicity of cancer stem cells. The PEDF signaling pathway could be a useful tool for controlling cancer stem cells' self-renewal and therefore control patient relapse, as PEDF enhances resistance in breast cancer patient cells' in vitro culture. We have designed a peptide consisting of the C-terminal part of this protein, which acts by blocking endogenous PEDF in cell culture assays. We demonstrate that it is possible to interfere with the self-renewal capacity of cancer stem cells, induce anoikis in vivo, and reduce resistance against docetaxel treatment in cancer patient cells in in vitro culture. We have also demonstrated that this modified PEDF protein produces a significant decrease in the percentage of expressed cancer stem cell markers.

R-spondin-3 promotes proliferation and invasion of breast cancer cells independently of Wnt signaling

We recently identified R-spondin-3 (RSPO3) as a novel driver of breast cancer associating with reduced patient survival, expanding its clinical value as potential therapeutic target that had been recognized mostly for colorectal cancer so far. (Pre)clinical studies exploring RSPO3 targeting in colorectal cancer approach this indirectly with Wnt inhibitors, or directly with anti-RSPO3 antibodies. Here, we address the clinical relevance of RSPO3 in breast cancer and provide insight in the oncogenic activities of RSPO3. Utilizing the RSPO3 breast cancer mouse model, we show that RSPO3 drives the aberrant expansion of luminal progenitor cells expressing cancer stem cell marker CD61, inducing proliferative, poorly differentiated and invasive tumors. Complementary studies with tumor organoids and human breast cancer cell lines demonstrate that RSPO3 consistently promotes the proliferation and invasion of breast cancer cells. Importantly, RSPO3 exerts these oncogenic effects independently of Wnt signaling, rejecting the therapeutic value of Wnt inhibitors in RSPO3-driven breast cancer. Instead, direct RSPO3 targeting effectively inhibited RSPO3-driven growth of breast cancer cells. Conclusively, our data indicate that RSPO3 exerts unfavorable oncogenic effects in breast cancer, enhancing proliferation and malignancy in a Wnt-independent fashion, proposing RSPO3 itself as a valuable therapeutic target in breast cancer.

Flavonostilbenes natural hybrids from Rhamnoneuron balansae as potential antitumors targeting ALDH1A1: molecular docking, ADMET, MM-GBSA calculations and molecular dynamics studies

Several studies have linked Cancer stem cells (CSCs) to cancer resistance development to chemotherapy and radiotherapy. ALDH1A1 is a key enzyme that regulates the gene expression of CSCs and creates an immunosuppressive tumor microenvironment. It was reported that quercetin and resveratrol were among the inhibitors of ALDH1A1. In early 2022, it was reported that new 11 flavonostilbenes (rhamnoneuronal D–N) were isolated from Rhamnoneuron balansae as potential antiaging natural products. Rhamnoneuronal H (5) could be envisioned as a natural hybrid of quercetin and resveratrol. It was therefore hypothesized that 5 and its analogous isolates rhamnoneuronal D–G (1–4) and rhamnoneuronal I–N (6–11) would have potential ALDH1A1 inhibitory activity. To this end, all isolates were subjected to molecular docking, MM-GBSA, ADMET, and molecular dynamics simulations studies to assess their potential as new leads for cancer treatment targeting ALDH1A1. In silico findings revealed that natural hybrid 5 has a similar binding affinity, judged by MM-GBSA, to the ALDH1A1 active site when compared to the co-crystalized ligand (−64.71 kcal/mole and −64.12 kcal/mole, respectively). Despite having lesser affinity than that of the co-crystalized ligand, the rest of the flavonostilbenes, except 2–4, displayed better binding affinities (−37.55 kcal/mole to −58.6 kcal/mole) in comparison to either resveratrol (−34.44 kcal/mole) or quercetin (−36.48 kcal/mole). Molecular dynamic simulations showed that the natural hybrids 1, 5–11 are of satisfactory stability up to 100 ns. ADMET outcomes indicate that these hybrids displayed acceptable properties and hence could represent an ideal starting point for the development of potent ALDH1A1 inhibitors for cancer treatment. Communicated by Ramaswamy H. Sarma

Regulation of self-renewal in ovarian cancer stem cells by fructose via chaperone-mediated autophagy

The chaperone-mediated autophagy (CMA) pathway is deregulated in different types of cancers; however, its role in cancer stem cells (CSCs) is unknown yet. Development of ovarian cancer, the most lethal gynecological type of cancer, involves the metastasis of CSCs to the abdominal cavity. This study aims to determine the role of CMA in ovarian CSCs. We found that the transcription factor EB (TFEB) and trehalose, a disaccharide that induces TFEB activation, enhance the expression of octamer-binding transcription factor 4 (OCT4) stem cell and lysosomal-associated membrane protein 2A (LAMP2A) CMA markers. However, trehalose did not increase the level of the LC3II macroautophagy marker in ovarian CSCs. In A2780 and SKOV3 ovarian CSCs, LAMP2A and heat shock protein 70 (HSC70) exhibited higher expression levels than in normal adherent cells. Our results showed that the silencing of the LAMP2A gene resulted in reduced sphere formation and enhanced GLUT5 expression in ovarian CSCs. Moreover, the treatment with fructose reduced sphere formation and enhanced the expression levels of LAMP2A, SOX2, and OCT4 in ovarian CSCs. The KEGG functional analysis revealed that differentially expressed genes were enriched in the ferroptosis pathway in A2780-spheroid (SP) cells after treatment with fructose. In A2780-SP and SKOV3-SP cells, the level of SLC7A11 decreased whereas FTH increased after treatment with fructose. Taken together, our results suggest that CMA is mediated in CSCs via fructose metabolism.

Abstract 5809: Tracking cancer stem cell expression of pancreatic and ampullary cancer organoids in response to chemotherapy

Abstract Background: Cancer stem cells (CSCs) have been described to confer resistance and aggressive disease biology in pancreatic ductal adenocarcinoma (PDAC) and ampullary carcinoma (AMPC). Patient derived cancer organoids (PCOs) are an important model of PDAC that maintain the molecular features of advanced disease and have been shown to predict drug response. This study aimed to determine the dynamics of CSC expression on the oligoclonal culture growth of three independent PCOs models. Methods: PCOs were assayed using low volume 10μL microformat in 96 well design (Ibidi, Inc). PCOs were treated with physiologic FOLFIRINOX or gemcitabine + nab-paclitaxel (nab-pac) including single agent controls. Response was assayed by growth using timecourse data from 0h to 72h using high content imaging (Cytation 5). At 72h, the PCOs were stained using four distinct CSC markers (CD44, CD133, CD117, and EpCAM). Images were aligned using thresholded root mean standard deviation of 75μm and tracked for normalized change in longest diameter. At 72h, CSC markers were tracked using % area positive from brightfield region of interest. Response was tracked by effect size Glass’s Delta (GΔ) defined as the difference between the mean of media control and treatment group normalized to standard deviation of media control. Results: PCOs maintained nuclear atypia and high nuclear-chromatin ratios consistent with matured xenografts. AMPC1 showed similar sensitivity with growth arrest by PCO Δ diameter for gem + nab-pac (mean = +2.2%, GΔ = 1.29) and FOLFIRINOX (mean = 0.0%, GΔ = 1.33). AMPC1 had CSC enrichment for CD44 after treatment with nab-pac (GΔ = 0.93), CD133 expression after treatment with gem + nab-pac (GΔ = 0.75), CD117 after treatment with nab-pac treatment (GΔ = 0.80), and EpCAM after treatment with gem + nab-pac (GΔ = 0.78). In AMPC1, interquartile analysis revealed increased CSC expression conferred reduced growth for CD133 (+72% v. 37%, p<0.005) and CD117 (+81% v. +25%, p<0.005). Normalized Δ diameter across PDAC PCOs revealed PDAC1 had reduced sensitivity to gem + nab-pac (mean = +16.0%, GΔ = 1.2) vs. PDAC2 (Mean = 2.1%, GΔ = 1.65). PDAC1 had CSC enrichment with increased % area of CD44+ after treatment with gem + nab-pac (GΔ = 1.13). In PDAC2, interquartile analysis revealed increased CSC expression conferred decreased growth rate for CD133 (+65% v. +36%, p< 0.005) and CD117 (+75% v. +38%, p<0.005). Conclusions: PDAC and AMPC PCOs reveal oligoclonal expression of CSC markers within a population. Increased CSC expression for CD133 and CD117 within a population consistently revealed decreased growth within an organoid population. CSC expression (CD44, CD133, CD117, and EpCAM) was found to be enriched under therapeutic challenge with gem and nab-pac containing chemotherapy. This work supports the oligoclonal and dynamic nature of CSCs for pancreaticobiliary cancers in response to therapeutic challenge. Citation Format: Eleanor Riedl, Austin Stram, MD Shahadat Hossan, Ethan S. Lin, Luke Koeppel, Jamie Warner, Sam Lubner, Stephanie McGregor, Wei Zhang, Jeremy Kratz. Tracking cancer stem cell expression of pancreatic and ampullary cancer organoids in response to chemotherapy. [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 5809.

Abstract 2443: Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the common malignancies worldwide. Poor prognosis of HCC patients is attributed to high frequency of tumor relapse and therapeutic resistance. Increasing evidence showed the existence of cancer stem cells (CSCs) which is crucial for tumor initiation and therapeutic resistance. However, there are limited kinase inhibitors available that show specific targeting against liver T-ICs for HCC treatment. To understand the mechanism for regulation of liver CSCs, we have employed RNA-Seq profiling to compare the gene expression profiles with focuses on protein kinases between (1) sorted CD133+ liver CSCs and CD133- differentiated counterparts isolated from two HCC cell lines (Huh7 and PLC8024) and cultured hepatospheres and adherent differentiated cells of HCC cell line PLC/PRF/5. This analysis revealed Serine/Threonine kinase 39 (STK39), also known as SPAK, to be the commonly up-regulated protein kinase common in both CD133+ liver CSCs and CSC enriched hepatospheres. Consistently, based on the TCGA data analysis, STK39 mRNA expression is positively correlated with the expression of liver CSC markers including CD24, CD133, and CD47. Using publicly available transcriptome datasets deposited in the NCBI Gene Expression Omnibus (GEO) including GSE14520 and GSE25097, we found that overexpression of STK39 was also observed in HCC tumor tissues (p<0.001) and correlated with poorer overall survival and disease-free survival. By lentiviral based overexpression and knockdown approaches, we demonstrated the role of STK39 in regulation of liver CSC properties, including self-renewal, tumorigenicity, cell invasiveness and expression of liver CSC markers. Specially, STK39 expression was highly elevated in sorafenib- and lenvatinib-resistant HCC cells, and its inhibition led to sensitization of HCC cells towards these treatments. In order to identify downstream target of STK39 for regulation of cancer stemness, tandem affinity purification coupled with mass spectrometry was employed. Upon analysis, PARP1, was identified to be the novel protein binding partner of STK39. Upon suppression of STK39 in HCC cells, phosphorylation of PARP1 at Threonine 368 (T368) was found to be highly inhibited, suggesting its role as downstream effector of STK39-mediated liver CSC functions. Furthermore, STK39 regulates PARP1-mediated chromatin decondensation, and thus opens chromatin structure at the promoter regions of SOX2/OCT4. In summary, STK39 regulates cancer stemness and drug resistance by directly modulating PARP1-mediated SOX2/OCT4 expression via chromatin remodelling. Targeting STK39/PARP1 signaling cascade alone or in combination with molecular targeted drugs including rucaparib and lenvatnib can be the potential therapeutic strategy for treatment of this deadly disease. Citation Format: Carmen Oi Ning Leung, Terence Kin-Wah Lee. Serine/threonine kinase 39 (STK39) regulates cancer stemness through regulating PARP-mediated SOX2/OCT4 expression in hepatocellular carcinoma [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 2443.

Abstract PD5-09: PD5-09 Oncogenic Pathways of Tumor Cells Signal to Stem-ness in TNBC: Relationship between Stem Cell Markers and PI3K-DDR Pathway Targeted Drugs

Abstract Background: The interest in cancer stem cells in TNBC is encouraged by the finding that CD44+/CD24- and ALDH1+ breast cancer stem cells (CSC) are enriched in TNBC and contribute to chemotherapy resistance and aggressive tumor progression (O’Conor et al., 2018; Ma et al. 2014). CD44+ CD24- phenotype can be utilized to determine TNBC patients’ prognosis (Shadbad et al., 2021; Wang et al., 2017). CD44 has also been known for its tumor-intrinsic function in TNBC (Kong et al., Cancer Res. 2020). We have reported a functional correlation between CD44+/CD24- cancer stem cells and 3D clonogenic growth in testing rational combinations of PI3K pathway inhibitors and Wnt-beta-catenin pathway inhibitors in TNBC (Aske et al., 2016; 2017). We also reported that doubling down on the PI3K pathway enhances the anti-tumor efficacy of PARP inhibitor in TNBC (De et al., 2014). Hypothesis: We hypothesized that the anti-tumor effect of PI3K pathway targeted drugs in combination with PARP inhibitor will regulate the expression of CSC in TNBC. Methods: To test our hypothesis, we examined the expression of stem cell markers CD44, CD44v6, and CD24 in MDA-MB-231, SUM149, and MDA-MB-468 TNBC cells in vitro and in vivo. IHC for CD44 and CD44v6 was standardized using tumor controls and identified in (1) BC TMAs, (2) TNBC cell lines, and (3) patients from our Avera cohort. FFPE sections of tumors from athymic mice (bearing xenograft tumors) treated with a combination of Apitolisib with Veliparib plus carboplatin were used to test stem cell markers by IHC. Staining was evaluated independently by a pathologist who was unaware of the study design. Mechanism-based in vitro studies were conducted to understand the mode of action of the drugs using flow cytometry and qRT-PCR. Results: CD44 was identified primarily in the membrane and to a lesser extent in the cytosol of tumor cells of BC patients (TMA and Avera cohort), TNBC cell lines, and xenograft-tumors. Apitolisib, in combination with Veliparib plus carboplatin, blocked the growth of established xenograft tumors by 80-90%, with a concomitant decrease in Ki67 levels. Membrane expression of CD44 inversely correlated to tumor sizes which significantly reduced in response to drug combinations. The relationship between the expression of CD44 and CD44v6 is being worked out, which will be presented at the meeting. Significance: Our findings showed that the anti-tumor effect of Veliparib plus carboplatin and Apitolisib effectively blocked the specific CD44v6 CSC population in TNBC xenograft tumors. Our results demonstrate the integral role of the PI3K-mTOR and the DDR pathways in the control of stem-ness orchestrating anti-tumor actions of PARP inhibitor and mTOR-PI3K dual inhibitors in TNBC. Citation Format: Nandini Dey, Jennifer Aske, Xiaoqian Lin, Adam Dale, Yuliang Sun, Pradip De. PD5-09 Oncogenic Pathways of Tumor Cells Signal to Stem-ness in TNBC: Relationship between Stem Cell Markers and PI3K-DDR Pathway Targeted Drugs [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-09.

Epithelial-mesenchymal transition and stemness of breast cancer cells: Effect of viscoelasticity of the substrate to mimics microenvironment

Metastasis is one of the greatest challenges in cancer treatment today. Normal mammary epithelial cells are optimally supported by interaction with a soft matrix (microenvironment) with elastic modulus of about 800 Pa. However, after transformation, breast tissue becomes progressively stiffer and tumour cells become significantly more contractile and hyper-responsive to matrix elasticity. In addition, importantly, the cancer cells penetrate into blood vessel and enter the circulation during metastasis. The modulus of fluid such as blood or mucus has very low stiffness of around 50 Pa. For this reason, the critical association between cancer cell phenotype and the change of matrix rigidity with an order of magnitude smaller should be emphasizing. This review highlights the current understanding of epithelial-mesenchymal transition and cancer stem cells in metastasis, and identified importance for investigation on artificial extracellular matrix with different viscoelastic properties, which is required to mimics in vivo microenvironment. The substrate damping coefficient (tand) as potential physical parameter emerged the important linkage to cellular motility, cancer stemness, and epithelial-mesenchymal transition induction. Although further investigation is required to clarify the efficacy of environmental stimuli (tand) for tumors exhibiting stem cell-like properties, this review indicates that the cancer cells incubated on softer substrate might lead to express cancer stem cell biomarkers exhibiting high expression.

279 (PB-103) Poster - Mammographic breast density correlated with worse prognosis of early breast cancer regardless of menopausal status

Background: Breast density (BD) is known to affect breast cancer risk, but it may be associated with breast cancer survival. The underlined mechanism remains unclear. Aldehyde dehydrogenase 1 (ALDH-1) and CD44+CD24− are the most consistently used biomarkers to identify and characterize breast cancer stem cells (CSCs). However, correlation between expression of CSCs and BD remains unclear. The interpretation of results from previous studies on mammographic BD and survival is complicated by the association between confounding factors and survival.

CD44+/CD24- Expression as predictors of ovarian cancer chemoresistance: immunohistochemistry and flow cytometry study

BackgroundThe conventional standard treatment for ovarian cancer is not very effective, and the disease is fatal for women. Cancer Stem Cells (CSCs) that express CD44+/CD24- can contribute to chemoresistance and a poor prognosis. We seek to investigate the expression of CSCs (CD44+/CD24-) in ovarian cancer and their predictive significance.MethodsThe ambispective cohort was performed on 64 patients (32 patients in each group) at four hospitals (Cipto Mangunkusumo, Tarakan, Fatmawati, and Dharmais Hospital). Debulking surgery was performed on the patients, followed by histopathological analysis. The patients had six rounds of chemotherapy and were under monitoring for six months. The therapeutic responses were evaluated using the RECIST criteria (Response Criteria in Solid Tumors) and categorized as chemoresistant or chemosensitive. Using immunohistochemistry, we directly assess the CSCs from ovarian cancer tissue and using flow cytometry to assess the CSCs from the blood.ResultsHigh CSCs expression and ovarian cancer chemoresistance were significantly related in both trials (p 0.05). A better outcome was obtained using CD44+/CD24- immunohistochemistry.ConclusionsWe conclude that there is a substantial association between high CSCs expression and chemoresistance in ovarian cancer and that CSCs immunohistochemistry has a higher predictive value.

A comparative study of metastatic potentials of three different cancer stem cell models

Metastasis is one of the major causes of cancer deaths. However, the mechanisms of cancer cells acquiring aggressiveness and metastatic potential are still under investigation. Although cancer stem cells (CSCs) have been suggested as tumor initiating cells responsible for cancer metastasis, no sufficiently practical models have been found because of less availability of CSCs. We have developed novel CSC models derived from mouse induced pluripotent stem cells (miPSCs) in the presence of conditioned media (CM) from different cancer cell lines. Here, we tried to establish the models of metastasis using three different CSC models, miPS-LLCev, miPS-BT549cm and miPS-Huh7cm cells. The metastatic abilities of these CSC models were evaluated by intraperitoneal injections into mice. The developed metastases were histologically analyzed and the differences in gene expression between parental and metastasized cells were assessed. The expression of CSC and stemness markers was maintained in the cells isolated from metastasis. The three types of CSCs were further analyzed by RNA-sequencing to identify the enriched cytoplasmic signaling pathways. As a result, the three CSC models exhibited different patterns of metastases while the metastasis of miPS-LLCev cells appeared the most aggressive demonstrating hepatocellular carcinoma, which developed from the inside of the liver, as well as pulmonary metastasis. Metastasis related “HIF-1 pathway” was nominated as the candidate of enriched pathway in miPS-LLCev and miPS-Huh7cm cells implying that these CSC models possessed distinguished metastatic potential. Therefore, we concluded that the CSC models developed in this study would provide good models of metastasis linked with the aggressiveness.

Inflammatory Breast Cancer: The Cytokinome of Post-Mastectomy Wound Fluid Augments Proliferation, Invasion, and Stem Cell Markers.

Inflammatory breast cancer (IBC) is an aggressive phenotype with a high recurrence and low survival rate. Approximately 90% of local breast cancer recurrences occur adjacent to the same quadrant as the initial cancer, implying that tumor recurrence may be caused by residual cancer cells and/or quiescent cancer stem cells (CSCs) in the tumor. We hypothesized that wound fluid (WF) collected after modified radical mastectomy (MRM) may activate cancer cells and CSCs, promoting epithelial mesenchymal transition (EMT) and invasion. Therefore, we characterized the cytokinome of WF drained from post-MRM cavities of non-IBC and IBC patients. The WF of IBC patients showed a significantly higher expression of various cytokines than in non-IBC patients. In vitro cell culture models of non-IBC and IBC cell lines were grown in media conditioned with and/without WF for 48 h. Afterwards, we assessed cell viability, the expression of CSCs and EMT-specific genes, and tumor invasion. Genes associated with CSCs properties and EMT markers were regulated in cells seeded in media conditioned by WF. IBC-WF exhibited a greater potential for inducing IBC cell invasion than non-IBC cells. The present study demonstrates the role of the post-surgical tumor cavity in IBC recurrence and metastasis.

Abstract 3200: Examining the role of obesity and leptin signaling in triple negative breast cancer

Abstract Individuals with a high body mass index (BMI) have an increased risk of developing many adult cancers, including breast cancer. In triple negative breast cancer (TNBC), a clinically aggressive subtype of breast cancer, increased BMI is associated with more aggressive tumor types and a higher risk of recurrence. The differences in outcomes between obese and non-obese breast cancer patients is a consequence of the complex interplay between social, environmental, and physiological factors that contribute to the etiology of breast cancer. Therefore, understanding the complex signaling events in the obese tumor microenvironment is essential. Adipose stem cells (ASCs) are a component of the breast microenvironment, and are a subset of mesenchymal stem cells. ASCs from obese patients (obASCs) secrete higher levels various cytokines and adipokines that induce a more invasive phenotype in triple negative breast cancer cells compared to ASCs from lean individuals. Leptin, an adipokine that is expressed proportionally to fat mass, has been implicated in many cancers. Increased leptin and leptin receptor expression is associated with worse prognosis. This study seeks to examine the role of leptin signaling in triple negative breast cancer. Previous work in conjunction with a collaborating lab has shown that leptin signaling promotes metastasis and increased expression of epithelial-mesenchymal transition (EMT) markers in triple negative breast cancer. This project expands upon this work through using both patient-derived cell lines and and patient-derived xenografts (PDX), and examines the role of leptin signaling both in vitro and in vivo. To determine the effects of obesity upon a PDX model, a high fat diet was used to induce obesity in vivo. Exposure to conditioned media harvested from obASCs increased the percentage of TNBC cells that expressed cancer stem cell markers, whereas exposure to a small peptide antagonist of the leptin receptor decreased the percentage of cells with CSC markers. PDX tumors implanted into diet-induced obesity mice had an increased volume compared to tumors implanted into lean controls. Conclusions: These molecular differences may contribute to the differences in cancer outcomes between obese and lean individuals with breast cancer, and further study of the crosstalk between obASCs and TNBC is critical. Citation Format: Courtney Brock, Maryl Wright, Khoa Nguyen, Katherine Hebert, Madlin Alzoubi, Thomas Cheng, Bridgette Collins-Burow, Matthew Burow. Examining the role of obesity and leptin signaling in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3200.