Bcl2:Beclin 1 complex: multiple mechanisms regulating autophagy/apoptosis toggle switch

This is the published version, also available here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3304572/.Cancer cells have developed novel mechanisms for evading chemotherapy-induced apoptosis and autophagy-associated cell death pathways. Upon the discovery that chemotherapeutics could target these cell death pathways in a manner that was not mutually exclusive, new discoveries about the interrelationship between these two pathways are emerging. Key proteins originally thought to be “autophagy-related proteins” are now found to be involved in either inducing or inhibiting apoptosis. Similarly, apoptosis inhibiting proteins can also block autophagy-associated cell death. One example is the complex formed by the autophagy protein, Beclin 1, and anti-apoptotic protein Bcl-2, which leads to inhibition of autophagy-associated cell death. Researchers have been investigating additional mechanisms that form/disrupt this complex in order to better design chemotherapeutics. This review will highlight the role Bcl-2 and Beclin 1 play in cancer development and drug resistance, as well as the role the Bcl-2:Beclin 1 complex in the switch between autophagy and apoptosis

Drug Resistance and Molecular Cancer Therapy: Apoptosis Versus Autophagy

This is the published version, also available electronically from http://www.intechopen.com/books/apoptosis/drug-resistance-and-molecular-cancer-therapy-apoptosis-versus-autophag

Overexpression of 17β-hydroxysteroid dehydrogenase type 10 increases pheochromocytoma cell growth and resistance to cell death

Background: 17β-hydroxysteroid dehydrogenase type 10 (HSD10) has been shown to play a protective role in cells undergoing stress. Upregulation of HSD10 under nutrient-limiting conditions leads to recovery of a homeostatic state. Across disease states, increased HSD10 levels can have a profound and varied impact, such as beneficial in Parkinson’s disease and harmful in Alzheimer’s disease. Recently, HSD10 overexpression has been observed in some prostate and bone cancers, consistently correlating with poor patient prognosis. As the role of HSD10 in cancer remains underexplored, we propose that cancer cells utilize this enzyme to promote cancer cell survival under cell death conditions. Methods: The proliferative effect of HSD10 was examined in transfected pheochromocytoma cells by growth curve analysis and a xenograft model. Fluctuations in mitochondrial bioenergetics were evaluated by electron transport chain complex enzyme activity assays and energy production. Additionally, the effect of HSD10 on pheochromocytoma resistance to cell death was investigated using TUNEL staining, MTT, and complex IV enzyme activity assays. Results: In this study, we examined the tumor-promoting effect of HSD10 in pheochromocytoma cells. Overexpression of HSD10 increased pheochromocytoma cell growth in both in vitro cell culture and an in vivo xenograft mouse model. The increases in respiratory enzymes and energy generation observed in HSD10-overexpressing cells likely supported the accelerated growth rate observed. Furthermore, cells overexpressing HSD10 were more resistant to oxidative stress-induced perturbation. Conclusions: Our findings demonstrate that overexpression of HSD10 accelerates pheochromocytoma cell growth, enhances cell respiration, and increases cellular resistance to cell death induction. This suggests that blockade of HSD10 may halt and/or prevent cancer growth, thus providing a promising novel target for cancer patients as a screening or therapeutic option

An Improved D-α-Tocopherol-Based Nanocarrier for Targeted Delivery of Doxorubicin with Reversal of Multidrug Resistance

Nanocarriers have recently emerged as an attractive platform for delivery of various types of therapeutics including anticancer agents. Previously, we developed an improved TPGS delivery system (PEG5K-VE2) which demonstrated improved colloidal stability and greater in vivo antitumor activity. Nevertheless, the application of this system is still limited by a relatively low drug loading capacity (DLC). In this study we report that incorporation of a fluorenylmethyloxycarbonyl (Fmoc) motif at the interfacial region of PEG5K-VE2 led to significant improvement of the system through the introduction of an additional mechanism of drug/carrier interaction. Doxorubicin (DOX) could be effectively loaded into PEG5K-Fmoc-VE2 micelles at a DLC of 39.9%, which compares favorably to most reported DOX nanoformulations. In addition, PEG5K-Fmoc-VE2/DOX mixed micelles showed more sustained release of DOX in comparison to the counterpart without Fmoc motif. MTT assay showed that PEG5K-Fmoc-VE2/DOX exerted significantly higher levels of cytotoxicity over DOX, Doxil as well as PEG5K-VE2/DOX in PC-3 and 4T1.2 cells. Cytotoxicity assay with NCI/ADR-RES, a drug resistant cell line, suggested that PEG5K-Fmoc-VE2 may have a potential to reverse the multidrug resistance, which was supported by its inhibition on P-gp ATPase. Pharmacokinetics (PK) and biodistribution studies showed an increased half-life in blood circulation and more effective tumor accumulation for DOX formulated in PEG5K-Fmoc-VE2 micelles. More importantly, DOX-loaded PEG5K-Fmoc-VE2 micelles showed an excellent safety profile with a MTD (~30 mg DOX/kg) that is about 3 times as much as that for free DOX. Finally, superior antitumor activity was demonstrated for PEG5K-Fmoc-VE2/DOX in both drug-sensitive (4T1.2 and PC-3) and drug-resistant (KB 8-5) tumor models compared to DOX, Doxil, and PEG5K-VE2/DOX

A PEG-Fmoc conjugate as a nanocarrier for paclitaxel

We report here that a simple, well-defined, and easy-to-scale up nanocarrier, PEG5000-lysyl-(α-Fmoc-ε-t-Boc-lysine)2 conjugate (PEG-Fmoc), provides high loading capacity, excellent formulation stability and low systemic toxicity for paclitaxel (PTX), a first-line chemotherapeutic agent for various types of cancers. 9-Fluorenylmethoxycarbonyl (Fmoc) was incorporated into the nanocarrier as a functional building block to interact with drug molecules. PEG-Fmoc was synthesized via a three-step synthetic route, and it readily interacted with PTX to form mixed nanomicelles of small particle size (25–30 nm). The PTX loading capacity was about 36%, which stands well among the reported micellar systems. PTX entrapment in this micellar system is achieved largely via an Fmoc/PTX π-π stacking interaction, which was demonstrated by fluorescence quenching studies and 13C-NMR. PTX formulated in PEG-Fmoc micelles demonstrated sustained release kinetics, and in vivo distribution study via near infrared fluorescence imaging demonstrated an effective delivery of Cy5.5-labled PTX to tumor sites. The maximal tolerated dose for PTX/PEG-Fmoc (MTD > 120 mg PTX/kg) is higher than those for most reported PTX formulations, and in vivo therapeutic study exhibited a significantly improved antitumor activity than Taxol, a clinically used formulation of PTX. Our system may hold promise as a simple, safe, and effective delivery system for PTX with a potential for rapid translation into clinical study

Overcoming chemo/radio-resistance of pancreatic cancer by inhibiting STAT3 signaling

Chemo/radio-therapy resistance to the deadly pancreatic cancer is mainly due to the failure to kill pancreatic cancer stem cells (CSCs). Signal transducer and activator of transcription 3 (STAT3) is activated in pancreatic CSCs and, therefore, may be a valid target for overcoming therapeutic resistance. Here we investigated the potential of STAT3 inhibition in sensitizing pancreatic cancer to chemo/radio-therapy. We found that the levels of nuclear pSTAT3 in pancreatic cancer correlated with advanced tumor grade and poor patient outcome. Liposomal delivery of a STAT3 inhibitor FLLL32 (Lip-FLLL32) inhibited STAT3 phosphorylation and STAT3 target genes in pancreatic cancer cells and tumors. Consequently, Lip-FLLL32 suppressed pancreatic cancer cell growth, and exhibited synergetic effects with gemcitabine and radiation treatment in vitro and in vivo. Furthermore, Lip-FLLL32 reduced ALDH1-positive CSC population and modulated several potential stem cell markers. These results demonstrate that Lip-FLLL32 suppresses pancreatic tumor growth and sensitizes pancreatic cancer cells to radiotherapy through inhibition of CSCs in a STAT3-dependent manner. By targeting pancreatic CSCs, Lip-FLLL32 provides a novel strategy for pancreatic cancer therapy via overcoming radioresistance

Tumor suppressive microRNA-137 negatively regulates Musashi-1 and colorectal cancer progression

Stem cell marker, Musashi-1 (MSI1) is over-expressed in many cancer types; however the molecular mechanisms involved in MSI1 over-expression are not well understood. We investigated the microRNA (miRNA) regulation of MSI1 and the implications this regulation plays in colorectal cancer. MicroRNA miR-137 was identified as a MSI1-targeting microRNA by immunoblotting and luciferase reporter assays. MSI1 protein was found to be highly expressed in 79% of primary rectal tumors (n=146), while miR-137 expression was decreased in 84% of the rectal tumor tissues (n=68) compared to paired normal mucosal samples. In addition to reduced MSI1 protein, exogenous expression of miR-137 inhibited cell growth, colony formation, and tumorsphere growth of colon cancer cells. Finally, in vivo studies demonstrated that induction of miR-137 can decrease growth of human colon cancer xenografts. Our results demonstrate that miR-137 acts as a tumor-suppressive miRNA in colorectal cancers and negatively regulates oncogenic MSI1

Identification and Validation of Novel Small Molecule Disruptors of HuR-mRNA Interaction

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3′-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR–ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ~6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR–ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers. NA-binding proteins (RBPs) are critical trans factors that associate with specific cis elements present in mRNAs, thereby regulating the fate of target mRNAs.1 The RBP Hu antigen R (HuR, also known as HuA; Hu references the patient's initials from whom an anti-HuR, autoinflammatory antibody was first isolated2) is a member of the embryonic lethal abnormal vision-like (ELAVL) protein family that binds to adenine- and uridine-rich elements (ARE) mainly located in the mRNA 3′-untranslated region (UTR).1,3,4 HuR is elevated in a broad range of cancer tissues compared with the corresponding normal tissues.5 In early reports, upregulated HuR in brain and colon cancers was linked to the enhanced expression of COX-2, VEGF, TGF-β, IL-8, and other cancer-associated proteins,6,7 Subsequent studies revealed that HuR was broadly overexpressed in virtually all malignancies tested, including cancers of the colon,5,8,9 prostate,10,11 breast,12 brain,6 ovaries,13 pancreas,14 and lung.15 Elevated cytoplasmic accumulation of HuR correlates with high-grade malignancy and serves as a prognostic factor of poor clinical outcome in those cancers.3,4,16 HuR is proposed to play a causal role in tumor development. Cultured carcinoma cells with elevated HuR produced significantly larger tumors than those arising from control populations in a mouse xenograft model,5 while reducing HuR by siRNA or microRNA led to decreased tumor size.5,17 HuR contains three RNA recognition motifs (RRM), of which RRM1 and RRM2 are involved in RNA binding, whereas RRM3 does not contribute to RNA binding but is needed for cooperative assembly of HuR oligomers on RNA.18 Many cytokine and proto-oncogene mRNAs have been identified as containing AREs within their 3′-UTRs, which confer a short mRNA half-life.19 Cytoplasmic binding of HuR to these ARE-containing mRNAs is generally accepted as leading to mRNA stabilization and increased translation.20,21 HuR promotes tumorigenesis by interacting with cancer-associated mRNAs which encode proteins implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis.3,4,16 HuR also promotes the translation of several target mRNAs encoding proteins that are involved in cancer treatment resistance.16,22–24 Taken together, these findings suggest that HuR is an attractive target for developing novel cancer therapies. RBPs have been considered “undruggable targets” due to the lack of a well-defined binding pocket for target mRNA. Indeed, there has globally been limited success in finding small molecules that directly disrupt the HuR interaction with AREs of target mRNAs, with limited reports indicating several active hits arising from screening for HuR inhibitors.25–27 Those reported hits are structurally independent, so they cannot provide information for later structure–activity relationship (SAR) analysis to design more potent and specific HuR inhibitors. Currently, the most potent hit reported (MS-444) acts via inhibition of HuR homodimerization, leading to disruption of the HuR–ARE interaction.25 Here, we try to identify HuR inhibitors, which competitively bind to HuR and directly disrupt the HuR–ARE interaction. In this study, we optimized a fluorescent polarization-based (FP-based) binding assay using human full-length HuR protein and an ARE region of Musashi1 (Msi1) 3′-UTR mRNA. HuR binds to and stabilizes the mRNA of Msi128 allowing for oncogenic overexpression of Msi1 and negative regulation of Numb and adenomatous polyposis coli (APC), which are involved in controlling Notch and Wnt signaling pathways.29 Using this FP-based HTS, we screened a library of ~6000 compounds and identified a set of HuR–ARE disruptors, which were validated by AlphaLISA assay, SPR, RNP IP, and luciferase reporter functional studies. The discovery of these inhibitors and related inactive compounds provides the impetus for rational design of more potent and specific HuR–ARE disruptors

Targeting the interaction between RNA-binding protein HuR and FOXQ1 suppresses breast cancer invasion and metastasis

This work is licensed under a Creative Commons Attribution 4.0 International License.Patients diagnosed with metastatic breast cancer have a dismal 5-year survival rate of only 24%. The RNA-binding protein Hu antigen R (HuR) is upregulated in breast cancer, and elevated cytoplasmic HuR correlates with high-grade tumors and poor clinical outcome of breast cancer. HuR promotes tumorigenesis by regulating numerous proto-oncogenes, growth factors, and cytokines that support major tumor hallmarks including invasion and metastasis. Here, we report a HuR inhibitor KH-3, which potently suppresses breast cancer cell growth and invasion. Furthermore, KH-3 inhibits breast cancer experimental lung metastasis, improves mouse survival, and reduces orthotopic tumor growth. Mechanistically, we identify FOXQ1 as a direct target of HuR. KH-3 disrupts HuR–FOXQ1 mRNA interaction, leading to inhibition of breast cancer invasion. Our study suggests that inhibiting HuR is a promising therapeutic strategy for lethal metastatic breast cancer

Natural product (L)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21WAF−1. We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (−)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (−)-gossypol with the RNA binding pocket of MSI1. We further showed that (−)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (−)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (−)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy