Unfolded protein response at the cross roads of tumourigenesis, oxygen sensing and drug resistance in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. Despite therapeutic advances, long term survival in patients diagnosed with advanced disease is low. Efforts to understand the mechanisms promoting disease progression will likely produce novel therapeutic targets. The unfolded protein response (UPR) is activated when unfolded protein accumulates in the endoplasmic reticulum (ER) upon cellular stress. Constitutive UPR activation is a characteristic of many malignancies. We discuss the accumulating evidence that describes a role for the UPR in ccRCC. Studies focused on UPR signalling may provide compelling avenues for therapeutic intervention in the future.

Maintaining Implementation through Dynamic Adaptations (MIDAS): protocol for a cluster-randomized trial of implementation strategies to optimize and sustain use of evidence-based practices in Veteran Health Administration (VHA) patients.

BACKGROUND: The adoption and sustainment of evidence-based practices (EBPs) is a challenge within many healthcare systems, especially in settings that have already strived but failed to achieve longer-term goals. The Veterans Affairs (VA) Maintaining Implementation through Dynamic Adaptations (MIDAS) Quality Enhancement Research Initiative (QUERI) program was funded as a series of trials to test multi-component implementation strategies to sustain optimal use of three EBPs: (1) a deprescribing approach intended to reduce potentially inappropriate polypharmacy; (2) appropriate dosing and drug selection of direct oral anticoagulants (DOACs); and (3) use of cognitive behavioral therapy as first-line treatment for insomnia before pharmacologic treatment. We describe the design and methods for a harmonized series of cluster-randomized control trials comparing two implementation strategies. METHODS: For each trial, we will recruit 8-12 clinics (24-36 total). All will have access to relevant clinical data to identify patients who may benefit from the target EBP at that clinic and provider. For each trial, clinics will be randomized to one of two implementation strategies to improve the use of the EBPs: (1) individual-level academic detailing (AD) or (2) AD plus the team-based Learn. Engage. Act. PROCESS: (LEAP) quality improvement (QI) learning program. The primary outcomes will be operationalized across the three trials as a patient-level dichotomous response (yes/no) indicating patients with potentially inappropriate medications (PIMs) among those who may benefit from the EBP. This outcome will be computed using month-by-month administrative data. Primary comparison between the two implementation strategies will be analyzed using generalized estimating equations (GEE) with clinic-level monthly (13 to 36 months) percent of PIMs as the dependent variable. Primary comparative endpoint will be at 18 months post-baseline. Each trial will also be analyzed independently. DISCUSSION: MIDAS QUERI trials will focus on fostering sustained use of EBPs that previously had targeted but incomplete implementation. Our implementation approaches are designed to engage frontline clinicians in a dynamic optimization process that integrates the use of actional clinical data and making incremental changes, designed to be feasible within busy clinical settings. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05065502 . Registered October 4, 2021-retrospectively registered.

Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer.

IRE1α is constitutively active in several cancers and can contribute to cancer progression. Activated IRE1α cleaves XBP1 mRNA, a key step in production of the transcription factor XBP1s. In addition, IRE1α cleaves select mRNAs through regulated IRE1α-dependent decay (RIDD). Accumulating evidence implicates IRE1α in the regulation of lipid metabolism. However, the roles of XBP1s and RIDD in this process remain ill-defined. In this study, transcriptome and lipidome profiling of triple negative breast cancer cells subjected to pharmacological inhibition of IRE1α reveals changes in lipid metabolism genes associated with accumulation of triacylglycerols (TAGs). We identify DGAT2 mRNA, encoding the rate-limiting enzyme in TAG biosynthesis, as a RIDD target. Inhibition of IRE1α, leads to DGAT2-dependent accumulation of TAGs in lipid droplets and sensitizes cells to nutritional stress, which is rescued by treatment with the DGAT2 inhibitor PF-06424439. Our results highlight the importance of IRE1α RIDD activity in reprograming cellular lipid metabolism.

The Q61H mutation decouples KRAS from upstream regulation and renders cancer cells resistant to SHP2 inhibitors.

Cancer cells bearing distinct KRAS mutations exhibit variable sensitivity to SHP2 inhibitors (SHP2i). Here we show that cells harboring KRAS Q61H are uniquely resistant to SHP2i, and investigate the underlying mechanisms using biophysics, molecular dynamics, and cell-based approaches. Q61H mutation impairs intrinsic and GAP-mediated GTP hydrolysis, and impedes activation by SOS1, but does not alter tyrosyl phosphorylation. Wild-type and Q61H-mutant KRAS are both phosphorylated by Src on Tyr32 and Tyr64 and dephosphorylated by SHP2, however, SHP2i does not reduce ERK phosphorylation in KRAS Q61H cells. Phosphorylation of wild-type and Gly12-mutant KRAS, which are associated with sensitivity to SHP2i, confers resistance to regulation by GAP and GEF activities and impairs binding to RAF, whereas the near-complete GAP/GEF-resistance of KRAS Q61H remains unaltered, and high-affinity RAF interaction is retained. SHP2 can stimulate KRAS signaling by modulating GEF/GAP activities and dephosphorylating KRAS, processes that fail to regulate signaling of the Q61H mutant.

Side population analysis in clear cell renal cell carcinoma.

Cancer stem cells have an important role in tumour biology. While their identity in haematological malignancies is clearly defined, stem cell identity remains elusive in some solid tumours. Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer, but the identity or existence of ccRCC stem cells remains unknown. We aimed to discern their existence using the widely utilised side population approach in ccRCC cell lines. In all cells tested, a well-defined side population was identified, and cell-based assays suggested stem-like properties. However, limiting dilution assays revealed comparable tumour initiating abilities and tumour histology of side and non-side populations, and single cell RNA-sequencing revealed minimal differences between these populations. The results indicate that the side population approach is not sufficient for cancer stem cell discovery in ccRCC.

The Need for Brevity During Shared Decision Making (SDM) for Cancer Screening: Veterans' Perspectives on an "Everyday SDM" Compromise.

Introduction. Detailed or "full" shared decision making (SDM) about cancer screening is difficult in the primary care setting. Time spent discussing cancer screening is time not spent on other important issues. Given time constraints, brief SDM that is incomplete but addresses key elements may be feasible and acceptable. However, little is known about how patients feel about abbreviated SDM. This study assessed patient perspectives on a compromise solution ("everyday SDM"): 1) primary care provided makes a tailored recommendation, 2) briefly presents qualitative information on key tradeoffs, and 3) conveys full support for decisional autonomy and desires for more information. Methods. We recruited a stratified random sample of Veterans from an academic Veterans Affairs medical center who were eligible for lung cancer screening, oversampling women and minority patients, to attend a 6-hour deliberative focus group. Experts informed participants about cancer screening, factors that influence screening benefits, and the role of patient preferences. Then, facilitator-led small groups elicited patient questions and informed opinions about the everyday SDM proposal, its acceptability, and their recommendations for improvement. Results. Thirty-six Veterans with a heavy smoking history participated (50% male, 83% white). There was a strong consensus that everyday SDM was acceptable if patients were the final deciders and could get more information on request. Participants broadly recommended that clinicians only mention downsides directly related to screening and avoid discussion of potential downstream harms (such as biopsies). Discussion. Although further testing in more diverse populations and different conditions is needed, these patients found the everyday SDM approach to be acceptable for routine lung cancer screening discussions, despite its use of an explicit recommendation and presentation of only qualitative information.

An Emerging Role for the Unfolded Protein Response in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and one of the leading causes of cancer-associated deaths in the world. It is characterised by dismal response rates to conventional therapies. A major challenge in treatment strategies for PDAC is the presence of a dense stroma that surrounds the tumour cells, shielding them from treatment. This unique tumour microenvironment is fuelled by paracrine signalling between pancreatic cancer cells and supporting stromal cell types including the pancreatic stellate cells (PSC). While our molecular understanding of PDAC is improving, there remains a vital need to develop effective, targeted treatments. The unfolded protein response (UPR) is an elaborate signalling network that governs the cellular response to perturbed protein homeostasis in the endoplasmic reticulum (ER) lumen. There is growing evidence that the UPR is constitutively active in PDAC and may contribute to the disease progression and the acquisition of resistance to therapy. Given the importance of the tumour microenvironment and cytokine signalling in PDAC, and an emerging role for the UPR in shaping the tumour microenvironment and in the regulation of cytokines in other cancer types, this review explores the importance of the UPR in PDAC biology and its potential as a therapeutic target in this disease.

NRAS Status Determines Sensitivity to SHP2 Inhibitor Combination Therapies Targeting the RAS-MAPK Pathway in Neuroblastoma.

Survival for high-risk neuroblastoma remains poor and treatment for relapsed disease rarely leads to long-term cures. Large sequencing studies of neuroblastoma tumors from diagnosis have not identified common targetable driver mutations other than the 10% of tumors that harbor mutations in the anaplastic lymphoma kinase (ALK) gene. However, at neuroblastoma recurrence, more frequent mutations in genes in the RAS-MAPK pathway have been detected. The PTPN11-encoded tyrosine phosphatase SHP2 is an activator of the RAS pathway, and we and others have shown that pharmacologic inhibition of SHP2 suppresses the growth of various tumor types harboring KRAS mutations such as pancreatic and lung cancers. Here we report inhibition of growth and downstream RAS-MAPK signaling in neuroblastoma cells in response to treatment with the SHP2 inhibitors SHP099, II-B08, and RMC-4550. However, neuroblastoma cell lines harboring endogenous NRAS Q61K mutation (which is commonly detected at relapse) or isogenic neuroblastoma cells engineered to overexpress NRASQ61K were distinctly resistant to SHP2 inhibitors. Combinations of SHP2 inhibitors with other RAS pathway inhibitors such as trametinib, vemurafenib, and ulixertinib were synergistic and reversed resistance to SHP2 inhibition in neuroblastoma in vitro and in vivo. These results suggest for the first time that combination therapies targeting SHP2 and other components of the RAS-MAPK pathway may be effective against conventional therapy-resistant relapsed neuroblastoma, including those that have acquired NRAS mutations. SIGNIFICANCE: These findings suggest that conventional therapy-resistant, relapsed neuroblastoma may be effectively treated via combined inhibition of SHP2 and MEK or ERK of the RAS-MAPK pathway.

A Hypoxia-Inducible HIF1-GAL3ST1-Sulfatide Axis Enhances ccRCC Immune Evasion via Increased Tumor Cell-Platelet Binding.

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer and the major cause of mortality for individuals with von Hippel-Lindau (VHL) disease. ccRCC is characterized most frequently by inactivation of VHL tumor suppressor protein that mediates degradation of the alpha subunit of the hypoxia-inducible factor (HIF) transcription factor family. HIF has been implicated in disease progression and the aim of this study was to identify novel HIF target genes that may contribute to ccRCC. We show that GAL3ST1, an enzyme that catalyzes the sulfonation of the plasma membrane sulfolipid sulfatide, is among the top 50 upregulated genes in ccRCC tissue relative to matched normal tissue. Increased expression of GAL3ST1 in primary ccRCC correlates with decreased survival. We show that GAL3ST1 is a HIF target gene whose expression is induced upon VHL loss leading to the accumulation of its enzymatic product sulfatide. Notably, platelets bind more efficiently to renal cancer cells with high GAL3ST1-sulfatide expression than to GAL3ST1-sulfatide-negative counterparts, which protects ccRCC cells against natural killer cell-mediated cytotoxicity. These results suggest that GAL3ST1 is a HIF-responsive gene that may contribute to ccRCC development via promoting cancer cell evasion of immune surveillance. IMPLICATIONS: Cancer development is in part dependent on evasion of immune response. We identify a HIF target gene product GAL3ST1 that may play a role in this critical process.

HIF-2α-pVHL complex reveals broad genotype-phenotype correlations in HIF-2α-driven disease.

It is definitively established that mutations in transcription factor HIF-2α are causative of both neuroendocrine tumors (class 1 disease) and polycythemia (class 2 disease). However, the molecular mechanism that underlies this emergent genotype-phenotype relationship has remained unclear. Here, we report the structure of HIF-2α peptide bound to pVHL-elongin B-elongin C (VBC) heterotrimeric complex, which shows topographical demarcation of class 1 and 2 mutations affecting residues predicted, and demonstrated via biophysical analyses, to differentially impact HIF-2α-pVHL interaction interface stability. Concordantly, biochemical experiments showed that class 1 mutations disrupt pVHL affinity to HIF-2α more adversely than class 2 mutations directly or indirectly via impeding PHD2-mediated hydroxylation. These findings suggest that neuroendocrine tumor pathogenesis requires a higher HIF-2α dose than polycythemia, which requires only a mild increase in HIF-2α activity. These biophysical data reveal a structural basis that underlies, and can be used to predict de novo, broad genotype-phenotype correlations in HIF-2α-driven disease.

Pesticides and public health: an analysis of the regulatory approach to assessing the carcinogenicity of glyphosate in the European Union.

The present paper scrutinises the European authorities' assessment of the carcinogenic hazard posed by glyphosate based on Regulation (EC) 1272/2008. We use the authorities' own criteria as a benchmark to analyse their weight of evidence (WoE) approach. Therefore, our analysis goes beyond the comparison of the assessments made by the European Food Safety Authority and the International Agency for Research on Cancer published by others. We show that not classifying glyphosate as a carcinogen by the European authorities, including the European Chemicals Agency, appears to be not consistent with, and in some instances, a direct violation of the applicable guidance and guideline documents. In particular, we criticise an arbitrary attenuation by the authorities of the power of statistical analyses; their disregard of existing dose-response relationships; their unjustified claim that the doses used in the mouse carcinogenicity studies were too high and their contention that the carcinogenic effects were not reproducible by focusing on quantitative and neglecting qualitative reproducibility. Further aspects incorrectly used were historical control data, multisite responses and progression of lesions to malignancy. Contrary to the authorities' evaluations, proper application of statistical methods and WoE criteria inevitably leads to the conclusion that glyphosate is 'probably carcinogenic' (corresponding to category 1B in the European Union).

Consequences of VHL Loss on Global DNA Methylome.

In clear-cell renal cell carcinoma (ccRCC), loss of von Hippel-Lindau (VHL) tumour suppressor gene and reduced oxygen tension promote stabilisation of hypoxia-inducible factor (HIF) family of transcription factors, which promote changes in the expression of genes that contribute to oncogenesis. Multiple studies have demonstrated significant perturbations in DNA methylation in ccRCC via largely unclear mechanisms that modify the transcriptional output of tumour cells. Here, we show that the methylation status of the CpG dinucleotide within the consensus hypoxia-responsive element (HRE) markedly influences the binding of HIF and that the loss of VHL results in significant alterations in the DNA methylome. Surprisingly, hypoxia, which likewise promotes HIF stabilisation and activation, has relatively few effects on global DNA methylation. Gene expression analysis of ccRCC patient samples highlighted expression of a group of genes whose transcription correlated with methylation changes, including hypoxic responsive genes such as VEGF and TGF. These results suggest that the loss of VHL alters DNA methylation profile across the genome, commonly associated with and contributing to ccRCC progression.

Recombinase-Mediated Cassette Exchange Using Adenoviral Vectors.

Site-specific recombinases are important tools for the modification of mammalian genomes. In conjunction with viral vectors, they can be utilized to mediate site-specific gene insertions in animals and in cell lines which are difficult to transfect. Here we describe a method for the generation and analysis of an adenovirus vector supporting a recombinase-mediated cassette exchange reaction and discuss the advantages and limitations of this approach.

Post-mortem computed tomography coaxial cutting needle biopsy to facilitate the detection of bacterioplankton using PCR probes as a diagnostic indicator for drowning.

We report for the first time the use of coaxial cutting needle biopsy, guided by post-mortem computed tomography (PMCT), to sample internal body tissues for bacterioplankton PCR analysis to investigate drowning. This technical report describes the biopsy technique, the comparison of the needle biopsy and the invasive autopsy sampling results, as well as the PMCT and autopsy findings. By using this new biopsy sampling approach for bacterioplankton PCR, we have developed on previous papers describing the minimally invasive PMCT approach for the diagnosis of drowning. When such a system is used, the operator must take all precautions to avoid contamination of the core biopsy samples due to the sensitivity of PCR-based analytic systems.

Alleviation of Epiphora by Canaliculosinostomy into the Caudal Maxillary Sinus in the Horse.

OBJECTIVE: To describe a technique of canaliculosinostomy into the caudal maxillary sinus to alleviate epiphora secondary to nasolacrimal duct obstruction and to report the long-term outcome in 5 horses. STUDY DESIGN: Case series. ANIMALS: Five client-owned horses. METHODS: Case records of all horses presented for chronic epiphora to a single equine hospital that underwent surgical treatment were reviewed. All included horses had a Jones test or dacryocystography to confirm nasolacrimal duct obstruction. All horses were anesthetized and canaliculosinostomy was created from the medial canthus of the eye into the caudal maxillary sinus using a Steinmann pin and Jacob's chuck. A Foley catheter was placed normograde through the stoma. The inflated bulb held the Foley in place in the sinus, while the proximal end was pulled through the upper eyelid and sutured to the skin on the head. The Foley catheter was maintained in place for 3 weeks and then removed under sedation. RESULTS: Five horses were included. There were no intraoperative difficulties or complications. One horse dislodged the Foley catheter 3 days postoperatively. No other postoperative complications occurred. Followup was available for all horses. One horse was euthanatized for unrelated reasons 10 weeks postoperative at which time epiphora was resolved. The remaining 4 horses had resolution of epiphora at followup (24-46 months postoperative). CONCLUSION: This report describes a simple technique for canaliculosinostomy into the caudal maxillary sinus and long-term outcome in 4 of 5 horses, all of which had resolution of epiphora. This technique can be used to resolve epiphora of various etiologies.

The multifaceted von Hippel-Lindau tumour suppressor protein.

Loss of von Hippel-Lindau protein (pVHL) is known to contribute to the initiation and progression of tumours associated with VHL disease as well as certain sporadic tumours including clear cell renal cell carcinoma (ccRCC). The VHL gene was first identified and cloned over 20 years ago and our understanding of its functions and effects has significantly increased since then. The best-known function of pVHL is its role in promoting the degradation of hypoxia-inducible factor α subunit (HIFα) as part of an E3 ubiquitin ligase complex. HIF stabilisation and transcriptional activation are also associated with various epigenetic alterations, indicating a potential role for VHL loss with changes in the epigenome. This review will highlight current knowledge regarding pVHL as well as discuss potentially novel roles of pVHL and how these may impact on cancer progression.

Mammary gland development is delayed in mice deficient for aminopeptidase N.

Development of the mammary gland requires the coordinated action of proteolytic enzymes during two phases of remodelling. Firstly, new ducts and side-branches thereof need to be established during pregnancy to generate an extensive ductal tree allowing the secretion and transport of milk. A second wave of remodelling occurs during mammary involution after weaning. We have analysed the role of the cell surface protease aminopeptidase N (Anpep, APN, CD13) during these processes using Anpep deficient and Anpep over-expressing mice. We find that APN deficiency significantly delays mammary gland morphogenesis during gestation. The defect is characterised by a reduction in alveolar buds and duct branching at mid-pregnancy. Conversely over-expression of Anpep leads to accelerated ductal development. This indicates that Anpep plays a critical role in the proteolytic remodelling of mammary tissue during adult mammary development.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype.

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression. METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter. RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2. CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Recombinase mediated cassette exchange into genomic targets using an adenovirus vector.

Recombinase mediated cassette exchange (RMCE) is a process in which site-specific recombinases exchange one gene cassette flanked by a pair of incompatible target sites for another cassette flanked by an identical pair of sites. Typically one cassette is present in the host genome, whereas the other gene cassette is introduced into the host cell by chemical or biological means. We show here that the frequency of cassette exchange is dependent on the relative and absolute quantities of the transgene cassette and the recombinase. We were able to successfully modify genomic targets not only by electroporation or chemically mediated gene transfer but also by using an adenovirus vector carrying both the transgene cassette to be inserted and the recombinase coding region. RMCE proceeds efficiently in cells in which the adenovirus vector is able to replicate. In contrast, insufficient quantities of the transgene cassette are produced in cells in which the virus cannot replicate. Additional transfection of the transgene cassette significantly enhances the RMCE frequency. This demonstrates that an RMCE system in the context of a viral vector allows the site directed insertion of a transgene into a defined genomic site.