Exogenous Oncostatin M induces Cardiac Dysfunction, Musculoskeletal Atrophy, and Fibrosis.

Musculoskeletal diseases such as muscular dystrophy, cachexia, osteoarthritis, and rheumatoid arthritis impair overall physical health and reduce survival. Patients suffer from pain, dysfunction, and dysmobility due to inflammation and fibrosis in bones, muscles, and joints, both locally and systemically. The Interleukin-6 (IL-6) family of cytokines, most notably IL-6, is implicated in musculoskeletal disorders and cachexia. Here we show elevated circulating levels of OSM in murine pancreatic cancer cachexia and evaluate the effects of the IL-6 family member, Oncostatin M (OSM), on muscle and bone using adeno-associated virus (AAV) mediated over-expression of murine OSM in wildtype and IL-6 deficient mice. Initial studies with high titer AAV-OSM injection yielded high circulating OSM and IL-6, thrombocytosis, inflammation, and 60% mortality without muscle loss within 4 days. Subsequently, to mimic OSM levels in cachexia, a lower titer of AAV-OSM was used in wildtype and Il6 null mice, observing effects out to 4 weeks and 12 weeks. AAV-OSM caused muscle atrophy and fibrosis in the gastrocnemius, tibialis anterior, and quadriceps of the injected limb, but these effects were not observed on the non-injected side. In contrast, OSM induced both local and distant trabecular bone loss as shown by reduced bone volume, trabecular number, and thickness, and increased trabecular separation. OSM caused cardiac dysfunction including reduced ejection fraction and reduced fractional shortening. RNA-sequencing of cardiac muscle revealed upregulation of genes related to inflammation and fibrosis. None of these effects were different in IL-6 knockout mice. Thus, OSM induces local muscle atrophy, systemic bone loss, tissue fibrosis, and cardiac dysfunction independently of IL-6, suggesting a role for OSM in musculoskeletal conditions with these characteristics, including cancer cachexia.

Sex specificity of pancreatic cancer cachexia phenotypes, mechanisms, and treatment in mice and humans: role of Activin.

BACKGROUND: Cachexia is frequent, deadly, and untreatable for patients with pancreatic ductal adenocarcinoma (PDAC). The reproductive hormone and cytokine Activin is a mediator of PDAC cachexia, and Activin receptor targeting was clinically tested for cancer cachexia therapy. However, sex-specific manifestations and mechanisms are poorly understood, constraining development of effective treatments. METHODS: Cachexia phenotypes, muscle gene/protein expression, and effects of the Activin blocker ACVR2B/Fc were assessed in LSL-KrasG12D/+ , LSL-Trp53R172H/+ , and Pdx-1-Cre (KPC) mice with autochthonic PDAC. Effects of PDAC and sex hormones were modelled by treating C2C12 myotubes with KPC-cell conditioned medium (CM) and estradiol. Muscle gene expression by RNAseq and change in muscle from serial CT scans were measured in patients with PDAC. RESULTS: Despite equivalent tumour latency (median 17 weeks) and mortality (24.5 weeks), male KPC mice showed earlier and more severe cachexia than females. In early PDAC, male gastrocnemius, quadriceps, and tibialis anterior muscles were reduced (-21.7%, -18.9%, and -20.8%, respectively, all P < 0.001), with only gastrocnemius reduced in females (-16%, P < 0.01). Sex differences disappeared in late PDAC. Plasma Activin A was similarly elevated between sexes throughout, while oestrogen and testosterone levels suggested a virilizing effect of PDAC in females. Estradiol partially protected myotubes from KPC-CM induced atrophy and promoted expression of the potential Activin inhibitor Fstl1. Early-stage female mice showed greater muscle expression of Activin inhibitors Fst, Fstl1, and Fstl3; this sex difference disappeared by late-stage PDAC. ACVR2B/Fc initiated in early PDAC preserved muscle and fat only in male KPC mice, with increases of 41.2%, 52.6%, 39.3%, and 348.8%, respectively, in gastrocnemius, quadriceps, tibialis, and fat pad weights vs. vehicle controls, without effect on tumour. No protection was observed in females. At protein and RNA levels, pro-atrophy pathways were induced more strongly in early-stage males, with sex differences less evident in late-stage disease. As with mass, ACVR2B/Fc blunted atrophy-associated pathways only in males. In patients with resectable PDAC, muscle expression of Activin inhibitors FSTL1, FSLT3, and WFIKKN2/GASP2 were higher in women than men. Overall, among 124 patients on first-line gemcitabine/nab-paclitaxel for PDAC, only men displayed muscle loss (P < 0.001); average muscle wasting in men was greater (-6.63 ± 10.70% vs. -1.62 ± 12.00% mean ± SD, P = 0.038) and more rapid (-0.0098 ± 0.0742%/day vs. -0.0466 ± 0.1066%/day, P = 0.017) than in women. CONCLUSIONS: Pancreatic ductal adenocarcinoma cachexia displays sex-specific phenotypes in mice and humans, with Activin a preferential driver of muscle wasting in males. Sex is a major modulator of cachexia mechanisms. Consideration of sexual dimorphism is essential for discovery and development of effective treatments.

RANKL Blockade Reduces Cachexia and Bone Loss Induced by Non-Metastatic Ovarian Cancer in Mice.

Tumor- and bone-derived soluble factors have been proposed to participate in the alterations of skeletal muscle size and function in cachexia. We previously showed that mice bearing ovarian cancer (OvCa) exhibit cachexia associated with marked bone loss, whereas bone-targeting agents, such as bisphosphonates, are able to preserve muscle mass in animals exposed to anticancer drugs. De-identified CT images and plasma samples from female patients affected with OvCa were used for body composition assessment and quantification of circulating cross-linked C-telopeptide type I (CTX-I) and receptor activator of NF-kB ligand (RANKL), respectively. Female mice bearing ES-2 tumors were used to characterize cancer- and RANKL-associated effects on muscle and bone. Murine C2C12 and human HSMM myotube cultures were used to determine the OvCa- and RANKL-dependent effects on myofiber size. To the extent of isolating new regulators of bone and muscle in cachexia, here we demonstrate that subjects affected with OvCa display evidence of cachexia and increased bone turnover. Similarly, mice carrying OvCa present high RANKL levels. By using in vitro and in vivo experimental models, we found that elevated circulating RANKL is sufficient to cause skeletal muscle atrophy and bone resorption, whereas bone preservation by means of antiresorptive and anti-RANKL treatments concurrently benefit muscle mass and function in cancer cachexia. Altogether, our data contribute to identifying RANKL as a novel therapeutic target for the treatment of musculoskeletal complications associated with RANKL-expressing non-metastatic cancers. © 2021 American Society for Bone and Mineral Research (ASBMR).

Tumor-derived IL-6 and trans-signaling among tumor, fat, and muscle mediate pancreatic cancer cachexia.

Most patients with pancreatic adenocarcinoma (PDAC) suffer cachexia; some do not. To model heterogeneity, we used patient-derived orthotopic xenografts. These phenocopied donor weight loss. Furthermore, muscle wasting correlated with mortality and murine IL-6, and human IL-6 associated with the greatest murine cachexia. In cell culture and mice, PDAC cells elicited adipocyte IL-6 expression and IL-6 plus IL-6 receptor (IL6R) in myocytes and blood. PDAC induced adipocyte lipolysis and muscle steatosis, dysmetabolism, and wasting. Depletion of IL-6 from malignant cells halved adipose wasting and abolished myosteatosis, dysmetabolism, and atrophy. In culture, adipocyte lipolysis required soluble (s)IL6R, while IL-6, sIL6R, or palmitate induced myotube atrophy. PDAC cells activated adipocytes to induce myotube wasting and activated myotubes to induce adipocyte lipolysis. Thus, PDAC cachexia results from tissue crosstalk via a feed-forward, IL-6 trans-signaling loop. Malignant cells signal via IL-6 to muscle and fat, muscle to fat via sIL6R, and fat to muscle via lipids and IL-6, all targetable mechanisms for treatment of cachexia.

Profiling of Adipose and Skeletal Muscle in Human Pancreatic Cancer Cachexia Reveals Distinct Gene Profiles with Convergent Pathways.

The vast majority of patients with pancreatic ductal adenocarcinoma (PDAC) suffer cachexia. Although cachexia results from concurrent loss of adipose and muscle tissue, most studies focus on muscle alone. Emerging data demonstrate the prognostic value of fat loss in cachexia. Here we sought to identify the muscle and adipose gene profiles and pathways regulated in cachexia. Matched rectus abdominis muscle and subcutaneous adipose tissue were obtained at surgery from patients with benign conditions (n = 11) and patients with PDAC (n = 24). Self-reported weight loss and body composition measurements defined cachexia status. Gene profiling was done using ion proton sequencing. Results were queried against external datasets for validation. 961 DE genes were identified from muscle and 2000 from adipose tissue, demonstrating greater response of adipose than muscle. In addition to known cachexia genes such as FOXO1, novel genes from muscle, including PPP1R8 and AEN correlated with cancer weight loss. All the adipose correlated genes including SCGN and EDR17 are novel for PDAC cachexia. Pathway analysis demonstrated shared pathways but largely non-overlapping genes in both tissues. Age related muscle loss predominantly had a distinct gene profiles compared to cachexia. This analysis of matched, externally validate gene expression points to novel targets in cachexia.

Identification of Potential Serum Protein Biomarkers and Pathways for Pancreatic Cancer Cachexia Using an Aptamer-Based Discovery Platform.

Patients with pancreatic ductal adenocarcinoma (PDAC) suffer debilitating and deadly weight loss, known as cachexia. Development of therapies requires biomarkers to diagnose, and monitor cachexia; however, no such markers are in use. Via Somascan, we measured ~1300 plasma proteins in 30 patients with PDAC vs. 11 controls. We found 60 proteins specific to local PDAC, 46 to metastatic, and 67 to presence of >5% cancer weight loss (FC ≥ |1.5|, p ≤ 0.05). Six were common for cancer stage (Up: GDF15, TIMP1, IL1RL1; Down: CCL22, APP, CLEC1B). Four were common for local/cachexia (C1R, PRKCG, ELANE, SOST: all oppositely regulated) and four for metastatic/cachexia (SERPINA6, PDGFRA, PRSS2, PRSS1: all consistently changed), suggesting that stage and cachexia status might be molecularly separable. We found 71 proteins that correlated with cachexia severity via weight loss grade, weight loss, skeletal muscle index and radiodensity (r ≥ |0.50|, p ≤ 0.05), including some known cachexia mediators/markers (LEP, MSTN, ALB) as well as novel proteins (e.g., LYVE1, C7, F2). Pathway, correlation, and upstream regulator analyses identified known (e.g., IL6, proteosome, mitochondrial dysfunction) and novel (e.g., Wnt signaling, NK cells) mechanisms. Overall, this study affords a basis for validation and provides insights into the processes underpinning cancer cachexia.

ACVR2B antagonism as a countermeasure to multi-organ perturbations in metastatic colorectal cancer cachexia.

BACKGROUND: Advanced colorectal cancer (CRC) is often accompanied by the development of liver metastases, as well as cachexia, a multi-organ co-morbidity primarily affecting skeletal (SKM) and cardiac muscles. Activin receptor type 2B (ACVR2B) signalling is known to cause SKM wasting, and its inhibition restores SKM mass and prolongs survival in cancer. Using a recently generated mouse model, here we tested whether ACVR2B blockade could preserve multiple organs, including skeletal and cardiac muscle, in the presence of metastatic CRC. METHODS: NSG male mice (8 weeks old) were injected intrasplenically with HCT116 human CRC cells (mHCT116), while sham-operated animals received saline (n = 5-10 per group). Sham and tumour-bearing mice received weekly injections of ACVR2B/Fc, a synthetic peptide inhibitor of ACVR2B. RESULTS: mHCT116 hosts displayed losses in fat mass ( - 79%, P < 0.0001), bone mass ( - 39%, P < 0.05), and SKM mass (quadriceps: - 22%, P < 0.001), in line with reduced muscle cross-sectional area ( - 24%, P < 0.01) and plantarflexion force ( - 28%, P < 0.05). Further, despite only moderately affected heart size, cardiac function was significantly impaired (ejection fraction %: - 16%, P < 0.0001; fractional shortening %: - 25%, P < 0.0001) in the mHCT116 hosts. Conversely, ACVR2B/Fc preserved fat mass ( + 238%, P < 0.001), bone mass ( + 124%, P < 0.0001), SKM mass (quadriceps: + 31%, P < 0.0001), size (cross-sectional area: + 43%, P < 0.0001) and plantarflexion force ( + 28%, P < 0.05) in tumour hosts. Cardiac function was also completely preserved in tumour hosts receiving ACVR2B/Fc (ejection fraction %: + 19%, P < 0.0001), despite no effect on heart size. RNA sequencing analysis of heart muscle revealed rescue of genes related to cardiac development and contraction in tumour hosts treated with ACVR2B/Fc. CONCLUSIONS: Our metastatic CRC model recapitulates the multi-systemic derangements of cachexia by displaying loss of fat, bone, and SKM along with decreased muscle strength in mHCT116 hosts. Additionally, with evidence of severe cardiac dysfunction, our data support the development of cardiac cachexia in the occurrence of metastatic CRC. Notably, ACVR2B antagonism preserved adipose tissue, bone, and SKM, whereas muscle and cardiac functions were completely maintained upon treatment. Altogether, our observations implicate ACVR2B signalling in the development of multi-organ perturbations in metastatic CRC and further dictate that ACVR2B represents a promising therapeutic target to preserve body composition and functionality in cancer cachexia.

Formation of colorectal liver metastases induces musculoskeletal and metabolic abnormalities consistent with exacerbated cachexia.

Advanced colorectal cancer (CRC) is often accompanied by development of liver metastases (LMs) and skeletal muscle wasting (i.e., cachexia). Despite plaguing the majority of CRC patients, cachexia remains unresolved. By using mice injected with Colon-26 mouse tumors, either subcutaneously (s.c.; C26) or intrasplenically to mimic hepatic dissemination of cancer cells (mC26), here we aimed to further characterize functional, molecular, and metabolic effects on skeletal muscle and examine whether LMs exacerbate CRC-induced cachexia. C26-derived LMs were associated with progressive loss of body weight, as well as with significant reductions in skeletal muscle size and strength, in line with reduced phosphorylation of markers of protein anabolism and enhanced protein catabolism. mC26 hosts showed prevalence of fibers with glycolytic metabolism and enhanced lipid accumulation, consistent with abnormalities of mitochondrial homeostasis and energy metabolism. In a comparison with mice bearing s.c. C26, cachexia appeared exacerbated in the mC26 hosts, as also supported by differentially expressed pathways within skeletal muscle. Overall, our model recapitulates the cachectic phenotype of metastatic CRC and reveals that formation of LMs resulting from CRC exacerbate cancer-induced skeletal muscle wasting by promoting differential gene expression signatures.

Differentially expressed alternatively spliced genes in skeletal muscle from cancer patients with cachexia.

BACKGROUND: Alternative splicing (AS) is a post-transcriptional gene regulatory mechanism that contributes to proteome diversity. Aberrant splicing mechanisms contribute to various cancers and muscle-related conditions such as Duchenne muscular dystrophy. However, dysregulation of AS in cancer cachexia (CC) remains unexplored. Our objectives were (i) to profile alternatively spliced genes (ASGs) on a genome-wide scale and (ii) to identify differentially expressed alternatively spliced genes (DASGs) associated with CC. METHODS: Rectus abdominis muscle biopsies obtained from cancer patients were stratified into cachectic cases (n = 21, classified based on International consensus diagnostic framework for CC) and non-cachectic controls (n = 19, weight stable cancer patients). Human transcriptome array 2.0 was used for profiling ASGs using the total RNA isolated from muscle biopsies. Representative DASG signatures were validated using semi-quantitative RT-PCR. RESULTS: We identified 8960 ASGs, of which 922 DASGs (772 up-regulated and 150 down-regulated) were identified at ≥1.4 fold-change and P < 0.05. Representative DASGs validated by semi-quantitative RT-PCR confirmed the primary findings from the human transcriptome arrays. Identified DASGs were associated with myogenesis, adipogenesis, protein ubiquitination, and inflammation. Up to 10% of the DASGs exhibited cassette exon (exon included or skipped) as a predominant form of AS event. We also observed other forms of AS events such as intron retention, alternate promoters. CONCLUSIONS: Overall, we have, for the first time, conducted global profiling of muscle tissue to identify DASGs associated with CC. The mechanistic roles of the identified DASGs in CC pathophysiology using model systems is warranted, as well as replication of findings in independent cohorts.

Small RNAome profiling from human skeletal muscle: novel miRNAs and their targets associated with cancer cachexia.

BACKGROUND: MicroRNAs (miRs) are small non-coding RNAs that regulate gene (mRNA) expression. Although the pathological role of miRs have been studied in muscle wasting conditions such as myotonic and muscular dystrophy, their roles in cancer cachexia (CC) are still emerging. OBJECTIVES: The objectives are (i) to profile human skeletal muscle expressed miRs; (ii) to identify differentially expressed (DE) miRs between cachectic and non-cachectic cancer patients; (iii) to identify mRNA targets for the DE miRs to gain mechanistic insights; and (iv) to investigate if miRs show potential prognostic and predictive value. METHODS: Study subjects were classified based on the international consensus diagnostic criteria for CC. Forty-two cancer patients were included, of which 22 were cachectic cases and 20 were non-cachectic cancer controls. Total RNA isolated from muscle biopsies were subjected to next-generation sequencing. RESULTS: A total of 777 miRs were profiled, and 82 miRs with read counts of ≥5 in 80% of samples were retained for analysis. We identified eight DE miRs (up-regulated, fold change of ≥1.4 at P < 0.05). A total of 191 potential mRNA targets were identified for the DE miRs using previously described human skeletal muscle mRNA expression data (n = 90), and a majority of them were also confirmed in an independent mRNA transcriptome dataset. Ingenuity pathway analysis identified pathways related to myogenesis and inflammation. qRT-PCR analysis of representative miRs showed similar direction of effect (P < 0.05), as observed in next-generation sequencing. The identified miRs also showed prognostic and predictive value. CONCLUSIONS: In all, we identified eight novel miRs associated with CC.

A Genome-Wide Association Study to Identify Potential Germline Copy Number Variants for Sporadic Breast Cancer Susceptibility.

Breast cancer (BC) predisposition in populations arises from both genetic and nongenetic risk factors. Structural variations such as copy number variations (CNVs) are heritable determinants for disease susceptibility. The primary objectives of this study are (1) to identify CNVs associated with sporadic BC using a genome-wide association study (GWAS) design; (2) to utilize 2 distinct CNV calling algorithms to identify concordant CNVs as a strategy to reduce false positive associations in the hypothesis-generating GWAS discovery phase, and (3) to identify potential candidate CNVs for follow-up replication studies. We used Affymetrix SNP Array 6.0 data profiled on Caucasian subjects (422 cases/348 controls) to call CNVs using algorithms implemented in Nexus Copy Number and Partek Genomics Suite software. Nexus algorithm identified CNVs associated with BC (731 autosomal CNVs with >5% frequency in the total sample and Q < 0.05). Thirteen CNVs were identified when Partek algorithm-called CNVs were overlapped with Nexus-identified CNVs; these CNVs showed concordances for frequency, effect size, and direction. Coding genes present within BC-associated CNVs were known to play a role in disease etiology and prognosis. Long noncoding RNAs identified within CNVs showed tissue-specific expression, indicating potential functional relevance of the findings. The identified candidate CNVs warrant independent replication.

Next generation sequencing profiling identifies miR-574-3p and miR-660-5p as potential novel prognostic markers for breast cancer.

BACKGROUND: Prognostication of Breast Cancer (BC) relies largely on traditional clinical factors and biomarkers such as hormone or growth factor receptors. Due to their suboptimal specificities, it is challenging to accurately identify the subset of patients who are likely to undergo recurrence and there remains a major need for markers of higher utility to guide therapeutic decisions. MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators of gene expression and have shown promise as potential prognostic markers in several cancer types including BC. RESULTS: In our study, we sequenced miRNAs from 104 BC samples and 11 apparently healthy normal (reduction mammoplasty) breast tissues. We used Case-control (CC) and Case-only (CO) statistical paradigm to identify prognostic markers. Cox-proportional hazards regression model was employed and risk score analysis was performed to identify miRNA signature independent of potential confounders. Representative miRNAs were validated using qRT-PCR. Gene targets for prognostic miRNAs were identified using in silico predictions and in-house BC transcriptome dataset. Gene ontology terms were identified using DAVID bioinformatics v6.7. A total of 1,423 miRNAs were captured. In the CC approach, 126 miRNAs were retained with predetermined criteria for good read counts, from which 80 miRNAs were differentially expressed. Of these, four and two miRNAs were significant for Overall Survival (OS) and Recurrence Free Survival (RFS), respectively. In the CO approach, from 147 miRNAs retained after filtering, 11 and 4 miRNAs were significant for OS and RFS, respectively. In both the approaches, the risk scores were significant after adjusting for potential confounders. The miRNAs associated with OS identified in our cohort were validated using an external dataset from The Cancer Genome Atlas (TCGA) project. Targets for the identified miRNAs were enriched for cell proliferation, invasion and migration. CONCLUSIONS: The study identified twelve non-redundant miRNAs associated with OS and/or RFS. These signatures include those that were reported by others in BC or other cancers. Importantly we report for the first time two new candidate miRNAs (miR-574-3p and miR-660-5p) as promising prognostic markers. Independent validation of signatures (for OS) using an external dataset from TCGA further strengthened the study findings.