Reduces Cancer Stem Research Articles

Doxycycline, an Inhibitor of Mitochondrial Biogenesis, Effectively Reduces Cancer Stem Cells (CSCs) in Early Breast Cancer Patients: A Clinical Pilot Study.

Background and objectives: Cancer stem cells (CSCs) have been implicated in tumor initiation, recurrence, metastatic spread and poor survival in multiple tumor types, breast cancers included. CSCs selectively overexpress key mitochondrial-related proteins and inhibition of mitochondrial function may represent a new potential approach for the eradication of CSCs. Because mitochondria evolved from bacteria, many classes of FDA-approved antibiotics, including doxycycline, actually target mitochondria. Our clinical pilot study aimed to determine whether short-term pre-operative treatment with oral doxycycline results in reduction of CSCs in early breast cancer patients.Methods: Doxycycline was administered orally for 14 days before surgery for a daily dose of 200 mg. Immuno-histochemical analysis of formalin-fixed paraffin-embedded (FFPE) samples from 15 patients, of which 9 were treated with doxycycline and 6 were controls (no treatment), was performed with known biomarkers of “stemness” (CD44, ALDH1), mitochondria (TOMM20), cell proliferation (Ki67, p27), apoptosis (cleaved caspase-3), and neo-angiogenesis (CD31). For each patient, the analysis was performed both on pre-operative specimens (core-biopsies) and surgical specimens. Changes from baseline to post-treatment were assessed with MedCalc 12 (unpaired t-test) and ANOVA.Results: Post-doxycycline tumor samples demonstrated a statistically significant decrease in the stemness marker CD44 (p-value < 0.005), when compared to pre-doxycycline tumor samples. More specifically, CD44 levels were reduced between 17.65 and 66.67%, in 8 out of 9 patients treated with doxycycline. In contrast, only one patient showed a rise in CD44, by 15%. Overall, this represents a positive response rate of nearly 90%. Similar results were also obtained with ALDH1, another marker of stemness. In contrast, markers of mitochondria, proliferation, apoptosis, and neo-angiogenesis, were all similar between the two groups.Conclusions: Quantitative decreases in CD44 and ALDH1 expression are consistent with pre-clinical experiments and suggest that doxycycline can selectively eradicate CSCs in breast cancer patients in vivo. Future studies (with larger numbers of patients) will be conducted to validate these promising pilot studies.

Chemosensitizing effect of honokiol in oral carcinoma stem cells via regulation of IL-6/Stat3 signaling.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers worldwide with poor prognosis. Numerous studies have attempted to explore alternative regimens aimed at reducing cancer stem cells (CSCs) without compromising the efficacy of conventional chemoradiotherapy. The present study sought to assess the effect of a natural compound honokiol on the reduction of elevated cancer stemness, metastatic capacity, and chemoresistance of oral carcinoma stem cells (OCSCs). Our results demonstrated that honokiol attenuated the cell survival and self-renewal of OCSCs in a dose-dependent manner. Moreover, honokiol downregulated the expression of 2 selective markers of OCSCs, ALDH1, and CD44, as well as the migration and invasion abilities, indicating its potential to suppress cancer stemness. We showed that honokiol reduced the secretion of IL-6 and phosphorylation of STAT3, and the honokiol-inhibited self-renewal, invasion and colony formation were reversed by administration of IL-6. Most importantly, our data demonstrated that honokiol was able to potentiate the effect of Cisplatin, leading to a lower proportion of OCSCs and the decreased cancer stemness features. Taken together, this study demonstrated the benefits of utilizing honokiol as an adjunct therapy for OSCC treatment.

Abstract 4461: Sirt6 deletion slows mouse mammary tumorigenesis

Abstract Studies show that the NAD+ dependent histone deacetylase Sirt6 exerts tumor suppressive effects in different tissues and organs by mechanisms that include the dampening of the Warburg effect and of Myc activity. In breast cancer (BC), Sirt6 was recently shown to blunt PI3K signaling and to reduce cancer stem cell like capacity in tumors with mutated PI3K. Here we sought define the effects of Sirt6 overexpression and of Sirt6 silencing in breast cancer cell lines, as well as the impact of a heterozygous Sirt6 deletion on polyoma middle T antigen (PyMT)-induced mouse mammary tumorigenesis, with the expectation of an accelerated mammary tumor development in PyMT+/-;Sirt6+/- as compared to PyMT+/-;Sirt6+/+ mice. SIRT6 was overexpressed in either wild type or catalytically inactive (H133Y) form, or silenced in BC cell lines and in non-tumorigenic MCF10A and HMLE cells, monitoring glycolytic enzyme activity (iHK, PFK, PK, LDH), lactate production, oxygen consumption rate, complex I, III, IV, and ATP synthase activity, and matrix metalloproteinase 9 (MMP9) expression. In vivo, we monitored the growth of MDA-MB-231 xenografts in which SIRT6 was silenced vs. control tumors. In addition, we crossed Sirt6+/- mice with MMTV-PyMT+/- mice (a kind gift of Dr. Thorsten Berger, University Health Network, Toronto, Canada) and comparatively monitored tumor latency and overall survival in PyMT+/-;Sirt6+/- vs. PyMT+/-;Sirt6+/+ mice. In cultured breast cancer cell lines, overexpression of catalytically active SIRT6 (but not of a catalytically inactive enzyme) reduced the activity of glycolytic enzymes as well as lactate production, while boosting OXPHOS and the ATP/AMP ratio. Opposite effects were obtained by SIRT6 silencing. Curiously, no effect of SIRT6 overexpression or of its silencing on glycolysis or OXPHOS was observed in non-tumorigenic mammary epithelial cells. SIRT6 was found to regulate MMP9 expression. Namely, in MDA-MB-231, SIRT6 overexpression increased, while SIRT6 silencing reduced MMP production. In vivo, subcutaneous xenografts of SIRT6-silenced MDA-MB-231 cells were found to grow slower than control cells. PyMT+/-;Sirt6+/- mice exhibited a markedly increased tumor latency and an increased overall survival as compared to the control PyMT+/-;Sirt6+/+ mice. The anticancer effect of Sirt6 heterozygous deletion did not reflect reduced glucose levels in Sirt6+/- mice, as the latter had normal blood glucose levels. In conclusion, our data show that reducing Sirt6 levels has antitumor effects in mouse breast cancer models, which would not be anticipated based on the available knowledge of Sirt6's biological role. In addition, by virtue of its ability to regulate MMP9 expression, SIRT6 could be a potential target for countering metastasis. Future studies should assess which molecular features predict the potential benefit of SIRT6 inhibition in breast cancer and test the anticancer activity of SIRT6 inhibitors. Citation Format: Pamela Becherini, Irene Caffa, Silvia Ravera, Patrizia Damonte, Francesco Piacente, Michele Cea, Antonia Cagnetta, Fiammetta Monacelli, Raul Mostoslavsky, Santina Bruzzone, Alberto Ballestrero, Patrizio Odetti, Alessio Nencioni. Sirt6 deletion slows mouse mammary tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4461.

Abstract LB-051: An Astragalus-based Chinese herbal medicine extraction inhibits cancer stem cell growth and sensitizes of drug-resistant human non-small cell lung cancer cells for targeted therapy

Abstract Chinese herbal medicine has been used extensively as a complementary intervention for many diseases including cancer, based on its relatively lower cytotoxicity and side-effects. The major criticism of its clinical use remains the scientific based evidence. Here, we described a precision-based translational approach to identify and validate an Astragalus-based herbal medicine (YQ1) as an antagonist of non-small cell lung cancer. Using various big data analysis approaches, we found that the L1000 profiles of YQ1was associated with anti-cancer stem cell signatures. It suggested that treatment of YQ1 might reduce cancer stem cell population. Experimentally, YQ1 treatment resulted in the significant reduction in lung cancer stem cell properties, including side population, sphere formation, stemness marker expression, and drug resistance. YQ1 may have anti-cancer stem cell ability via suppressing Wnt/β-catenin signaling to inhibit the self-renewal or/and proliferation of lung cancer stem cells. Notably, the combination of YQ1 and gefitinib significantly suppresses tumorigenesis in vivo, reflected by the down-regulation of EGFR signaling and β-catenin. Collectively, we provided preclinical evidence for using YQ1 as a cancer stem cell inhibitor to potentiate anti-tumor effects when combined with tyrosine-kinase inhibitors to significantly improve therapeutic efficacy in NSCLC patients. Citation Format: Tse-Hung Huang, Hsuan-Min Hsu, Chi-Ying F. Huang. An Astragalus-based Chinese herbal medicine extraction inhibits cancer stem cell growth and sensitizes of drug-resistant human non-small cell lung cancer cells for targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-051.

Abstract 4410: miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization

Abstract Background: Cancer stem cells (CSCs) have inherent or developed drug resistance for which chemotherapy fails to eliminate all tumor cells, a property that is critically contributing to recurrence and metastasis. Here, we combine miR-34a, a well-known tumor suppressor miRNA that controls proliferation, apoptosis and self-renewal, with Paclitaxel (PTX), a commonly used first-line chemotherapy drug. Based on our preliminary observations (Bonetti et al., manuscript under revision), we hypothesized a combinatorial approach, coupling miRNA- and chemo- treatment, aimed at reducing CSCs in vitro and recurrence in vivo using triple-negative breast cancer (TNBC) as a model. Methodology: As a model, we exploited SUM159pt cells (TNBC mesenchymal-like, with CSC features) to mimic aggressive breast cancer, which harbor a vector for pLUC, which is induced by luciferin in vivo, to monitor tumor growth in real time. Furthermore, we used i) pSlik34a, with tunable expression of miR-34a by doxycycline; ii) treatment with PTX to select for resistant cells (RC); and combined the two, by treating RC with doxycycline. CSC content was evaluated by ALDH (Aldehyde Dehydrogenase) activity, sphere-forming efficiency (SFE) and qRT-PCR of known markers. A similar approach was followed for in vivo studies, with NSG female injected through tail vein injection with 500,000 SUM159/pLUC/pSlik34a cells, and separated into three groups: not treated (NT), PTX alone, and PTX+34a. Results: As observed for aggressive breast cancer, treatment of SUM159 with PTX generated resistant cells (RC) with increased CSC content (ALDH activity and spheres forming ability). When miR-34a was induced in the RC, cells changed morphology, slowed proliferation rate, and decreased CSC content (ALDH activity and spheres). Moreover, a second PTX treatment almost completely abolished the ALDH activity and SFE. The combined effect was evaluated in vivo, following by luminescence the growth of metastases after lung colonization, with PTX+34a mice reducing lung colonization than the PTX treatment or miR-34a treatment alone. Conclusions: Overall, these results indicate that miR-34a sensitize PTX-resistant cells reducing cancer stem-cell capacities, and in this preclinical setting limited the aggressive phenotype of breast cancer, including proliferation, drug resistance, relapse and metastasis causing in delay of lung colonization in vivo. Citation Format: Montserrat Climent, Paola Bonetti, Francesco Nicassio. miRNA-34a sensitizes triple-negative breast cancer cells to paclitaxel reducing cancer stem cell population and lung colonization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4410.

Abstract 68: Targeting macrophage that forms a chemotherapy-induced niche significantly reduces cancer stem cells and tumors

Abstract Failure in cancer therapy is often caused by relapse resulting largely from chemo-resistant cancer stem cells (CSCs). Most colorectal cancer patients, after receiving chemoradiotherapy, still harbor treatment-resistant residual tumor cells that are akin to colorectal adenoma. We therefore exploited the APCMin adenoma model that allowed us to study the natural and dynamic interaction between CSCs and their microenvironment in vivo following therapeutic stress. We observed that macrophages were directly recruited from blood vessel to the CSC niche in response to chemotherapy, playing a role in protecting and further promoting activation of CSCs. Reduction in the Ly6c-CD11bintCX3CR1+ MHCII+ population of tumor associated macrophages (TAMs) by celecoxib or clodrosome was correlated with a decline in slow-cycling drug-resistant CSCs and tumorigenesis. Molecularly, we found that COX-2 inhibitor celecoxib suppressed Akt and Wnt signaling, downstream of macrophage-dependent PGE2-EP pathway, thus reducing CSC survival and activation. Taken together, in the naturally formed adenoma reflecting human familial adenomatous polyposis, Chemotherapy-induced recruitment of TAMs to the CSC niche protected and promoted activation of surviving CSCs via PGE2-EP mediated Akt-Wnt signaling, leading to tumor regrowth. Targeting the TAMs will benefit clinical treatment of colorectal cancer. Citation Format: Xi He, Paloma I. Giles, Edward H. Lin, Linheng Li. Targeting macrophage that forms a chemotherapy-induced niche significantly reduces cancer stem cells and tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 68.

Combined Gemcitabine and Metronidazole Is a Promising Therapeutic Strategy for Cancer Stem-like Cholangiocarcinoma.

Metronidazole (MNZ) is a common antibiotic that exerts disulfiram-like effects when taken together with alcohol. However, the relationship between MNZ and aldehyde dehydrogenase (ALDH) activity remains unclear. This study investigated whether MNZ reduces cancer stemness by suppressing ALDH activity and accordingly reducing the malignancy of cholangiocarcinoma (CCA). We developed gemcitabine (GEM)-resistant TFK-1 cells and originally established CCA cell line from a patient with GEM-resistant CCA. Using these cell lines, we analyzed the impacts of MNZ for cancer stem cell markers, invasiveness, and chemosensitivity. MNZ reduced ALDH activity in GEM-resistant CCA cells, leading to decreased invasiveness and enhanced chemosensitivity. MNZ diminished the invasiveness by inducing mesenchymal-epithelial transition and enhancing chemosensitivity by increasing ENT1 (equilibrative nucleoside transporter 1) and reducing RRM1 (ribonucleotide reductase M1). MNZ reduced cancer stemness in GEM-resistant CCA cells. Combined GEM and MNZ would be a promising therapeutic strategy for cancer stem-like CAA.

Jade family PHD finger 3 (JADE3) increases cancer stem cell-like properties and tumorigenicity in colon cancer

Jade family PHD finger 3 (JADE3) plays a role in inducing histone acetylation during transcription, and is involved in the progression of several human cancers; however, its role in colon cancer remains unclear. Herein, we found that JADE3 was markedly upregulated in colon cancer tissues and significantly correlated with cancer progression, and predicted shorter patient survival. Further, JADE3 was expressed much higher in colon cancer cell lines that are enriched with a stem-like signature. Overexpression of JADE3 increased, while silencing JADE3 reduced cancer stem cell-like traits in colon cancer cells in vitro and in vivo. Importantly, silencing of JADE3 strongly impaired the tumor initiating capacity of colon cancer cells in vivo. Furthermore, JADE3 interacted with the promoters of colon stem cell marker LGR5 and activated its transcription, by increasing the occupancy of p300 acetyltransferase and histone acetylation on the promoters. Finally, we found that JADE3 expression was substantially induced by Wnt/β-catenin signaling. These findings suggest an oncogenic role of JADE3 by regulating cancer stem cell-like traits in the colon cancer, and therefore JADE3 might be a potential therapeutic target for the treatment of colon cancer.

Polyene phosphatidylcholine overcomes oxaliplatin resistance in human gastric cancer BGC823 cells

Intrinsic or acquired resistance to oxaliplatin (L-OHP) is a major reason of treatment failure in gastric cancer and limits therapeutic success. Here we generated an oxaliplatin resistant gastric cancer cell line, BGC823/L-OHP, to investigate the effect of a hepatoprotective compound, polyene phosphatidylcholine (PPC), on conquest of oxaliplatin resistance. BGC823/L-OHP cells showed less sensitive to L-OHP directed growth inhibition than the parental BGC823 cells. PPC treatment significantly increased anti-proliferative activity of L-OHP on resistant cells and promoted L-OHP triggered apoptosis, indicating that drug resistance was overcome. Mechanistically, L-OHP incubation stimulated upregulation of an ABC family protein, ABCF2, and the expression was inhibited by PPC. Moreover, expression levels of the stemness factor Nanog and its regulator TLR4 were notably enhanced in BGC823/L-OHP cells and reduced by PPC treatment. To conclude, PPC can overcome oxaliplatin resistance in gastric cancer cells via promoting apoptosis, inhibiting ABCF2, as well via reducing cancer stem cell-like features. The combination therapeutic strategy could serve to increase oxaliplatin effectiveness in the clinic.

Inhibition of the HDAC6/Sp1 pathway by azaindolylsulfonamide reduces cancer stemness and restores treatment susceptibility in glioblastoma

Inhibition of the HDAC6/Sp1 pathway by azaindolylsulfonamide reduces cancer stemness and restores treatment susceptibility in glioblastoma

The use of alpha-fetoprotein for the treatment of autoimmune diseases and cancer.

Alpha-fetoprotein is a shuttle protein that delivers nutrients through receptor-mediated endocytosis to embryotic cells. In adults, alpha-fetoprotein can shuttle drugs into alpha-fetoprotein receptor-positive myeloid-derived suppressor, regenerating and also cancer cells. Drugs with high-binding affinity to alpha-fetoprotein can activate or deplete targeted cells. Myeloid-derived suppressor cells activation leads to immune suppression that can be used for treating autoimmune diseases. On the other hand, toxins delivered by alpha-fetoprotein can damage myeloid-derived suppressor cells and consequently unleash innate and adaptive immunity to destroy cancer cells. Innate immunity natural killers reduce cancer stem cells and metastases. The new alpha-fetoprotein drug noncovalent complexes for immunotherapy change the local immune balance and has potential in oncology, autoimmune and infectious diseases treatment, inflammation, transplantation, vaccination, etc.

Sulforaphane reduces YAP/∆Np63α signaling to reduce cancer stem cell survival and tumor formation.

Epidermal squamous cell carcinoma (SCC) is among the most common cancers. SCC can be treated by surgical excision, but recurrence of therapy-resistant disease is a major problem. We recently showed that YAP1, the Hippo signaling transcription adaptor protein, and ∆Np63α, a key epidermal stem cell survival protein, form a complex to drive epidermal cancer stem cell survival. In the present study, we demonstrate that YAP1 and ∆Np63α are important sulforaphane cancer prevention targets. We show that sulforaphane treatment increases YAP1 phosphorylation and proteolytic degradation. The loss of YAP1 is associated with a reduction in ∆Np63α level and a reduction in ECS cell survival, spheroid formation, invasion and migration. Loss of YAP1 and ∆Np63α is mediated by the proteasome and can be inhibited by lactacystin treatment. YAP1 or ∆Np63α knockdown replicates the responses to sulforaphane, and restoration of YAP1 or ∆Np63α antagonizes sulforaphane action. Sulforaphane suppresses ECS cell tumor formation and this is associated with reduced levels of YAP1 and ∆Np63α. These studies suggest that YAP1 and ∆Np63α may be important sulforaphane cancer preventive targets in epidermal squamous cell carcinoma.

Exostosin 1 regulates cancer cell stemness in doxorubicin-resistant breast cancer cells.

Cancer stem cells (CSCs) are associated with cancer recurrence following radio/chemotherapy owing to their high resistance to therapeutic intervention. In this study, we investigated the role of exostoxin 1 (EXT1), an endoplasmic reticulum (ER)-residing type II transmembrane glycoprotein, in cancer cell stemness. DNA microarray analysis revealed that doxorubicin-resistant MCF7/ADR cells have high levels of EXT1 expression compared to its parental cell line, MCF7. These cells showed significantly higher populations of CSCs and larger populations of aldehyde dehydrogenase (ALDH+) and CD44+/CD24-cells, as compared to MCF7 cells. siRNA-mediated knockdown of EXT1 in MCF7/ADR cells significantly reduced cancer stem cell markers, populations of ALDH+and CD44+/CD24- cells, mRNA and protein expression for CD44, and mammosphere number. Furthermore, epithelial mesenchymal transition (EMT) markers and migratory behavior were also repressed with reduced EXT1. In an in vitro soft agar colony formation assay, EXT1 knockdown by short hairpin RNA (shRNA) reduced the colony formation ability of these cells. Based on these results, we suggest that EXT1 could be a promising novel target to overcome cancer cell stemness in anthracycline-based therapeutic resistance.

Combination therapy in combating cancer.

Combination therapy, a treatment modality that combines two or more therapeutic agents, is a cornerstone of cancer therapy. The amalgamation of anti-cancer drugs enhances efficacy compared to the mono-therapy approach because it targets key pathways in a characteristically synergistic or an additive manner. This approach potentially reduces drug resistance, while simultaneously providing therapeutic anti-cancer benefits, such as reducing tumour growth and metastatic potential, arresting mitotically active cells, reducing cancer stem cell populations, and inducing apoptosis. The 5-year survival rates for most metastatic cancers are still quite low, and the process of developing a new anti-cancer drug is costly and extremely time-consuming. Therefore, new strategies that target the survival pathways that provide efficient and effective results at an affordable cost are being considered. One such approach incorporates repurposing therapeutic agents initially used for the treatment of different diseases other than cancer. This approach is effective primarily when the FDA-approved agent targets similar pathways found in cancer. Because one of the drugs used in combination therapy is already FDA-approved, overall costs of combination therapy research are reduced. This increases cost efficiency of therapy, thereby benefiting the “medically underserved”. In addition, an approach that combines repurposed pharmaceutical agents with other therapeutics has shown promising results in mitigating tumour burden. In this systematic review, we discuss important pathways commonly targeted in cancer therapy. Furthermore, we also review important repurposed or primary anti-cancer agents that have gained popularity in clinical trials and research since 2012.

Salinomycin Abolished STAT3 and STAT1 Interactions and Reduced Telomerase Activity in Colorectal Cancer Cells.

Colorectal cancer is the third leading cause of cancer-related mortality in most developed countries. This mortality is mainly due to the metastatic progression to the liver with frequent recurrence. Colorectal cancer remains a therapeutic challenge and this has intensified the search for new drug targets. In an effort to establish a novel targeted-therapy, we studied the molecular mechanisms of cancer stem cell inhibitor salinomycin. Co-immunoprecipitation was performed to examine STAT3-STAT1 protein interactions. Telomerase activity was measured by polymerase chain reaction (PCR) and ELISA assays. Apoptosis and cell stress arrays were analyzed to identify key proteins responding to salinomycin treatments. IL-6 and TNF-α induced STAT3 and STAT1 interactions, however the interactions were abolished by salinomycin challenge. Salinomycin reduced cancer stem cell phenotype and decreased telomerase activity of colorectal cancer cells. Our work uncovers a new mechanism through which salinomycin inhibits cancer stemness suggesting a novel targeted-therapy for metastatic colorectal cancer.

Abstract B47: Combating cancer with combination therapy in underserved populations

Abstract Combination therapy is an emerging treatment modality that combines two or more agents to produce a therapeutic effect. The combined use of therapeutic agents enhance efficacy compared to the monotherapy approach because it characteristically targets main key pathways with a synergistic or an additive effect. This approach potentially reduces drug-resistance, while simultaneously producing therapeutic anti-cancer benefits, such as the potential in reducing tumor growth and metastasis, arresting the cell cycle, reducing cancer stem cell populations, and inducing apoptosis. The 5-year survival rate for most cancers are still quite low, and the process of developing a new anti-cancer drug is long and extremely time consuming. Therefore, new strategies that target the survival pathways while providing efficient and effective results, as well as affordability, are being considered. One such approach incorporates the testing of therapeutic agents initially used for the treatment of different diseases on cancer. This approach is effective primarily when the FDA approved agent targets similar pathways found in cancer. Because one of the drugs used in combination therapy is already FDA approved and requires less funding to research, overall costs of combination therapy research are reduced. This increases cost efficiency of therapy and reduces the price of treatment, benefiting the “medically underserved”. In addition, an approach that combines repurposed pharmaceutical agents with other therapeutics have shown promising results in mitigating tumor size and volume. Note: This abstract was not presented at the conference. Citation Format: Tina Homayouni, Reza Bayat Mokhtari, Albina Tyker, Parandis Kazemi, Zhenya Morgatskaya, Narges Baluch, Sara Dhalla, Shreya Rekhi, Sushil Kumar, Herman Yeger. Combating cancer with combination therapy in underserved populations. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B47.

Discovery of furan carboxylate derivatives as novel inhibitors of ATP-citrate lyase via virtual high-throughput screening

The enzyme ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl CoA, the starting material for de novo lipid and cholesterol biosynthesis. The dysfunction and upregulation of ACL in numerous cancers makes it an attractive target for developing anticancer therapies. ACL inhibition by shRNA knockdown limits cancer cell proliferation and reduces cancer stemness. We designed and implemented a dual docking protocol to select virtual ACL inhibitors that were scored among the top 10 percentiles by both the Autodock Vina and the Glamdock algorithms. Via this in silico screens of a focused furoic acid library, we discovered four subtypes of furans and benzofurans as novel ACL inhibitors. The hit rate of our in silico protocol was 45.8% with 11 of 24 virtual hits confirmed as active in an in vitro ACL enzymatic assay. The IC50 of the most potent ACL inhibitor A1 is 4.1μM. Our results demonstrated remarkable hit rate by the dual docking approach and provided novel chemical scaffolds for the development of ACL inhibitors for the treatment of cancer.

MiR34a/GOLPH3 Axis abrogates Urothelial Bladder Cancer Chemoresistance via Reduced Cancer Stemness.

Rationale: Chemoresistance and subsequent recurrence of human urothelial bladder cancer (UBC) is partially driven by a subpopulation of tumor initiating cells, namely cancer stem cells (CSCs). However, the underlying molecular mechanism in chemotherapy-induced CSCs enrichment and following chemoresistance and recurrence remains largely unclear.Methods: Gemcitabine and cisplatin (GC) chemoresistant cell lines (T24 GC 3rd and 5637 GC 3rd cells) and the chemo-sensitive UBC cell lines T24 and 5637 were established in vivo for the investigation of acquired resistance mechanisms. The role of miR34a/GOLPH3 axis in regulating UBC chemoresistance and recurrence was evaluated in cell and animal models. The expression levels of miR34a/GOLPH3 axis and CSCs markers were assayed in specimens of UBC. The association of GOLPH3 with clinicopathologic features and prognosis was analysed.Results: RT-PCR and western blotting confirmed that the expression levels of miR34a were decreased and GOLPH3 were increased in GC chemoresistant UBC cell lines. Downregulation of miR34a resulted in the overexpression of GOLPH3, which is a target gene of miR34a confirmed by luciferase experiment. The ectopic expression of miR34a decreased the stem cell properties of chemoresistant UBC cells and re-sensitized these cells to GC treatment in vitro and in vivo. Moreover, miR34a/GOLPH3 axis has obvious clinical relevance with prognosis and recurrence in human UBC patients with standard GC chemotherapy.Conclusion: Our results suggest that miR34a/GOLPH3 axis exert key role in CSCs involved UBC drug resistance and recurrence and warrant further development as a promising therapeutic approach in treating drug-resistant UBC.

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking. The activities are mutually exclusive, as the closed conformation has GTP binding/GTPase activity, and the open conformation transamidase activity. We recently showed that GTP binding, but not transamidase activity, is required for TG2-dependent cancer stem cell invasion, migration and tumor formation. However, we were surprised that transamidase site-specific inhibitors reduce cancer stem cell survival. We now show that compounds NC9, VA4 and VA5, which react exclusively at the TG2 transamidase site, inhibit both transamidase and GTP-binding activities. Transamidase activity is inhibited by direct inhibitor binding at the transamidase site, and GTP binding is blocked because inhibitor interaction at the transamidase site locks the protein in the extended/open conformation to disorganize/inactivate the GTP binding/GTPase site. These findings suggest that transamidase site-specific inhibitors can inhibit GTP binding/signaling by driving a conformation change that disorganizes the TG2 GTP binding to reduce TG2-dependent signaling, and that drugs designed to target this site may be potent anti-cancer agents.

Design and synthesis of emodin derivatives as novel inhibitors of ATP-citrate lyase

Aberrant cellular metabolism drives cancer proliferation and metastasis. ATP citrate lyase (ACL) plays a critical role in generating cytosolic acetyl CoA, a key building block for de novo fatty acid and cholesterol biosynthesis. ACL is overexpressed in cancer cells, and siRNA knockdown of ACL limits cancer cell proliferation and reduces cancer stemness. We characterized a new class of ACL inhibitors bearing the key structural feature of the natural product emodin. Structure-activity relationship (SAR) study led to the identification of 1d as a potent lead that demonstrated dose-dependent inhibition of proliferation and cancer stemness of the A549 lung cancer cell line. Computational modeling indicates this class of inhibitors occupies an allosteric binding site and blocks the entrance of the substrate citrate to its binding site.