Cancer cachexia (CC) results in impaired muscle function and quality of life and is the primary cause of death for ~20-30% of cancer patients. We demonstrated mitochondrial degeneration as a precursor to CC in male mice, however, if such alterations occur in females is currently unknown. The purpose of this study was to elucidate muscle alterations in CC development in female tumor-bearing mice. 60 female C57BL/6J mice were injected with PBS or Lewis Lung Carcinoma at 8-week age, and tumors developed for 1, 2, 3, or 4 weeks to assess the time course of cachectic development. In vivo muscle contractile function, protein fractional synthetic rate (FSR), protein turnover, and mitochondrial health were assessed. 3- and 4-week tumor-bearing mice displayed a dichotomy in tumor growth and were reassigned to High Tumor (HT) and Low Tumor (LT) groups. HT mice exhibited lower soleus, TA, and fat weights compared to PBS. HT mice showed lower peak isometric torque and slower one-half relaxation time compared to PBS. HT mice had lower FSR compared to PBS while E3 ubiquitin ligases were greater in HT compared to other groups. Bnip3 (mitophagy) and pMitoTimer red puncta (mitochondrial degeneration) were greater in HT while Pgc1α1 and Tfam (mitochondrial biogenesis) were lower in HT compared to PBS. We demonstrate alterations in female tumor-bearing mice where HT exhibited greater protein degradation, impaired muscle contractility, and mitochondrial degeneration compared to other groups. Our data provide novel evidence for a distinct cachectic development in tumor-bearing female mice compared to previous male studies.

microRNAs (miRs) are linked to various human diseases including Type 2 Diabetes Mellitus (T2DM) and emerging evidence suggests miRs may serve as potential therapeutic targets. Lower miR-16 content is consistent across different models of T2DM; however, the role of miR-16 in muscle metabolic health is still elusive. Therefore, the purpose of this study was to investigate how deletion of miR-16 in mice affects skeletal muscle metabolic health and contractile function in both sexes. This study was conducted using both in vitro (1) and in vivo (2) experiments. (1) We utilized C2C12 myoblasts to test if inhibition or overexpression of miR-16 affected insulin-mediated glucose handling. (2) We generated muscle-specific miR-16 knockout (KO) mice fed a high-fat diet (HFD) to assess how miR-16 content impacts metabolic and contractile properties including glucose tolerance, insulin sensitivity, muscle contractile function, protein anabolism, and mitochondrial network health. (1) Although inhibition of miR-16 induced impaired insulin signaling (p=0.002) and glucose uptake (p=0.014), overexpression of miR-16 did not attenuate lipid overload-induced insulin resistance using the diacylglycerol analog 1‐oleoyl‐2‐acetyl‐sn‐glycerol. (2) miR-16 deletion induced both impaired muscle contractility (p=0.031-0.033), and mitochondrial network health (p=0.008-0.018) in both sexes. However, while males specifically exhibited impaired insulin sensitivity following miR-16 deletion (p=0.030), female KO mice showed pronounced glucose intolerance (p=0.046), corresponding with lower muscle weights (p=0.015), and protein hyperanabolism (p=0.023). Our findings suggest distinct sex differences in muscle adaptation in response to miR-16 deletion and miR-16 may serve as a key regulator for metabolic dysregulation in T2DM.

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Background: Cancer-cachexia (CC) is experienced by 80% of cancer patients, representing 40% of cancer-related deaths. Evidence suggests biological sex dimorphism is associated with CC. Assessments of the female transcriptome in CC are lacking and direct comparisons between biological sex are scarce. The purpose of this study was to define the time course of LLC-induced CC in females using transcriptomics, while directly comparing the effects of biological sex. Methods: Eight-week-old female mice were injected with LLC cells (1x106) or sterile PBS to the hind flank. Tumors developed for 1, 2, 3 or 4-weeks. Due to dimorphism between tumor weight in 3- and 4-weeks of development, these were reorganized as low-tumor weight (LT, tumor-weight ≤1.2g), or high-tumor weight (HT, tumor-weight ≥2g). Gastrocnemius muscle was collected for RNA-sequencing (RNA-seq). Differentially expressed genes (DEGs) were defined as FDR<0.05. Data were further compared to RNA-seq of male mice from a previous study. Results: Global gene expression of female gastrocnemius muscle reveals consistent DEGs at all timepoints, all associated with type-II interferon signaling (FDR<0.05). Early transcriptomic upregulation of extracellular-matrix pathways was noted at 1wk (p<0.05), JAK-STAT pathway was upregulated in 2wk, LT, and HT. Type II interferon signaling was downregulated in 1wk, LT, and HT (p<0.05). A second major transcriptomic downregulation in oxidative phosphorylation, electron transport chain and TCA cycle were noted in cachectic (HT) muscle only (p<0.05). Male-female comparison of cachectic groups revealed 69% of DEGs were distinct between sex (FDR<0.05). Comparison of the top 10-up and down DEGs revealed downregulation of type-II Interferon genes was unique to female, while males show upregulation of interferon-signaling pathways. Conclusion: We demonstrate biphasic disruptions in transcriptome of female LLC tumor-bearing mice: an early phase associated with ECM remodeling and a late phase, accompanied by onset of systemic cachexia, affecting overall skeletal muscle energy metabolism. Comparison of cachectic female-male mice reveals ~2/3 of DEGs are biological sex specific, providing evidence of dimorphic mechanisms of cachexia between sexes. Alterations to Type-II Interferon signaling appears specific to CC development in females, suggesting a new biological sex-specific marker of CC. Our data support biological sex dimorphisms in development of CC.

Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under‐investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Female and male C57BL6/J mice were injected with phosphate‐buffered saline or Lewis Lung Carcinoma (LLC) at 8‐week of age, and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor‐bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on cocultured C2C12 and 3T3 cells exposed to LLC conditioned media. Immunohistochemistry and quantitative polymerase chain reaction (qPCR) analysis were used to investigate fibrosis and fibrosis‐related signaling in skeletal muscle. Collagen deposition in skeletal muscle was increased in the 1wk, LT, and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general, female mice displayed earlier alterations in extracellular matrix (ECM)‐related genes beginning at 1wk post‐LLC injection. Whereas this was not seen in males. While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early‐stage alterations in ECM‐related genes contrary to what was noted in female mice.

AIM Cachexia is characterized by losses in lean body mass and its progression results in worsened quality of life and exacerbated outcomes in cancer patients. However, the role and impact of fibrosis during the early stages and development of cachexia in under-investigated. The purpose of this study was to determine if fibrosis occurs during cachexia development, and to evaluate this in both sexes. Methods Female and male C57BL6/J mice were injected with PBS or Lewis Lung Carcinoma (LLC) at 8‐week of age and tumors were allowed to develop for 1, 2, 3, or 4 weeks. 3wk and 4wk female tumor‐bearing mice displayed a dichotomy in tumor growth and were reassigned to high tumor (HT) and low tumor (LT) groups. In vitro analyses were also performed on co-cultured C2C12 and 3T3 exposed to LLC conditioned media. Immunohistochemistry and quantitative PCR analysis were used to investigate fibrosis and fibrosis related signaling in skeletal muscle. Results Collagen deposition in skeletal muscle was increased in the 1wk, LT and HT groups in female mice. However, collagen deposition was only increased in the 4wk group in male mice. In general female mice displayed earlier alterations in ECM related genes beginning at 1 wk post-LLC injection. Whereas this was not seen in males. Conclusions While overall tumor burden is tightly correlated to cachexia development in both sexes, fibrotic development is not. Male mice did not exhibit early-stage alterations in ECM related genes contrary to what was noted in female mice.

Background: Cancer-cachexia is clinically defined by involuntary weight loss >5% in <6 months, primarily affecting skeletal muscle. Here, we aimed to identify sex differences in the onset of colorectal cancer-cachexia with specific consideration to skeletal muscle contractile and metabolic functions. Methods: Eight-weeks old BALB/c mice (69 male, 59 female) received subcutaneous C26 allografts or PBS vehicle. Tumors developed for 10-, 15-, 20-, or 25-days. Muscles and organs were collected, in vivo muscle contractility, protein synthesis rate, mitochondrial function, and protein turnover markers were assessed. One-way ANOVA within sex and trend analysis between sexes were performed, p<0.05. Results: Gastrocnemius and TA muscles became atrophic in male mice at 25-days, while female mice exhibited no significant differences in muscle weights at endpoints despite presenting hallmarks of cancer-cachexia (fat loss, hepatosplenomegaly). We observed lowered muscle contractility and protein synthesis concomitantly to muscle mass decay in males, with higher proteolytic markers in muscles of both sexes. mRNA of Opa1 was lower in TA, while Bnip3 was higher in gastrocnemius after 25-days in male mice, with no significance effect in female mice. Conclusions: Our data suggest relative protections to skeletal muscle in females compared to males despite other canonical signs of cancer-cachexia and increased protein degradation markers; suggesting we should place onus upon non-muscle tissues during early-stages of cancer-cachexia in females. We noted potential protective mechanisms relating to skeletal muscle contractile and mitochondrial functions. Our findings, underline possible heterogeneity in onset of cancer-cachexia between biological sexes, suggesting the need for sex-specific approaches to treat cancer-cachexia.

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