Evaluation of the validity and screening performance of a revised single-item fear of cancer recurrence screening measure (FCR-1r).

OBJECTIVE: Fear of cancer recurrence (FCR) is highly prevalent among cancer survivors, but irregularly identified in practice. Single-item FCR measures suitable for integration into broader psychosocial screening are needed. This study evaluated the validity of a revised version of the original FCR-1 (FCR-1r) and screening performance alongside the Edmonton Symptom Assessment System - Revised (ESAS-r) anxiety item. METHODS: The FCR-1r was adapted from the FCR-1 and modelled on the ESAS-r. Associations between FCR-1r and FCR Inventory-Short Form (FCRI-SF) scores determined concurrent validity. Relationships of FCR-1r scores with variables related (e.g., anxiety, intrusive thoughts) and unrelated (e.g., employment/marital status) to FCR determined convergent and divergent validity respectively. A Receiver-Operating Characteristic analysis examined screening performance and cut-offs for the FCR-1r and ESAS-r anxiety item. RESULTS: 107 participants were recruited in two studies (Study 1, July-October 2021, n = 54; Study 2: November 2021-May 2022, n = 53). The FCR-1r demonstrated concurrent validity against the FCRI-SF (r = 0.83, p < 0.0001) and convergent validity versus the Generalised Anxiety Disorder-7 (r = 0.63, p < 0.0001) and Impact of Event Scale-Revised Intrusion subscale (r = 0.55, p < 0.0001). It did not correlate with unrelated variables (e.g., employment/marital status), indicating divergent validity. An FCR-1r cut-off ≥5/10 had 95% sensitivity and 77% specificity for detecting clinical FCR (area under the curve (AUC) = 0.91, 95% CI 0.85-0.97, p < 0.0001); ESAS-r anxiety cut-off ≥4 had 91% sensitivity and 82% specificity (AUC = 0.87, 95% CI 0.77-0.98, p < 0.0001). CONCLUSIONS: The FCR-1r is a valid and accurate tool for FCR screening. Further evaluation of the screening performance of the FCR-1r versus the ESAS-r anxiety item in routine care is needed.

Study protocol for the evaluation of Fear-Less: a stepped-care program for fear of cancer recurrence in survivors with early-stage disease.

BACKGROUND: Fear of cancer recurrence (FCR) is a significant unmet need amongst cancer survivors and is consistently associated with psychological distress and impaired quality of life. Psychological interventions for FCR, such as ConquerFear, have demonstrated efficacy in reducing FCR and improving emotional wellbeing. Unfortunately, there are barriers to the uptake of evidence-based FCR treatments in clinical practice. A stepped-care FCR treatment model may overcome these barriers and has demonstrated potential in people with advanced melanoma. This study aims to evaluate the acceptability, feasibility, and impact of a stepped-care FCR treatment model (Fear-Less) in people with other cancer types, who have completed treatment with curative intent. METHODS: Sixty people with early-stage cancer (defined as individuals who have received treatment with curative intent and with no metastatic disease) will be screened for FCR using the Fear of Cancer Recurrence Inventory-Short Form (FCRI-SF). Individuals reporting moderate FCR (FCRI-SF between 13 and 21) will be offered a clinician-guided self-management resource; those reporting high FCR (FCRI-SF ≥ 22) will be offered individual therapy according to the ConquerFear protocol. Participants will complete purpose-built evaluation surveys assessing their FCR screening and intervention experiences. Clinicians will also complete a survey regarding their experiences of the treatment model. Fear-Less will be evaluated in terms of (1) acceptability (i.e., patient and clinician experience), (2) feasibility (i.e., referral uptake, treatment adherence, and time taken to screen and deliver interventions), and (3) impact (i.e., pre- to post-intervention FCR changes). DISCUSSION: The Fear-Less stepped-care model is a novel framework for screening FCR and stratifying survivors to the appropriate level of treatment. Our study will provide an indication of whether Fear-Less is a feasible and acceptable FCR model of care amongst survivors with early-stage disease and inform further investigations of this model. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR); ACTRN12622000818730 .

An Evolutionarily Conserved uORF Regulates PGC1α and Oxidative Metabolism in Mice, Flies, and Bluefin Tuna.

Mitochondrial abundance and function are tightly controlled during metabolic adaptation but dysregulated in pathological states such as diabetes, neurodegeneration, cancer, and kidney disease. We show here that translation of PGC1α, a key governor of mitochondrial biogenesis and oxidative metabolism, is negatively regulated by an upstream open reading frame (uORF) in the 5' untranslated region of its gene (PPARGC1A). We find that uORF-mediated translational repression is a feature of PPARGC1A orthologs from human to fly. Strikingly, whereas multiple inhibitory uORFs are broadly present in fish PPARGC1A orthologs, they are completely absent in the Atlantic bluefin tuna, an animal with exceptionally high mitochondrial content. In mice, an engineered mutation disrupting the PPARGC1A uORF increases PGC1α protein levels and oxidative metabolism and confers protection from acute kidney injury. These studies identify a translational regulatory element governing oxidative metabolism and highlight its potential contribution to the evolution of organismal mitochondrial function.

TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.

Because injured mitochondria can accelerate cell death through the elaboration of oxidative free radicals and other mediators, it is striking that proliferator gamma coactivator 1-alpha (PGC1α), a stimulator of increased mitochondrial abundance, protects stressed renal cells instead of potentiating injury. Here we report that PGC1α's induction of lysosomes via transcription factor EB (TFEB) may be pivotal for kidney protection. CRISPR and stable gene transfer showed that PGC1α knockout tubular cells were sensitized to the genotoxic stressor cisplatin whereas transgenic cells were protected. The biosensor mtKeima unexpectedly revealed that cisplatin blunts mitophagy both in cells and mice. PGC1α not only counteracted this effect but also raised basal mitophagy, as did the downstream mediator nicotinamide adenine dinucleotide (NAD+). PGC1α did not consistently affect known autophagy pathways modulated by cisplatin. Instead RNA sequencing identified coordinated regulation of lysosomal biogenesis via TFEB. This effector pathway was sufficiently important that inhibition of TFEB or lysosomes unveiled a striking harmful effect of excess PGC1α in cells and conditional mice. These results uncover an unexpected effect of cisplatin on mitophagy and PGC1α's exquisite reliance on lysosomes for kidney protection. Finally, the data illuminate TFEB as a novel target for renal tubular stress resistance.

De novo NAD+ biosynthetic impairment in acute kidney injury in humans.

Nicotinamide adenine dinucleotide (NAD+) extends longevity in experimental organisms, raising interest in its impact on human health. De novo NAD+ biosynthesis from tryptophan is evolutionarily conserved yet considered supplanted among higher species by biosynthesis from nicotinamide (NAM). Here we show that a bottleneck enzyme in de novo biosynthesis, quinolinate phosphoribosyltransferase (QPRT), defends renal NAD+ and mediates resistance to acute kidney injury (AKI). Following murine AKI, renal NAD+ fell, quinolinate rose, and QPRT declined. QPRT+/- mice exhibited higher quinolinate, lower NAD+, and higher AKI susceptibility. Metabolomics suggested an elevated urinary quinolinate/tryptophan ratio (uQ/T) as an indicator of reduced QPRT. Elevated uQ/T predicted AKI and other adverse outcomes in critically ill patients. A phase 1 placebo-controlled study of oral NAM demonstrated a dose-related increase in circulating NAD+ metabolites. NAM was well tolerated and was associated with less AKI. Therefore, impaired NAD+ biosynthesis may be a feature of high-risk hospitalizations for which NAD+ augmentation could be beneficial.

PGC1α in the kidney.

Acute kidney injury (AKI) arising from diverse etiologies is characterized by mitochondrial dysfunction. The peroxisome proliferator-activated receptor γ coactivator-1alpha (PGC1α), a master regulator of mitochondrial biogenesis, has been shown to be protective in AKI. Interestingly, reduction of PGC1α has also been implicated in the development of diabetic kidney disease and renal fibrosis. The beneficial renal effects of PGC1α make it a prime target for therapeutics aimed at ameliorating AKI, forms of chronic kidney disease (CKD), and their intersection. This review summarizes the current literature on the relationship between renal health and PGC1α and proposes areas of future interest.

Gα12 is required for renal cystogenesis induced by Pkd1 inactivation.

Mutation of PKD1, encoding the protein polycystin-1 (PC1), is the main cause of autosomal dominant polycystic kidney disease (ADPKD). The signaling pathways downstream of PC1 in ADPKD are still not fully understood. Here, we provide genetic evidence for the necessity of Gα12 (encoded by Gna12, hereafter Gα12) for renal cystogenesis induced by Pkd1 knockout. There was no phenotype in mice with deletion of Gα12 (Gα12-/-). Polyinosine-polycytosine (pI:pC)-induced deletion of Pkd1 (Mx1Cre+Pkd1f/fGα12+/+) in 1-week-old mice resulted in multiple kidney cysts by 9 weeks, but the mice with double knockout of Pkd1 and Gα12 (Mx1Cre+Pkd1f/fGα12-/-) had no structural and functional abnormalities in the kidneys. These mice could survive more than one year without kidney abnormalities except multiple hepatic cysts in some mice, which indicates that the effect of Gα12 on cystogenesis is kidney specific. Furthermore, Pkd1 knockout promoted Gα12 activation, which subsequently decreased cell-matrix and cell-cell adhesion by affecting the function of focal adhesion and E-cadherin, respectively. Our results demonstrate that Gα12 is required for the development of kidney cysts induced by Pkd1 mutation in mouse ADPKD.

PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection.

The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischaemia. Acute kidney injury (AKI) affects 3% of all hospitalized patients. Here we show that the mitochondrial biogenesis regulator, PGC1α, is a pivotal determinant of renal recovery from injury by regulating nicotinamide adenine dinucleotide (NAD) biosynthesis. Following renal ischaemia, Pgc1α(-/-) (also known as Ppargc1a(-/-)) mice develop local deficiency of the NAD precursor niacinamide (NAM, also known as nicotinamide), marked fat accumulation, and failure to re-establish normal function. Notably, exogenous NAM improves local NAD levels, fat accumulation, and renal function in post-ischaemic Pgc1α(-/-) mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulate the effects of NAM supplementation, including more local NAD and less fat accumulation with better renal function after ischaemia. PGC1α coordinately upregulates the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuates the de novo pathway. NAM enhances NAD via the enzyme NAMPT and augments production of the fat breakdown product ß-hydroxybutyrate, leading to increased production of prostaglandin PGE2 (ref. 5), a secreted autacoid that maintains renal function. NAM treatment reverses established ischaemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of ß-hydroxybutyrate signalling or prostaglandin production similarly abolishes PGC1α-dependent renoprotection. Given the importance of mitochondrial health in ageing and the function of metabolically active organs, the results implicate NAM and NAD as key effectors for achieving PGC1α-dependent stress resistance.

Gene control of tyrosine kinase TIE2 and vascular manifestations of infections.

Ligands of the endothelial-enriched tunica interna endothelial cell kinase 2 (Tie2) are markedly imbalanced in severe infections associated with vascular leakage, yet regulation of the receptor itself has been understudied in this context. Here, we show that TIE2 gene expression may constitute a novel vascular barrier control mechanism in diverse infections. Tie2 expression declined rapidly in wide-ranging models of leak-associated infections, including anthrax, influenza, malaria, and sepsis. Forced Tie2 suppression sufficed to attenuate barrier function and sensitize endothelium to permeability mediators. Rapid reduction of pulmonary Tie2 in otherwise healthy animals attenuated downstream kinase signaling to the barrier effector vascular endothelial (VE)-cadherin and induced vascular leakage. Compared with wild-type littermates, mice possessing one allele of Tie2 suffered more severe vascular leakage and higher mortality in two different sepsis models. Common genetic variants that influence TIE2 expression were then sought in the HapMap3 cohort. Remarkably, each of the three strongest predicted cis-acting SNPs in HapMap3 was also associated with the risk of acute respiratory distress syndrome (ARDS) in an intensive care unit cohort of 1,614 subjects. The haplotype associated with the highest TIE2 expression conferred a 28% reduction in the risk of ARDS independent of other major clinical variables, including disease severity. In contrast, the most common haplotype was associated with both the lowest TIE2 expression and 31% higher ARDS risk. Together, the results implicate common genetic variation at the TIE2 locus as a determinant of vascular leak-related clinical outcomes from common infections, suggesting new tools to identify individuals at unusual risk for deleterious complications of infection.

Mitochondrial biogenesis in the acutely injured kidney.

Mitochondrial dysfunction within the tubular epithelium has been implicated in the pathogenesis of acute kidney injury. Inflammatory, ischemic, or toxic insults dysregulate mitochondrial dynamics, resulting in mitochondrial swelling, fission, and apoptosis. The coordinated processes of generating healthy mitochondria and clearing damaged organelles may contribute to the preservation and restoration of mitochondrial homeostasis. Emerging literature suggests that a master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor-γ-coactivator-1α (PGC-1α), is highly expressed in the tubular epithelium of the healthy kidney, and its induction during the post-injury period may contribute to functional recovery from acute kidney injury.

H2O2 activates G protein, α 12 to disrupt the junctional complex and enhance ischemia reperfusion injury.

The epithelial cell tight junction separates apical and basolateral domains and is essential for barrier function. Disruption of the tight junction is a hallmark of epithelial cell damage and can lead to end organ damage including renal failure. Herein, we identify Gα12 activation by H(2)O(2) leading to tight junction disruption and demonstrate a critical role for Gα12 activation during bilateral renal ischemia/reperfusion injury. Madin-Darby canine kidney (MDCK) cells with inducible Gα12 (Gα12-MDCK) and silenced Gα12 (shGα12-MDCK) were subjected to ATP depletion/repletion and H(2)O(2)/catalase as models of tight junction disruption and recovery by monitoring transepithelial resistance. In ATP depleted cells, barrier disruption and recovery was not affected by Gα12, but reassembly was accelerated by Gα12 depletion. In contrast, silencing of Gα12 completely protected cells from H(2)O(2)-stimulated barrier disruption, a response that rapidly occurred in control cells. H(2)O(2) activated Src and Rho, and Src inhibition (by PP2), but not Rho (by Y27632), protected cells from H(2)O(2)-mediated barrier disruption. Immunofluorescent and biochemical analysis showed that H(2)O(2) led to increased tyrosine phosphorylation of numerous proteins and altered membrane localization of tight junction proteins through Gα12/Src signaling pathway. Gα12 and Src were activated in vivo during ischemia/reperfusion injury, and transgenic mice with renal tubular QLα12 (activated mutant) expression were delayed in recovery and showed more extensive injury. Conversely, Gα12 knockout mice were nearly completely protected from ischemia/reperfusion injury. Taken together, these studies reveal that ROS stimulates Gα12 to activate injury pathways and identifies a therapeutic target for ameliorating ROS mediated injury.

Gα12 activation in podocytes leads to cumulative changes in glomerular collagen expression, proteinuria and glomerulosclerosis.

Glomerulosclerosis is a common pathological finding that often progresses to renal failure. The mechanisms of chronic kidney disease progression are not well defined, but may include activation of numerous vasoactive and inflammatory pathways. We hypothesized that podocytes are susceptible to filtered plasma components, including hormones and growth factors that stimulate signaling pathways leading to glomerulosclerosis. Gα12 couples to numerous G-protein-coupled receptors (GPCRs) and regulates multiple epithelial responses, including proliferation, apoptosis, permeability and the actin cytoskeleton. Herein, we report that genetic activation of Gα12 in podocytes leads to time-dependent increases in proteinuria and glomerulosclerosis. To mimic activation of Gα12 pathways, constitutively active Gα12 (QL) was conditionally expressed in podocytes using Nphs2-Cre and LacZ/floxed QLα12 transgenic mice. Some QLα12(LacZ+/Cre+) mice developed proteinuria at 4-6 months, and most were proteinuric by 12 months. Proteinuria increased with age, and by 12-14 months, many demonstrated glomerulosclerosis with ultrastructural changes, including foot process fusion and both mesangial and subendothelial deposits. QLα12(LacZ+/Cre+) mice showed no changes in podocyte number, apoptosis, proliferation or Rho/Src activation. Real-time PCR revealed no significant changes in Nphs1, Nphs2, Cd2ap or Trpc6 expression, but Col4a2 message was increased in younger and older mice, while Col4a5 was decreased in older mice. Confocal microscopy revealed disordered collagen IVα1/2 staining in older mice and loss of α5 without changes in other collagen IV subunits. Taken together, these studies suggest that Gα12 activation promotes glomerular injury without podocyte depletion through a novel mechanism regulating collagen (α)IV expression, and supports the notion that glomerular damage may accrue through persistent GPCR activation in podocytes.

PGC-1α promotes recovery after acute kidney injury during systemic inflammation in mice.

Sepsis-associated acute kidney injury (AKI) is a common and morbid condition that is distinguishable from typical ischemic renal injury by its paucity of tubular cell death. The mechanisms underlying renal dysfunction in individuals with sepsis-associated AKI are therefore less clear. Here we have shown that endotoxemia reduces oxygen delivery to the kidney, without changing tissue oxygen levels, suggesting reduced oxygen consumption by the kidney cells. Tubular mitochondria were swollen, and their function was impaired. Expression profiling showed that oxidative phosphorylation genes were selectively suppressed during sepsis-associated AKI and reactivated when global function was normalized. PPARγ coactivator-1α (PGC-1α), a major regulator of mitochondrial biogenesis and metabolism, not only followed this pattern but was proportionally suppressed with the degree of renal impairment. Furthermore, tubular cells had reduced PGC-1α expression and oxygen consumption in response to TNF-α; however, excess PGC-1α reversed the latter effect. Both global and tubule-specific PGC-1α-knockout mice had normal basal renal function but suffered persistent injury following endotoxemia. Our results demonstrate what we believe to be a novel mechanism for sepsis-associated AKI and suggest that PGC-1α induction may be necessary for recovery from this disorder, identifying a potential new target for future therapeutic studies.

Regulation of integrin expression by Gα12: An additional potential mechanism modulating cell attachment.

Integrins regulate cell attachment and migration through interactions with specific proteins in the extra-cellular matrix. Heterotrimeric G proteins are essential signal transduction proteins that intersect with integrin signaling to regulate fundamental cellular behaviors. Although integrin and G protein signaling often act in concert, how these mechanisms interact in epithelial cells has not been extensively studied. We recently reported Gα12 regulation of epithelial cell attachment and migration on collagen-I through α2ß1 integrins (Kong et al. Mol Biol Cell 2009). Activated Gα12 inhibited α2ß1 integrin functions through an inside-out signaling mechanism that involved Rho, Src and protein phosphatases without affecting α2 or ß1 expression. Activated Gα12 prevented tubulogenesis in 3D-MDCK cell cultures and promoted the formation of cystic structures. Herein, we extend these findings to show Gα12-stimulated transcriptional changes in integrin expression that affect MDCK cell attachment. Based on results from a microarray with MDCK cells expressing constitutively active Gα12 (QLα12), we confirmed with real time PCR that expressing QLα12 led to a 4-fold inhibition of α6 mRNA expression. Cell surface expression and total α6 protein was reduced by FACS and immunofluorescence. QLα12 expressing MDCK cells also revealed less attachment to laminin-5, an α6 integrin ligand. Taken together, G proteins regulate integrins through canonical signaling pathways and potentially regulate integrin expression levels to modulate cellular responses in a variety of pathophysiologic conditions including polycystic kidney disease.

Polycystin-1 protein level determines activity of the Galpha12/JNK apoptosis pathway.

Mutations in PKD1 are the most common cause of autosomal dominant polycystic kidney disease (ADPKD). The protein product of PKD1 (polycystin-1 (PC1)) is a large transmembrane protein with a short intracellular C terminus that interacts with numerous signaling molecules, including Galpha(12). Cyst formation in ADPKD results from numerous cellular defects, including abnormal cilia, changes in polarity, and dysregulated apoptosis and proliferation. Recently, we reported increased apoptosis in Madin-Darby canine kidney (MDCK) cells through Galpha(12) stimulation of JNK and degradation of the anti-apoptotic protein Bcl-2 (Yanamadala, V., Negoro, H., Gunaratnam, L., Kong, T., and Denker, B. M. (2007) J. Biol. Chem. 282, 24352-24363). Herein, we confirm this pathway in Galpha(12)-silenced MDCK cells and utilize MDCK cell lines harboring either overexpressed or silenced PC1 to demonstrate that PC1 expression levels determine activity of the JNK/Bcl-2 apoptosis pathway. PC1-overexpressing MDCK cells were resistant to thrombin/Galpha(12)-stimulated apoptosis, JNK activation, and Bcl-2 degradation. In contrast, PC1-silenced MDCK cells displayed enhanced thrombin-induced apoptosis, JNK activity, and Bcl-2 degradation. In pulldown experiments, PC1 bound to Galpha(12), but not the related Galpha(13) subunit, and thrombin-stimulated MDCK cells led to increased interaction of Galpha(12) with the PC1 C terminus. In transient transfection assays, a PC1 C-terminal mutant lacking the G protein-binding domain was uncoupled from PC1-inhibited apoptosis. PC1 expression levels may be increased or decreased in ADPKD, and these findings suggest a mechanism in which levels of PC1 expression modulate Galpha(12)/JNK-stimulated apoptosis. Taken together, these findings are consistent with a set point model in which PC1 expression levels regulate specific G protein signaling pathways important to cyst development.

G alpha 12 inhibits alpha2 beta1 integrin-mediated Madin-Darby canine kidney cell attachment and migration on collagen-I and blocks tubulogenesis.

Regulation of epithelial cell attachment and migration are essential for normal development and maintenance of numerous tissues. G proteins and integrins are critical signaling proteins regulating these processes, yet in polarized cells little is known about the interaction of these pathways. Herein, we demonstrate that G alpha 12 inhibits interaction of MDCK cells with collagen-I, the major ligand for alpha2 beta1 integrin. Activating G alpha 12 (QL point mutation or stimulating endogenous G alpha 12 with thrombin) inhibited focal adhesions and lamellipodia formation and led to impaired cell migration. Consistent with G alpha 12-regulated attachment to collagen-I, G alpha 12-silenced MDCK cells revealed a more adherent phenotype. Inhibiting Rho kinase completely restored normal attachment in G alpha 12-activated cells, and there was partial recovery with inhibition of Src and protein phosphatase pathways. G alpha 12 activation led to decreased phosphorylation of focal adhesion kinase and paxillin with displacement of alpha2 integrin from the focal adhesion protein complex. Using the MDCK cell 3D-tubulogenesis assay, activated G alpha 12 inhibited tubulogenesis and led to the formation of cyst-like structures. Furthermore, G alpha 12-silenced MDCK cells were resistant to thrombin-stimulated cyst development. Taken together, these studies provide direct evidence for G alpha 12-integrin regulation of epithelial cell spreading and migration necessary for normal tubulogenesis.