WNT-1 Signaling Inhibits Apoptosis by Activating β-Catenin/T Cell Factor–Mediated Transcription

Wnt signaling plays a critical role in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of Wnt signaling, little is known regarding Wnt signaling modification of the cell death machinery. Given that numerous oncogenes transform cells by providing cell survival function, we hypothesized that Wnt signaling may inhibit apoptosis. Here, we report that cells expressing Wnt-1 were resistant to cancer therapy–mediated apoptosis. Wnt-1 signaling inhibited the cytochrome c release and the subsequent caspase-9 activation induced by chemotherapeutic drugs, including both vincristine and vinblastine. Furthermore, we found that Wnt-1–mediated cell survival was dependent on the activation of β-catenin/T cell factor (Tcf) transcription. Inhibition of β-catenin/Tcf transcription by expression of the dominant-negative mutant of Tcf-4 blocked Wnt-1–mediated cell survival and rendered cells sensitive to apoptotic stimuli. These results provide the first demonstration that Wnt-1 inhibits cancer therapy–mediated apoptosis and suggests that Wnt-1 may exhibit its oncogenic potential through a mechanism of anti-apoptosis

Inhibitors of histone deacetylase and MCL-1 synergistically reduce proliferation in malignant melanoma

Melanoma is a skin cancer that arises in melanocytes; it is the fifth most common cancer in the United States with approximately 100,000 new cases per year. Current treatments for malignant melanoma are surgical excision, radiation therapy and systemic therapy; however, the five-year survival rate for patients with stage IV is 29.8%. There is an urgent unmet clinical need to investigate novel treatments for these patients. Panobinostat is an orally available histone deacetylase inhibitor used in several hematologic malignancies, but it was ineffective as a single agent against melanoma in Phase 1. To address the insufficiency of options for melanoma patients, we treated a panel of cultured melanoma cell lines with panobinostat and the novel preclinical MCL-1 inhibitor AZD5991. We hypothesized the addition of AZD5991 (currently in phase 3) would enhance the antiproliferative effect of panobinostat in vitro. MTT and ATP-based proliferation assays demonstrated a significant reduction in proliferation when treated with either panobinostat or AZD5991. Isobologram analysis revealed that much lower concentrations of each drug was required to increase caspase 3/7 activity, induce a panel of Growth and DNA Damage (GADD) gene transcripts, and reduce proliferation when the drugs were added in combination. These in vitro studies revealed that panobinostat and AZD5991 synergistically inhibit melanoma growth. Increased caspase activation and the accumulation of GADD transcripts suggests apoptosis is a key feature of the antiproliferative mechanism. Ongoing studies are focused to further characterize panobinostat/AZD5991- induced cell death and to validate our cell culture observations in patient-derived xenograft models

Strategy for tumor selective disruption of androgen receptor function in the spectrum of prostate cancer

Purpose: Testosterone suppression in prostate cancer (PC) is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for ARdependent growth in various hormone-dependent and castration resistant PC models. The amino terminal domain of AR docks at two sites on ELK1 to co-activate essential growth genes. This study explores the ability of small molecules to disrupt the ELK1-AR interaction in the spectrum of PC, inhibiting AR activity in a manner that would predict functional tumor selectivity. Experimental design: Small molecule drug discovery and extensive biological characterization of a lead compound. Results: We have discovered a lead molecule (KCI807) that selectively disrupts ELK1-dependent promoter activation by wild-type and variant ARs without interfering with ELK1 activation by ERK. KCI807 has an obligatory flavone scaffold and functional hydroxyl groups on C5 and C3'. KCI807 binds to AR, blocking ELK1 binding, and selectively blocks recruitment of AR to chromatin by ELK1. KCI807 primarily affects a subset of AR target growth genes selectively suppressing AR-dependent growth of PC cell lines with a better inhibitory profile than enzalutamide. KCI807 also inhibits in vivo growth of castration/enzalutamide-resistant cell line-derived and patient-derived tumor xenografts. In the rodent model, KCI807 has a plasma half-life of 6h and maintenance of its antitumor effect is limited by self-induced metabolism at its 3'-hydroxyl. Conclusions: The results offer a mechanism-based therapeutic paradigm for disrupting the AR growth-promoting axis in the spectrum of prostate tumors while reducing global suppression of testosterone actions. KCI807 offers a good lead molecule for drug development

Novel Lobophorins Inhibit Oral Cancer Cell Growth and Induce Atf4- and Chop-Dependent Cell Death in Murine Fibroblasts

As part of the International Cooperative Biodiversity Groups (ICBG) Program, we were interested in identifying biologically active unfolded protein response (UPR) inducing compounds from marine microorganisms isolated from Costa Rican biota. With this aim in mind we have now generated more than 33,000 unique prefractionated natural product extracts from marine and terrestrial organisms that have been submitted to the Center of Chemical Genomics (CCG) at the University of Michigan for high throughput screening (HTS). An effective complementary cell-based assay to identify novel modulators of UPR signaling was used for screening extracts. Active fractions were iteratively subjected to reverse-phase HPLC chromatographic analysis, and together with lobophorin A, B, E, and F (1–4), three new lobophorin congeners, designated as CR1 (5), CR2 (6), and CR3 (7) were isolated. Herein, we report that secondary assays revealed that the new lobophorins induced UPR-associated gene expression, inhibited oral squamous cell carcinoma cell growth, and led to UPR-dependent cell death in murine embryonic fibroblast (MEF) cells.International Cooperative Biodiversity Groups/[U01 TW007404]/ICBG/Estados UnidosNational Institutes of Health/[DK042394]/NIH/Estados UnidosNational Institutes of Health/[DK088227]/NIH/Estados UnidosNational Institutes of Health/[HL052173]/NIH/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis

Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells.National Institutes of Health/[DE019678]/NIH/Estados UnidosInternational Cooperative Biodiversity Groups/[U01 TW007404]/ICBG/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Básicas::Centro de Investigaciones en Productos Naturales (CIPRONA)UCR::Vicerrectoría de Docencia::Ciencias Básicas::Facultad de Ciencias::Escuela de Químic

Modulation of NF-kappaB and induction of endoplasmic reticulum stress potentiate chemotherapy -induced apoptosis in oral squamous cell carcinoma.

Squamous cell carcinoma is the major cancer diagnosed in the head and neck and oral cavity. Head and neck squamous cell carcinoma (HNSCC) is a tremendous public health challenge; it is the third most prevalent cancer with only breast and colorectal cancers being more common. Despite technological advances in surgery, radiotherapy and chemotherapy for patients who suffer from head and neck cancer, the survival-rate has remained un-improved in the last two decades indicating our ability to treat patients has reached a plateau. Recent surges in the number of young people who develop HNSCC, and our limited ability to satisfactorily care for those who suffer from this disease have fueled an intense search for new treatment strategies. An increasing body of evidence has indicated that malignant transformation of oral keratinocytes can be modulated by a wide variety of genetic mutations and misregulated cell signaling networks. Many signaling intermediates from these pathways, such as NF-kappaB, are also known to modulate resistance to chemotherapy-induced tumor cell death. The work presented in this dissertation is focused toward the elucidation of novel gene therapy and chemotherapy strategies to manipulate the signaling machinery in malignant oral keratinocytes to potentiate or directly induce cell death. Upon completion of this work we have: (1) provided a molecular basis for gene therapy treatment of head and neck cancer with a super repressor of IkappaBalpha to inhibit NF-kappaB-mediated survival and chemoresistance; (2) demonstrated that the proteasome inhibitor PS-341 (Velcade) induces ER stress and reactive oxygen species to kill HNSCC cell in vitro; and (3) elucidated a mechanism by which ATF-4, induced following PS-341-mediated ER stress, transcriptionally regulates the pro-apoptotic protein Noxa prior to cell death in cisplatin-resistant head and neck cancer cells. Furthermore, we have demonstrated that proteasome inhibition induces cell death through two distinct apoptotic mechanisms. PS-341 simultaneously induced caspase 12-dependent stress-specific apoptosis, and also activated the intrinsic (mitochondrion-mediated) apoptosis pathway. Our work has established that NF-kappaB and ER stress can be modulated to potentiate chemotherapy-induced tumor cell death in head and neck squamous cell carcinoma.Ph.D.DentistryHealth and Environmental SciencesMedicineOncologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/125082/2/3186632.pd

The Mechanisms of Zinc Action as a Potent Anti-Viral Agent: The Clinical Therapeutic Implication in COVID-19

The pandemic of COVID-19 was caused by a novel coronavirus termed as SARS-CoV2 and is still ongoing with high morbidity and mortality rates in the whole world. The pathogenesis of COVID-19 is highly linked with over-active immune and inflammatory responses, leading to activated cytokine storm, which contribute to ARDS with worsen outcome. Currently, there is no effective therapeutic drug for the treatment of COVID-19. Zinc is known to act as an immune modulator, which plays an important role in immune defense system. Recently, zinc has been widely considered as an anti-inflammatory and anti-oxidant agent. Accumulating numbers of studies have revealed that zinc plays an important role in antiviral immunity in several viral infections. Several early clinical trials clearly indicate that zinc treatment remarkably decreased the severity of the upper respiratory infection of rhinovirus in humans. Currently, zinc has been used for the therapeutic intervention of COVID-19 in many different clinical trials. Several clinical studies reveal that zinc treatment using a combination of HCQ and zinc pronouncedly reduced symptom score and the rates of hospital admission and mortality in COVID-19 patients. These data support that zinc might act as an anti-viral agent in the addition to its anti-inflammatory and anti-oxidant properties for the adjuvant therapeutic intervention of COVID-19