RNA Sequencing of Carboplatin- and Paclitaxel-Resistant Endometrial Cancer Cells Reveals New Stratification Markers and Molecular Targets for Cancer Treatment.

Despite advances in surgical technique and adjuvant treatment, endometrial cancer has recently seen an increase in incidence and mortality in the USA. The majority of endometrial cancers can be cured by surgery alone or in combination with adjuvant chemo- or radiotherapy; however, a subset of patients experience recurrence for reasons that remain unclear. Recurrence is associated with chemoresistance to carboplatin and paclitaxel and consequentially, high mortality. Understanding the pathways involved in endometrial cancer chemoresistance is paramount for the identification of biomarkers and novel molecular targets for this disease. Here, we generated the first matched pairs of carboplatin-sensitive/carboplatin-resistant and paclitaxel-sensitive/paclitaxel-resistant endometrial cancer cells and subjected them to bulk RNA sequencing analysis. We found that 45 genes are commonly upregulated in carboplatin- and paclitaxel-resistant cells as compared to controls. Of these, the leukemia inhibitory factor, (LIF), the protein tyrosine phosphatase type IVA, member 3 (PTP4A3), and the transforming growth factor beta 1 (TGFB1) showed a highly significant correlation between expression level and endometrial cancer overall survival (OS) and can stratify the 545 endometrial cancer patients in the TCGA cohort into a high-risk and low-risk-cohorts. Additionally, four genes within the 45 upregulated chemoresistance-associated genes are ADAMTS5, MICAL2, STAT5A, and PTP4A3 codes for proteins for which small-molecule inhibitors already exist. We identified these proteins as molecular targets for chemoresistant endometrial cancer and showed that treatment with their correspondent inhibitors effectively killed otherwise chemoresistant cells. Collectively, these findings underline the utility of matched pair of chemosensitive and chemoresistant cancer cells to identify markers for endometrial cancer risk stratification and to serve as a pharmacogenomics model for identification of alternative chemotherapy approaches for treatment of patients with recurrent disease

Stressing the Ubiquitin-Proteasome System without 20S Proteolytic Inhibition Selectively Kills Cervical Cancer Cells

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate, and for specific signaling pathways, notably HPV E6-targeted degradation of p53 and PDZ proteins. Natural compounds with antioxidant properties including flavonoids and triterpenoids hold promise as anticancer agents by interfering with ubiquitin-dependent protein degradation. An increasing body of evidence indicates that their α-β unsaturated carbonyl system is the molecular determinant for inhibition of ubiquitin-mediated protein degradation up-stream of the catalytic sites of the 20S proteasome. Herein we report the identification and characterization of a new class of chalcone-based, potent and cell permeable chemical inhibitors of ubiquitin-dependent protein degradation, and a lead compound RAMB1. RAMB1 inhibits ubiquitin-dependent protein degradation without compromising the catalytic activities of the 20S proteasome, a mechanism distinct from that of Bortezomib. Treatment of cervical cancer cells with RAMB1 triggers unfolded protein responses, including aggresome formation and Hsp90 stabilization, and increases p53 steady state levels. RAMB1 treatment results in activation of lysosomal-dependent degradation pathways as a mechanism to compensate for increasing levels of poly-ubiquitin enriched toxic aggregates. Importantly, RAMB1 synergistically triggers cell death of cervical cancer cells when combined with the lysosome inhibitor Chloroquine

Cellular and Molecular Networking Within the Ecosystem of Cancer Cell Communication via Tunneling Nanotubes

Intercellular communication is vital to the ecosystem of cancer cell organization and invasion. Identification of key cellular cargo and their varied modes of transport are important considerations in understanding the basic mechanisms of cancer cell growth. Gap junctions, exosomes, and apoptotic bodies play key roles as physical modalities in mediating intercellular transport. Tunneling nanotubes (TNTs)—narrow actin-based cytoplasmic extensions—are unique structures that facilitate direct, long distance cell-to-cell transport of cargo, including microRNAs, mitochondria, and a variety of other sub cellular components. The transport of cargo via TNTs occurs between malignant and stromal cells and can lead to changes in gene regulation that propagate the cancer phenotype. More notably, the transfer of these varied molecules almost invariably plays a critical role in the communication between cancer cells themselves in an effort to resist death by chemotherapy and promote the growth and metastases of the primary oncogenic cell. The more traditional definition of “Systems Biology” is the computational and mathematical modeling of complex biological systems. The concept, however, is now used more widely in biology for a variety of contexts, including interdisciplinary fields of study that focus on complex interactions within biological systems and how these interactions give rise to the function and behavior of such systems. In fact, it is imperative to understand and reconstruct components in their native context rather than examining them separately. The long-term objective of evaluating cancer ecosystems in their proper context is to better diagnose, classify, and more accurately predict the outcome of cancer treatment. Communication is essential for the advancement and evolution of the tumor ecosystem. This interplay results in cancer progression. As key mediators of intercellular communication within the tumor ecosystem, TNTs are the central topic of this article

Dienone Compounds: Targets and Pharmacological Responses

The biological responses to dienone compounds with a 1,5-diaryl-3-oxo-1,4-pentadienyl pharmacophore have been studied extensively. Despite their expected general thiol reactivity, these compounds display considerable degrees of tumor cell selectivity. Here we review in vitro and preclinical studies of dienone compounds including b-AP15, VLX1570, RA-9, RA-190, EF24, HO-3867, and MCB-613. A common property of these compounds is their targeting of the ubiquitin-proteasome system (UPS), known to be essential for the viability of tumor cells. Gene expression profiling experiments have shown induction of responses characteristic of UPS inhibition, and experiments using cellular reporter proteins have shown that proteasome inhibition is associated with cell death. Other mechanisms of action such as reactivation of mutant p53, stimulation of steroid receptor coactivators, and induction of protein cross-linking have also been described. Although unsuitable as biological probes due to widespread reactivity, dienone compounds are cytotoxic to apoptosis-resistant tumor cells and show activity in animal tumor models.Funding Agencies|Department of Defense Ovarian Cancer Research ProgramUnited States Department of Defense [OC160377]; Minnesota Ovarian Cancer Alliance; Randy Shaver Cancer Research Funds; NIHUnited States Department of Health &amp; Human ServicesNational Institutes of Health (NIH) - USA [NIGMS R01-GM130800]</p

Deubiquitinating Enzymes in Coronaviruses and Possible Therapeutic Opportunities for COVID-19

Following the outbreak of novel severe acute respiratory syndrome (SARS)-coronavirus (CoV)2, the majority of nations are struggling with countermeasures to fight infection, prevent spread and improve patient survival. Considering that the pandemic is a recent event, no large clinical trials have been possible and since coronavirus specific drug are not yet available, there is no strong consensus on how to treat the coronavirus disease 2019 (COVID-19) associated viral pneumonia. Coronaviruses code for an important multifunctional enzyme named papain-like protease (PLP), that has many roles in pathogenesis. First, PLP is one of the two viral cysteine proteases, along with 3-chymotripsin-like protease, that is responsible for the production of the replicase proteins required for viral replication. Second, its intrinsic deubiquitinating and deISGylating activities serve to antagonize the hosts immune response that would otherwise hinder infection. Both deubiquitinating and deISGylating functions involve the removal of the small regulatory polypeptides, ubiquitin and ISG15, respectively, from target proteins. Ubiquitin modifications can regulate the innate immune response by affecting regulatory proteins, either by altering their stability via the ubiquitin proteasome pathway or by directly regulating their activity. ISG15 is a ubiquitin-like modifier with pleiotropic effects, typically expressed during the host cell immune response. PLP inhibitors have been evaluated during past coronavirus epidemics, and have showed promising results as an antiviral therapy in vitro. In this review, we recapitulate the roles of PLPs in coronavirus infections, report a list of PLP inhibitors and suggest possible therapeutic strategies for COVID-19 treatment, using both clinical and preclinical drugs.Funding agencies: The Department of Defense Ovarian Cancer Research Program GrantOC160377, the Minnesota Ovarian Cancer Alliance, the Randy Shaver Cancer Research Funds and the NIH grant1R01GM130800-01A1 to Martina Bazzaro. This work was supported by the Swedish Research Council to PadraigD’Arcy. This work was supported by Rotary Club Forlì to Valentino Clemente.</p

Drug Development Targeting the Ubiquitin-Proteasome System (UPS) for the Treatment of Human Cancers

Cancer cells are characterized by a higher rate of protein turnover and greater demand for protein homeostasis compared to normal cells. In this scenario, the ubiquitin-proteasome system (UPS), which is responsible for the degradation of over 80% of cellular proteins within mammalian cells, becomes vital to cancer cells, making the UPS a critical target for the discovery of novel cancer therapeutics. This review systematically categorizes all current reported small molecule inhibitors of the various essential components of the UPS, including ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), ubiquitin ligases (E3s), the 20S proteasome catalytic core particle (20S CP) and the 19S proteasome regulatory particles (19S RP), as well as their mechanism/s of action and limitations. We also discuss the immunoproteasome which is considered as a prospective therapeutic target of the next generation of proteasome inhibitors in cancer therapies

Targeting Mitochondrial Metabolism in Clear Cell Carcinoma of the Ovaries

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells; however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs

Simultaneous inhibition of deubiquitinating enzymes (DUBs) and autophagy synergistically kills breast cancer cells

Breast cancer is one of the leading causes of cancer death among women in the United States. Patients expressing the estrogen and progesterone receptor (ER and PR) and human epidermal growth factor 2 (HER-2) tumor markers have favorable prognosis and efficacious therapeutic options. In contrast, tumors that are negative for these markers (triple-negative) have a disproportionate share of morbidity and mortality due to lack of a validated molecular target. Deubiquitinating enzymes (DUBs) are a critical component of ubiquitin-proteasome-system degradation and have been shown to be differentially expressed and activated in a number of cancers, including breast, with their aberrant activity linked to cancer prognosis and clinical outcome. We evaluated the effect of the DUB inhibitors b-AP15 and RA-9 alone and in combination with early- and late-stage lysosomal inhibitors on cell viability in a panel of triple negative breast cancer (TNBC) cell lines. Our results indicate small-molecule DUB inhibitors have a profound effect on TNBC viability and lead to activation of autophagy as a cellular mechanism to compensate for ubiquitin-proteasome-system stress. Treatment with sub-optimal doses of DUB and lysosome inhibitors synergistically kills TNBC cells. This supports the evaluation of DUB inhibition, in combination with lysosomal inhibition, as a therapeutic approach for the treatment of TNBC

Targeting Mitochondria for Treatment of Chemoresistant Ovarian Cancer

Ovarian cancer is the leading cause of death from gynecologic malignancy in the Western world. This is due, in part, to the fact that despite standard treatment of surgery and platinum/paclitaxel most patients recur with ultimately chemoresistant disease. Ovarian cancer is a unique form of solid tumor that develops, metastasizes and recurs in the same space, the abdominal cavity, which becomes a unique microenvironment characterized by ascites, hypoxia and low glucose levels. It is under these conditions that cancer cells adapt and switch to mitochondrial respiration, which becomes crucial to their survival, and therefore an ideal metabolic target for chemoresistant ovarian cancer. Importantly, independent of microenvironmental factors, mitochondria spatial redistribution has been associated to both tumor metastasis and chemoresistance in ovarian cancer while specific sets of genetic mutations have been shown to cause aberrant dependence on mitochondrial pathways in the most aggressive ovarian cancer subtypes. In this review we summarize on targeting mitochondria for treatment of chemoresistant ovarian cancer and current state of understanding of the role of mitochondria respiration in ovarian cancer. We feel this is an important and timely topic given that ovarian cancer remains the deadliest of the gynecological diseases, and that the mitochondrial pathway has recently emerged as critical in sustaining solid tumor progression