Reduces Cancer Stem Cell Research Articles

A pH-triggered N-oxide polyzwitterionic nano-drug loaded system for the anti-tumor immunity activation research

Triple negative breast cancer (TNBC) has the characteristics of low immune cell infiltration, high expression of tumor programmed death ligand 1 (PD-L1), and abundant cancer stem cells. Systemic toxicity of traditional chemotherapy drugs due to poor drug selectivity, and chemotherapy failure due to tumor drug resistance and other problems, so it is particularly important to find new cancer treatment strategies for TNBC with limited treatment options. Both the anti-tumor natural drugs curcumin and ginsenoside Rg3 can exert anti-tumor effects by inducing immunogenic cell death (ICD) of tumor cells, reducing PD-L1 expression, and reducing cancer stem cells. However, they have the disadvantages of poor water solubility, low bioavailability, and weak anti-tumor effect of single agents. We used vinyl ether bonds to link curcumin (Cur) with N-O type zwitterionic polymers and at the same time encapsulated ginsenoside Rg3 to obtain hyperbranched zwitterionic drug-loaded micelles OPDEA-PGED-5HA@Cur@Rg3 (PPH@CR) with pH response. In vitro cell experiments and in vivo animal experiments have proved that PPH@CR could not only promote the maturation of dendritic cells (DCs) and increase the CD4+ T cells and CD8+ T cells by inducing ICD in tumor cells but also reduce the expression of PD-L1 in tumor tissues, and reduce cancer stem cells and showed better anti-tumor effects and good biological safety compared with free double drugs, which is a promising cancer treatment strategy.

SMARCC2 silencing suppresses oncogenic activation through modulation of chromatin accessibility in breast cancer

SWI/SNF chromatin remodeling complexes play a key role in gene transcription as epigenetic regulators and are typically considered to act as tumor suppressors in cancers. Compared to other cancer-related components of the SWI/SNF complex, research on SMARCC2, a component of the initial BAF core, has been relatively limited. This study aimed to elucidate the role of SMARCC2 in breast cancer by employing various in vitro and in vivo methods including cell proliferation assays, mammosphere formation, and xenograft models, complemented by RNA-seq, ATAC-seq, and ChIP analyses. The results showed that SMARCC2 silencing surprisingly led to the suppression of breast tumorigenesis, indicating a pro-tumorigenic function for SMARCC2 in breast cancer, which contrasts with the roles of other SWI/SNF subunits. In addition, SMARCC2 depletion reduces cancer stem cell features of breast cancer cells. Mechanistic study showed that SMARCC2 silencing downregulated the oncogenic Ras-PI3K signaling pathway, likely by directly regulating the chromatin accessibility of the enhancers of the key genes such as PIK3CB. Together, these results expand our understanding of the SWI/SNF complex's role in cancer development and identify SMARCC2 as a promising new target for breast cancer therapies.

Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Small molecule inhibition of TFF3 overcomes tamoxifen resistance and enhances taxane efficacy in ER+ mammary carcinoma

Even though tamoxifen has significantly improved the survival of estrogen receptor positive (ER+) mammary carcinoma (MC) patients, the development of drug resistance with consequent disease recurrence has limited its therapeutic efficacy. Trefoil factor-3 (TFF3) has been previously reported to mediate anti-estrogen resistance in ER+MC. Herein, the efficacy of a small molecule inhibitor of TFF3 (AMPC) in enhancing sensitivity and mitigating acquired resistance to tamoxifen in ER+MC cells was investigated. AMPC induced apoptosis of tamoxifen-sensitive and resistant ER+MC cells and significantly reduced cell survival in 2D and 3D culture in vitro. In addition, AMPC reduced cancer stem cell (CSC)-like behavior in ER+MC cells in a BCL2-dependent manner. Synergistic effects of AMPC and tamoxifen were demonstrated in ER+MC cells and AMPC was observed to improve tamoxifen efficacy in tamoxifen-sensitive cells and to re-sensitize cells to tamoxifen in tamoxifen-resistant ER+MC in vitro and in vivo. Additionally, tamoxifen-resistant ER+MC cells were concomitantly resistant to anthracycline, platinum and fluoropyrimidine drugs, but not to Taxanes. Taxane treatment of tamoxifen-sensitive and resistant ER+MC cells increased TFF3 expression indicating a combination vulnerability for tamoxifen-resistant ER+MC cells. Taxanes increased CSC-like behavior of tamoxifen-sensitive and resistant ER+MC cells which was reduced by AMPC treatment. Taxanes synergized with AMPC to promote apoptosis and reduce CSC-like behavior in vitro and in vivo. Hence, AMPC restored the sensitivity of tamoxifen and enhanced the efficacy of Taxanes in tamoxifen-resistant ER+MC. In conclusion, pharmacological inhibition of TFF3 may serve as an effective combinatorial therapeutic strategy for the treatment of tamoxifen-resistant ER+MC.

Modulation of Viability, Proliferation, and Stemness by Rosmarinic Acid in Medulloblastoma Cells: Involvement of HDACs and EGFR.

Medulloblastoma (MB) is a heterogeneous group of malignant pediatric brain tumors, divided into molecular groups with distinct biological features and prognoses. Currently available therapy often results in poor long-term quality of life for patients, which will be afflicted by neurological, neuropsychiatric, and emotional sequelae. Identifying novel therapeutic agents capable of targeting the tumors without jeopardizing patients' quality of life is imperative. Rosmarinic acid (RA) is a plant-derived compound whose action against a series of diseases including cancer has been investigated, with no side effects reported so far. Previous studies have not examined whether RA has effects in MB. Here, we show RA is cytotoxic against human Daoy (IC50 = 168μM) and D283 (IC50 = 334μM) MB cells. Exposure to RA for 48h reduced histone deacetylase 1 (HDAC1) expression while increasing H3K9 hyperacetylation, reduced epidermal growth factor (EGFR) expression, and inhibited EGFR downstream targets extracellular-regulated kinase (ERK)1/2 and AKT in Daoy cells. These modifications were accompanied by increased expression of CDKN1A/p21, reduced expression of SOX2, and a decrease in proliferative rate. Treatment with RA also reduced cancer stem cell markers expression and neurosphere size. Taken together, our findings indicate that RA can reduce cell proliferation and stemness and induce cell cycle arrest in MB cells. Mechanisms mediating these effects may include targeting HDAC1, EGFR, and ERK signaling, and promoting p21 expression, possibly through an increase in H3K9ac and AKT deactivation. RA should be further investigated as a potential anticancer agent in experimental MB.

Reduction of Cancer Stem Cells and Invasiveness of Human Melanoma and Breast Cancer by Cucurbitacin B from Lagenaria siceraria

Cucurbitacins are secondary metabolites that are commonly found in the Cucurbitacae family. Many biological properties have been reported for cucurbitacins, including anti-inflammatory, antioxidant, antiviral, anti-malaria, and anticancer properties. While studies for the anticancer property of cucurbitacins focus mostly on the cell-cycle progression and apoptosis, no study has considered the effect of cucurbitacin on other cancer behaviors. Here, we report cell-proliferation-based drug testing on random herbal extracts leading to the identification of cucurbitacin B as an anticancer compound. Interestingly, cucurbitacin B had no effect on the proliferation of rat embryonic myoblast cells. We also found that cucurbitacin B significantly reduced the invasiveness of at least two highly metastatic breast cancer and melanoma cells. Using known cancer stem-cell markers, we observed a significant reduction of the melanoma stem cells. Molecularly, cucurbitacin B caused reduction of the metastasis-promoting gene Snail in melanoma and one of the cancer stem cell markers, ALDH1A1 (aldehyde dehydrogenase 1 A1), in breast cancer. Finally, we report the potential toxicity of cucurbitacin B in developing skin tissue and the olfactory organ using zebrafish embryo. In summary, our study suggests the potential use of cucurbitacin B for cancer metastasis and relapse treatment.

Inhalant cannabidiol impedes tumor growth through decreased tumor stemness and impaired angiogenic switch in NCI-H1437-induced human lung cancer model.

Lung cancer remains the most chronic form of cancer and the leading cause of cancer mortality in the world. Despite significant improvements in the treatment of lung cancer, the current therapeutic interventions are only partially effective, necessitating the continued search for better, novel alternative treatments. Angiogenesis and cancer stem cells play a central role in the initiation and propagation of cancers. Tumor angiogenesis is triggered by an angiogenic switch when pro-angiogenic factors exceed anti-angiogenic components. Although many anti-angiogenic agents are used in cancer treatment, there are therapeutic limitations with significant side effects. In recent years, cannabinoids have been investigated extensively for their potential anti-neoplastic effects. Our previous findings showed that cannabidiol (CBD) could impede tumor growth in mouse models of melanoma and glioblastoma. Importantly, CBD has been suggested to possess anti-angiogenic activity. In this study, we tested, for the first time, inhalant CBD in the treatment of heterotopic lung cancer and whether such potential effects could reduce cancer stem cell numbers and inhibit tumor angiogenesis. We implanted NCI H1437 human lung cancer cells in nude mice and treated the mice with inhalant CBD or placebo. The outcomes were measured by tumor size and imaging, as well as by immunohistochemistry and flow cytometric analysis for CD44, VEGF, and P-selectin. Our findings showed that CBD decreased tumor growth rate and suppressed expression of CD44 and the angiogenic factors VEGF and P-selectin. These results suggest, for the first time, that inhalant CBD can impede lung cancer growth by suppressing CD44 and angiogenesis.

A phase1b study of navicixizumab plus FOLFIRI in second-line treatment of patients with metastatic colorectal cancer.

111 Background: Navicixizumab is an anti-delta-like ligand 4 (DLL4)/vascular endothelial growth factor bispecific antibody which has shown promising preclinical and clinical activity in multiple tumor types. The blockade of DLL4, a transmembrane protein acting as a ligand for Notch receptors 1 and 4, causes tumor differentiation and reduction in cancer stem cells, as well as repolarization of the tumor microenvironment. Methods: We conducted a phase 1b single-arm dose escalation study combining navicixizumab with FOLFIRI or FOLFOX as second line treatment for patients with metastatic colorectal cancer (CRC). Patients were dosed either at 3 or 4 mg/kg intravenously once every 2 weeks (Q2W). The primary endpoint was the maximum tolerated dose of navicixizumab in combination with FOLFOX or FOLFIRI. Results: Between December 2016 and August 2018, 15 patients received navicixizumab plus FOLFIRI. Median patient age was 55 years, and nine of 15 patients were female (60%). Three patients were enrolled in the 3 mg/kg dose-escalation cohort, 2 patients were enrolled in the 4-mg/kg dose-escalation cohort, and 10 patients were enrolled in the 3 mg/kg dose expansion cohort. No DLTs were observed in this study. The median duration of navicixizumab therapy was 127 days. Hypertension was the most frequent all-grade treatment-related adverse event reported (12 of 15 patients, 80%), followed by fatigue (8 of 15 patients, 53.3%). Grade 3 hypertension was reported in 3 patients (20%). Eight of the 15 patients (53.3%) experienced at least one serious adverse event, resulting in discontinuation of study for 6 (40%) patients. One patient had a bowel perforation. One patient (3 mg/kg cohort) experienced a treatment-related grade 5 cardiopulmonary arrest resulting in death. The best overall response was partial response in three patients (20%). Eleven patients (73.3%) had stable disease and one (6.7%) experienced disease progression. Conclusions: This study was stopped early to focus on the development of navicixizumab in ovarian cancer. Although the disease control rate was promising, further evaluation is needed to determine whether navicixizumab delivers efficacy greater than chemotherapy alone and assess tolerability of this regimen. [Table: see text]

NRP1 inhibition modulates radiosensitivity of medulloblastoma by targeting cancer stem cells

BackgroundMedulloblastoma (MB) is the most common pediatric malignant brain tumor. Despite current therapies, the morbidity and recurrent risk remains significant. Neuropilin-1 receptor (NRP1) has been implicated in the tumor progression of MB. Our recent study showed that NRP1 inhibition stimulated MB stem cells differentiation. Consequently, we hypothesized that targeting NRP1 in medulloblastoma could improve current treatments.MethodsNRP1 inhibition with a novel peptidomimetic agent, MR438, was evaluated with radiotherapy (RT) in MB models (DAOY, D283-Med and D341-Med) in vitro on cancer stem-like cells as well as in vivo on heterotopic and orthotopic xenografts.ResultsWe show that NRP1 inhibition by MR438 radiosensitizes MB stem-like cells in vitro. In heterotopic DAOY models, MR438 improves RT efficacy as measured by tumor growth and mouse survival. In addition, clonogenic assays after tumor dissociation showed a significant reduction in cancer stem cells with the combination treatment. In the same way, a benefit of the combined therapy was observed in the orthotopic model only for a low cumulative irradiation dose of 10 Gy but not for 20 Gy.ConclusionsFinally, our results demonstrated that targeting NRP1 with MR438 could be a potential new strategy and could limit MB progression by decreasing the stem cell number while reducing the radiation dose.

Crosstalk between heat shock factor 1 and signal transducer and activator of transcription 3 mediated by interleukin-8 autocrine signaling maintains the cancer stem cell phenotype in liver cancer.

Liver cancer stem cells (LCSCs) cause therapeutic refractoriness and relapse in hepatocellular carcinoma. Heat shock factor 1 (HSF1) plays versatile roles in multiple cancers. However, the role of HSF1 in LCSCs is not well understood. This study investigated the function and signal mechanisms of HSF1 in maintaining LCSC phenotypes. We established two LCSC lines, HepG2-R and HuH-7-R. Constitutive activation of HSF1 was observed in these LCSCs. Specific short hairpin RNAs (shRNAs) and chemical inhibitors were used to identify the relationship between HSF1 expression and LCSCs phenotypes. We revealed a concomitant activation modality involving HSF1 and STAT3 in LCSCs and liver cancer tissues. We also found that liver cancer patients whose HSF1 and STAT3 mRNA expression levels were high presented with unfavorable clinicopathological characteristics. Moreover, the secretion of interleukin-8 (IL-8) was elevated in the LCSC medium and was directly regulated by HSF1 at the transcriptional level. In turn, IL-8 activated HSF1 and STAT3 signaling, and a neutralizing IL-8 antibody inhibited HSF1 and STAT3 activity, reduced cancer stem cell marker expression, and decreased LCSC microsphere formation. Simultaneous intervention with HSF1 and STAT3 led to synergistically suppressed stemness acquisition and growth suppression in the LCSCs in vivo and in vitro. Our study indicates that IL-8 mediates the crosstalk between the HSF1 and Stat3 signaling pathways in LCSCs and that the combined targeting of HSF1 and STAT3 is a promising treatment strategy for patients with advanced liver cancer.

STEM-06. NUCLEAR STRUCTURE MAINTAINS GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma stem cells (GSCs) contribute to therapeutic resistance, promote tumor angiogenesis, and evade anti-tumor immune responses. We and others have shown that GSCs display differential regulation of their epigenetic landscapes and metabolic profiles and contribute to the striking cellular heterogeneity and complex tumor microenvironments. Here, we find that GSCs undergo dramatic changes in chromatin organization and nuclear morphology upon differentiation. Leveraging unbiased RNA sequencing and protein profiling screens comparing GSCs and differentiated glioma cells, we identified nuclear membrane molecular regulation that defines nuclear size and lipid regulation. Genetic or pharmacologic targeting of nuclear envelope function reduced cancer stem cell proliferation, self-renewal, and in vivo tumor growth. Collectively, we define a novel role of the nuclear envelope in maintaining chromatin state and cancer stem cell self-renewing, informing the development of novel potential therapeutic paradigms for glioblastoma.

The transglutaminase 2 cancer cell survival factor maintains mTOR activity to drive an aggressive cancer phenotype.

Transglutaminase 2 (TG2) is an important cancer stem-like cell survival protein that is highly expressed in epidermal squamous cell carcinoma and drives an aggressive cancer phenotype. In the present study, we show that TG2 knockdown or inactivation results in a reduction in mammalian target of rapamycin(mTOR) level and activity in epidermal cancer stem-like cells which are associated with reduced spheroid formation, invasion, and migration, and reduced cancer stem cell and epithelial-mesenchymal transition(EMT) marker expression. Similar changes were observed in both cultured cells and tumors. mTOR knockdown or treatment with rapamycin phenocopies the reduction in spheroid formation, invasion, and migration, and cancer stem cell and EMT marker expression. Moreover, mTOR appears to be a necessary mediator of TG2 action, as a forced expression of constitutively active mTOR in TG2 knockdown cells partially restores the aggressive cancer phenotype and cancer stem cell and EMT marker expression. Tumor studies show that rapamycin reduces tumor growth and cancer stem cell marker expression and EMT. These studies suggest that TG2 stimulates mTOR activity to stimulate cancer cell stemness and EMT and drive aggressive tumor growth.

Homo and Heterotypic Cellular Cross-Talk in Epithelial Ovarian Cancer Impart Pro-Tumorigenic Properties through Differential Activation of the Notch3 Pathway.

Simple SummaryThe omental metastatic spreading of epithelial ovarian cancer is spearheaded by complex cell–cell interactions present in the fluidic microenvironment (ascites), which is yet to be fully decoded. Using a unique co-culture model of SNFT (SKOV3 cells expressing a Notch3 luciferase reporter-sensor) and NIH3T3 cells (differentially overexpressing Jagged1 ligand), we demonstrated that incremental Jagged1 expression led to proportional Notch3 activation in SNFT. Differential Notch3 activation was also evident from co-culture of SNFT with other EOC cell lines and ascites-derived cancer-associated fibroblasts of HGSOC patients expressing varying levels of Jagged1. Amongst the top five modulated genes identified by the gene profiler array, both p21 and VEGFA showed enhanced expression (for p21)/secretion (for VEGFA) in SNFT when induced with Jagged1. Secreted VEGFA further reduced CSC differentiation in platinum-resistant A2780 cells. Pronounced VEGFA expression associated with Notch3 up-regulation in metastatic HGSOC tumors delineates an unknown role of the Notch3/VEGFA axis in EOC progression.An active fluidic microenvironment governs peritoneal metastasis in epithelial ovarian cancer (EOC), but its critical functional/molecular cues are not fully understood. Utilizing co-culture models of NIH3T3 cells (differentially overexpressing Jagged1) and SKOV3 cells expressing a Notch3 luciferase reporter-sensor (SNFT), we showed that incremental expression of Jagged1 led to proportional Notch3 activation in SNFT. With no basal luciferase activity, this system efficiently recorded dose-dependent Notch3 activation by rh-Jag1 peptide and the non-appearance of such induction in co-culture with NIH3T3Δjag1 cells indicates its sensitivity and specificity. Similar Notch3 modulation was shown for the first time in co-cultures with HGSOC patients’ ascites-derived cancer-associated fibroblasts and Jagged1-expressing EOC cell lines. NIH3T3J1-A and OVCAR3 co-cultured SNFT cells showed maximum proliferation, invasion, and cisplatin resistance among all the heterotypic/homotypic cellular partners. VEGFA and CDKN1A are the two most upregulated genes identified across co-cultures by the gene profiler array. Co-culture induced VEGFA secretion from SNFT cells which also reduced cancer stem cell differentiation in platinum-resistant A2780 cells. rh-Jag1-peptide promoted enhanced nuclear-cytoplasmic p21 expression. Additionally, metastatic HGSOC tumors had higher VEGFA than corresponding primary tumors. This study thus demonstrates the tumoral and non-tumoral cell-mediated differential Notch3 activation imparting its tumorigenic effects through two critical molecular regulators, VEGFA and p21, during EOC progression.

CXCR4 knockdown enhances sensitivity of paclitaxel via the PI3K/Akt/mTOR pathway in ovarian carcinoma.

Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy. EOC control remains difficult, and EOC patients show poor prognosis regarding metastasis and chemotherapy resistance. The aim of this study was to estimate the effect of CXCR4 knockdown-mediated reduction of cancer stem cells (CSCs) and epithelial–mesenchymal transition (EMT) stemness and enhancement of chemotherapy sensitivity in EOC. Mechanisms contributing to these effects were also explored. Our data showed distinct contribution of CXCR4 overexpression by dependent PI3K/Akt/mTOR signaling pathway in EOC development. CXCR4 knockdown resulted in a reduction in CSCs and EMT formation and enhancement of chemotherapy sensitivity in tumor cells, which was further advanced by blocking CXCR4-PI3K/Akt/mTOR signaling. This study also documented the critical role of silencing CXCR4 in sensitizing ovarian CSCs to chemotherapy. Thus, targeting CXCR4 to suppress EOC progression, specifically in combination with paclitaxel (PTX) treatment, may have clinical application value.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Butein induces intrinsic pathway of apoptosis, vimentin proteolysis, and inhibition of cancer stem cell population in a human papillary thyroid cancer cell line

Epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) play an essential role in metastasis of papillary thyroid cancer (PTC). Further mesenchymal marker vimentin is linked with metastasis and cancer stem cell generation. Hence, inhibition of EMT and effective elimination of CSCs offers a novel target for the development of new therapeutic agents.The present study observed that at lower concentration, butein, a major bioactive chalcone, significantly inhibits NPA (papillary thyroid cancer cell line) cell migration and reduces extracellular acidification rate (ECAR) an indicator of enhanced glycolysis, required for cell migration. Additionally, at lower concentrations, butein treatment also suppresses vimentin phosphorylation, an essential step in cell migration, proving its potential against cell migration. Phosphorylation of vimentin is crucial in the protection of vimentin from caspase-mediated proteolysis. Interestingly, butein activates caspase-3 for the apoptosis execution at higher concentration; hence, total levels of vimentin were investigated. Butein induces caspase-3 mediated proteolysis of vimentin. Vimentin and glycolysis are essential for maintaining CSCs; therefore, aldeflour assay and side population assay were performed to investigate the effect of butein on CSCs. Our data suggest butein mediates the reduction in CSCs population.Here we report a novel mechanism of butein mediated inhibition of NPA cells migration by suppressing vimentin phosphorylation and its subsequent proteolysis. Collectively our data suggest the potential of butein as an innovative anticancer therapeutic agent for PTC management.

Effect of aerobic exercise on acquired gefitinib resistance in lung adenocarcinoma

Lung adenocarcinoma patients with epidermal growth factor receptor (EGFR)-activating mutations respond well to tyrosine kinase inhibitors but typically develop resistance. Current therapies mainly target differentiated cells, not cancer stem cells (CSCs), but CSCs affect the occurrence, invasion, metastasis and treatment sensitivity of malignant tumours. Recently, aerobic exercise has emerged as adjuvant therapy for cancer. Aerobic exercise can accelerate blood circulation, improve tissue oxygen supply, reduce the stress level of patients, improve the antioxidant capacity of the body, and facilitate the degradation of hypoxia-inducible factor-1 (HIF-1) in tumour tissues, thus weakening its maintenance effect on CSCs. In this study, we successfully established lung adenocarcinoma cell lines with gefitinib resistance. Long-term gefitinib induction could increase the level of oxidative stress in lung adenocarcinoma cells and reduce the antioxidant capacity, resulting in the high expression of HIF-1 and ALDH1 and leading to the enrichment of CSCs, and a decreased response to gefitinib. This may be one of the important reasons for gefitinib-acquired resistance in lung adenocarcinoma. In the case of drug resistance, effective aerobic exercise could reduce ROS, activate SOD, inhibit HIF-1 and ALDH1, and cause a reduction in CSCs to sensitise cells to gefitinib again and ultimately inhibit the malignant proliferation of tumours. Therefore, in the treatment of lung adenocarcinoma, the inhibitory effect of aerobic exercise on oxidative stress can enhance the response of drug-resistant cells to gefitinib and can be used as an effective adjunct measure in the treatment of lung adenocarcinoma.

Abstract 1977: Combined inhibition of EGFR and PI3Kinase signaling in EGFR amplified triple negative breast cancer cells induces apoptosis and reduces cancer stem cells

Abstract Signaling from the Epidermal Growth Factor Receptor (EGFR) family is commonly dysregulated in various cancer types by amplification or activating mutations. For example, HER2 amplification occurs in 15-20% of breast cancers, and is a well-defined driver of tumorigenesis. EGFR is amplified in approximately 2% of breast cancer patients with unknown significance. It is amplified more frequently in TNBC (5.7-6.7%) and ER-/HER2+ cancers (5.8-6.5%). Basal (4.3-8.1%) and HER2 enriched (3.9-45.4%) molecular subtypes show the highest amplification than other molecular subtypes. EGFR amplification leads to shorter overall survival among breast cancer patients compared to those without amplified EGFR. Approximately 71% of EGFR amplified tumors have activated PI3K signaling, indicating a possible dual-inhibition therapeutic approach targeting both EGFR and PI3K. In order to study the role of EGFR amplification and PI3K aberrations in TNBC, we used the TNBC cell lines BT20 (amplified EGFR, PIK3CA activating mutation), MDA-MB-468 (amplified EGFR, PTEN deletion), and MDA-MB-231 (no amplification of EGFR or PI3K pathway mutations). EGFR amplification in both MDA-MB-468 and BT20 cell lines was confirmed by western blotting and Fluorescent in situ Hybridization; MDA-MB-231 did not have EGFR amplification. Activating mutations in both alleles of PIK3CA in BT20 was confirmed by sequencing. Loss of PTEN in MDA-MB-468 was confirmed by immunoblotting. Inhibition of EGFR with erlotinib resulted in decreased ERK signaling in BT20 and MDA-MB-468, but not in MDA-MB-231. Inhibition of the PI3K with Ly294002 or BKM120 decreased activation of AKT and mTOR signaling in BT20 and MDA-MB-468, but not in MDA-MB-231. The combination of erlotinib and PI3K inhibitors more dramatically reduced mTOR and AKT signaling in the BT20 and MDA-MB-468 cells. Combination of erlotinib and PI3K inhibitors reduced cell viability in these 3 cell lines, but inhibition was greater in MDA-MB-468 and BT20 compared to MDA-MB-231, and only MDA-MB-468 and BT20 cells had an increased fraction of apoptotic cells, signifying that combination therapy is most effective in cells with amplified EGFR and aberrant PI3K. It has been previously shown that HER2/HER3 activation plays a role in self renewal of tumor initiating cells. We demonstrate that the combination of erlotinib and BKM120 reduced the cancer stem cell population as measured by an aldeflour assay and by mammosphere formation. In summary, these data demonstrate that EGFR amplification in TNBC drives cell viability through EGFR and PI3K signaling, and EGFR/PI3K inhibitor combination causes apoptosis as well as reduction in the cancer stem cell population. We present evidence therefore that dual inhibition of EGFR/PI3K presents as a potential targetable pathway in TNBC with EGFR amplification and aberrant PI3K signaling. Citation Format: David John Wisniewski, Donna Voeller, Stanley Lipkowitz. Combined inhibition of EGFR and PI3Kinase signaling in EGFR amplified triple negative breast cancer cells induces apoptosis and reduces cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1977.

Abstract 3091: Relevance of iNOS expression in bladder cancer stem cells

Abstract Background: Bladder cancer (BC) is one of the most common tumors of the male urogenital tract and an important worldwide cause of death. Increasing evidence has indicated the presence of cancer stem cells (CSCs) in many types of tumors, associated with aggressiveness, chemoresistance and relapse. The expression of inducible nitric oxide (NO) synthase (iNOS) in human BC is a poor prognostic factor associated with increased invasion and tumor recurrence. Although there is evidence supporting the role of NO/iNOS in the promotion and progression of BC, the possibility that there are CSCs dependent on endogenous NO generation was not yet been clearly defined. In this study we evaluate the role of NO in CSC maintenance, modulating its production, using pharmacological inhibitors or silencing iNOS expression in a murine BC model. Also, we analyzed the correlation between iNOS expression and stemness-related genes (SG) in human BC samples with different invasion grades. Methods: iNOS pharmacological inhibitors (pan-NOS or iNOS specific) or shRNA were used on murine BC model with different iNOS expression and invasiveness grade: MB49 [iNOS+, non-muscle invasive (NMI)] and MB49-I [iNOS++, muscle invasive (MI)], in order to analyze cell proliferation, number of CSC determined as spheres forming efficiency (SFE) and SG (Sox 2, Oct4 and Nanog expression) by qPCR. iNOS expression and SG were also analyzed in human BC samples (16 NMI low grade (LG), 10 NMI high grade (HG) and 17 MI). Results: The number of CSCs was higher in MB49-I than NMI MB49 line (p<0.05), in concordance with the higher expression of iNOS and Sox2, Oct4 and Nanog (p<0.001). iNOS inhibition diminished CSC in both cell lines (p<0.05 vs MB49; p<0.001 vs MB49-I). Inhibition of SG was only observed in MB49-I line (p<0.05). Moreover, the reduction of CSC and SG were more evident in MB49-I-shiNOS (p<0.0001 vs MB49-I scr; p<0.001 vs MB49-I-scr). The in vivo tumorigenic potential of cells derived from MB49-I spheres were higher than the cells growing in monolayers. MB49-I spheres cells treated with iNOS inhibitors did not developed tumors in vivo. Furthermore, MB49-I shiNOS spheres showed an incipient growth and regressed about 5 weeks post inoculation, in correlation with low iNOS and SG expression. Regarding human samples, iNOS was expressed in 78% of MI, 55% HG and 50% LGNMI (p<0.05 vs NMI). SG expression were significantly higher in MI than in NMI LG. Sox2 was expressed 82% in MI, 25% LG and 40% HG in NMI (p=0.0002). 76% of MI samples were positive for Nanog and Oct4 (p<0.05 vs NMI LG). A positive correlation between iNOS and SG expression was found (sox2: p<0.0001; Oct4: p<0.0001 and Nanog: p<0.05). Conclusion: Together, this results show that iNOS plays an important role in maintenance of CSC and in vivo BC growth by regulating stemness and self-renewal ability. Thus, iNOS identification and inhibition could be a potential target to eradicate CSC, responsible for tumor recurrences. Citation Format: Denise Belgorosky, Yanina Langle, Julie Girouard, Jovane Hamelin-Morrissette, Lina Marino, Eduardo I. Agüero, Héctor Malagrino, Carlos Reyes-Moreno, Ana M. Eiján. Relevance of iNOS expression in bladder cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3091.

Abstract 968: Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation

Abstract Ovarian cancer is one of the deadliest malignancies in women, and up to 70% of patients with epithelial ovarian cancer have FAK overexpression, amplification, or activation. FAK plays an important role in cellular migration, growth factor signaling, cell cycle progression, cellular survival and chemoresistance. This biomarker is also significantly associated with higher tumor stage, metastasis, and shorter overall survival in patients with ovarian cancer. Inhibition of FAK is therefore emerging as a promising treatment target. APG-2449 is a clinical stage FAK/ALK/ROS1 multi-kinase inhibitor. In this study we investigated antitumor activity of APG-2449 combined with standard-of-care chemotherapeutics in ovarian cancer in the preclinical setting. In a mouse xenograft tumor model derived from ovarian cancer cell line OVCAR-3, which was resistant to platinum-based therapies, APG-2449 combined with paclitaxel, and paclitaxel plus carboplatin, synergistically enhanced antitumor activity, whereas the chemotherapeutics showed no activity. Synergistic antitumor activity was also observed in multiple patient-derived xenograft (PDX) models derived from women with chemoinsensitive ovarian cancer, which also frequently expresses high levels of FAK. By comparing gene expression profiles of PDX tumors obtained from responders and nonresponders to the combined therapy, we identified CD44 (a marker for cancer stem cells) as a potentially predictive biomarker. Western blot analysis confirmed higher protein levels of CD44 in pretreated tumors of responders. Interestingly, downregulation of CD44 levels was observed in combination-treated tumors, suggesting that these combinations reduced cancer stem cell populations in ovarian cancer. Accordingly, in ovarian cancer cells exposed to APG-2449 alone or combined with paclitaxel, numbers of cells positive for CD44 or aldehyde dehydrogenase 1 (ALDH1; another marker for cancer stem cells) decreased in a dose-dependent manner. In summary, our data suggest that FAK inhibition by APG-2449 sensitizes ovarian tumors to chemotherapeutics in preclinical tumor models of ovarian cancer. The synergistic antitumor activity was mediated by downregulation of CD44+ or ALDH1+ cancer stem cell populations. These findings encourage clinical development of APG-2449 in combination with chemotherapies for treatment of ovarian cancer. Citation Format: Ran Tao, Douglas D. Fang, Yuanbao Li, Kaixiang Zhang, Chunhua Xu, Xu Fang, Qixin Wang, Dajun Yang, Yifan Zhai. Focal adhesion kinase (FAK) inhibitor APG-2449 sensitizes ovarian tumors to chemotherapy via CD44 downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 968.