Long-term Musculoskeletal Consequences of Chemotherapy in Pediatric Mice.

Thanks to recent progress in cancer research, most children treated for cancer survive into adulthood. Nevertheless, the long-term consequences of anticancer agents are understudied, especially in the pediatric population. We and others have shown that routinely administered chemotherapeutics drive musculoskeletal alterations, which contribute to increased treatment-related toxicity and long-term morbidity. Yet, the nature and scope of these enduring musculoskeletal defects following anticancer treatments and whether they can potentially impact growth and quality of life in young individuals remain to be elucidated. Here, we aimed at investigating the persistent musculoskeletal consequences of chemotherapy in young (pediatric) mice. Four-week-old male mice were administered a combination of 5-FU, leucovorin, irinotecan (a.k.a., Folfiri) or the vehicle for up to 5 wk. At time of sacrifice, skeletal muscle, bones, and other tissues were collected, processed, and stored for further analyses. In another set of experiments, chemotherapy-treated mice were monitored for up to 4 wk after cessation of treatment. Overall, the growth rate was significantly slower in the chemotherapy-treated animals, resulting in diminished lean and fat mass, as well as significantly smaller skeletal muscles. Interestingly, 4 wk after cessation of the treatment, the animals exposed to chemotherapy showed persistent musculoskeletal defects, including muscle innervation deficits and abnormal mitochondrial homeostasis. Altogether, our data support that anticancer treatments may lead to long-lasting musculoskeletal complications in actively growing pediatric mice and support the need for further studies to determine the mechanisms responsible for these complications, so that new therapies to prevent or diminish chemotherapy-related toxicities can be identified.

Mechanisms of Ovarian Cancer-Associated Cachexia.

Cancer-associated cachexia occurs in 50% to 80% of cancer patients and is responsible for 20% to 30% of cancer-related deaths. Cachexia limits survival and treatment outcomes, and is a major contributor to morbidity and mortality during cancer. Ovarian cancer is one of the leading causes of cancer-related deaths in women, and recent studies have begun to highlight the prevalence and clinical impact of cachexia in this population. Here, we review the existing understanding of cachexia pathophysiology and summarize relevant studies assessing ovarian cancer-associated cachexia in clinical and preclinical studies. In clinical studies, there is increased evidence that reduced skeletal muscle mass and quality associate with worse outcomes in subjects with ovarian cancer. Mouse models of ovarian cancer display cachexia, often characterized by muscle and fat wasting alongside inflammation, although they remain underexplored relative to other cachexia-associated cancer types. Certain soluble factors have been identified and successfully targeted in these models, providing novel therapeutic targets for mitigating cachexia during ovarian cancer. However, given the relatively low number of studies, the translational relevance of these findings is yet to be determined and requires more research. Overall, our current understanding of ovarian cancer-associated cachexia is insufficient and this review highlights the need for future research specifically aimed at exploring mechanisms of ovarian cancer-associated cachexia by using unbiased approaches and animal models representative of the clinical landscape of ovarian cancer.

Depleting Ly6G Positive Myeloid Cells Reduces Pancreatic Cancer-Induced Skeletal Muscle Atrophy.

Immune cells can mount desirable anti-cancer immunity. However, some immune cells can support cancer disease progression. The presence of cancer can lead to production of immature myeloid cells from the bone marrow known as myeloid-derived suppressor cells (MDSCs). The immunosuppressive and pro-tumorigenic effects of MDSCs are well understood. Whether MDSCs are involved in promoting cancer cachexia is not well understood. We orthotopically injected the pancreas of mice with KPC cells or PBS. One group of tumor-bearing mice was treated with an anti-Ly6G antibody that depletes granulocytic MDSCs and neutrophils; the other received a control antibody. Anti-Ly6G treatment delayed body mass loss, reduced tibialis anterior (TA) muscle wasting, abolished TA muscle fiber atrophy, reduced diaphragm muscle fiber atrophy of type IIb and IIx fibers, and reduced atrophic gene expression in the TA muscles. Anti-ly6G treatment resulted in greater than 50% Ly6G+ cell depletion efficiency in the tumors and TA muscles. These data show that, in the orthotopic KPC model, anti-Ly6G treatment reduces the number of Ly6G+ cells in the tumor and skeletal muscle and reduces skeletal muscle atrophy. These data implicate Ly6G+ cells, including granulocytic MDSCs and neutrophils, as possible contributors to the development of pancreatic cancer-induced skeletal muscle wasting.

FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness.

BACKGROUND: Skeletal muscle wasting is a devastating consequence of cancer that affects up to 80% of cancer patients and associates with reduced survival. Herein, we investigated the biological significance of Forkhead box P1 (FoxP1), a transcriptional repressor that we demonstrate is up-regulated in skeletal muscle in multiple models of cancer cachexia and in cachectic cancer patients. METHODS: Inducible, skeletal muscle-specific FoxP1 over-expressing (FoxP1iSkmTg/Tg ) mice were generated through crossing conditional Foxp1a transgenic mice with HSA-MCM mice that express tamoxifen-inducible Cre recombinase under control of the skeletal muscle actin promoter. To determine the requirement of FoxP1 for cancer-induced skeletal muscle wasting, FoxP1-shRNA was packaged and targeted to muscles using AAV9 delivery prior to inoculation of mice with Colon-26 Adenocarcinoma (C26) cells. RESULTS: Up-regulation of FoxP1 in adult skeletal muscle was sufficient to induce features of cachexia, including 15% reduction in body mass (P < 0.05), and a 16-27% reduction in skeletal muscle mass (P < 0.05) that was characterized by a 20% reduction in muscle fibre cross-sectional area of type IIX/B muscle fibres (P = 0.020). Skeletal muscles from FoxP1iSkmTg/Tg mice also showed significant damage and myopathy characterized by the presence of centrally nucleated myofibres, extracellular matrix expansion, and were 19-26% weaker than controls (P < 0.05). Transcriptomic analysis revealed FoxP1 as a potent transcriptional repressor of skeletal muscle gene expression, with enrichment of pathways related to skeletal muscle structure and function, growth signalling, and cell quality control. Because FoxP1 functions, at least in part, as a transcriptional repressor through its interaction with histone deacetylase proteins, we treated FoxP1iSkmTg/Tg mice with Trichostatin A, and found that this completely prevented the loss of muscle mass (p = 0.007) and fibre atrophy (P < 0.001) in the tibialis anterior. In the context of cancer, FoxP1 knockdown blocked the cancer-induced repression of myocyte enhancer factor 2 (MEF2)-target genes critical to muscle differentiation and repair, improved muscle ultrastructure, and attenuated muscle fibre atrophy by 50% (P < 0.05). CONCLUSIONS: In summary, we identify FoxP1 as a novel repressor of skeletal muscle gene expression that is increased in cancer cachexia, whose up-regulation is sufficient to induce skeletal muscle wasting and weakness, and required for the normal wasting response to cancer.

MEF2c-Dependent Downregulation of Myocilin Mediates Cancer-Induced Muscle Wasting and Associates with Cachexia in Patients with Cancer.

Skeletal muscle wasting is a devastating consequence of cancer that contributes to increased complications and poor survival, but is not well understood at the molecular level. Herein, we investigated the role of Myocilin (Myoc), a skeletal muscle hypertrophy-promoting protein that we showed is downregulated in multiple mouse models of cancer cachexia. Loss of Myoc alone was sufficient to induce phenotypes identified in mouse models of cancer cachexia, including muscle fiber atrophy, sarcolemmal fragility, and impaired muscle regeneration. By 18 months of age, mice deficient in Myoc showed significant skeletal muscle remodeling, characterized by increased fat and collagen deposition compared with wild-type mice, thus also supporting Myoc as a regulator of muscle quality. In cancer cachexia models, maintaining skeletal muscle expression of Myoc significantly attenuated muscle loss, while mice lacking Myoc showed enhanced muscle wasting. Furthermore, we identified the myocyte enhancer factor 2 C (MEF2C) transcription factor as a key upstream activator of Myoc whose gain of function significantly deterred cancer-induced muscle wasting and dysfunction in a preclinical model of pancreatic ductal adenocarcinoma (PDAC). Finally, compared with noncancer control patients, MYOC was significantly reduced in skeletal muscle of patients with PDAC defined as cachectic and correlated with MEF2c. These data therefore identify disruptions in MEF2c-dependent transcription of Myoc as a novel mechanism of cancer-associated muscle wasting that is similarly disrupted in muscle of patients with cachectic cancer. SIGNIFICANCE: This work identifies a novel transcriptional mechanism that mediates skeletal muscle wasting in murine models of cancer cachexia that is disrupted in skeletal muscle of patients with cancer exhibiting cachexia.

IL-8 Released from Human Pancreatic Cancer and Tumor-Associated Stromal Cells Signals through a CXCR2-ERK1/2 Axis to Induce Muscle Atrophy.

Tumor-derived cytokines are known to drive the catabolism of host tissues, including skeletal muscle. However, our understanding of the specific cytokines that initiate this process remains incomplete. In the current study, we conducted multiplex analyte profiling of cytokines in conditioned medium (CM) collected from human pancreatic cancer (PC) cells, human tumor-associated stromal (TAS) cells, and their co-culture. Of the factors identified, interleukin-8 (IL-8) is released at high levels from PC cells and PC/TAS co-culture and has previously been associated with low muscle mass in cancer patients. We, therefore, treated C2C12 myotubes with IL-8 which led to the activation of ERK1/2, STAT, and Smad signaling, and induced myotube atrophy. Moreover, the treatment of mice with IL-8 also induced significant muscle wasting, confirming the in vivo relevance of IL-8 on muscle. Mechanistically, IL-8-induced myotube atrophy is inhibited by treatment with the CXCR2 antagonist, SB225002, or by treatment with the ERK1/2 inhibitor, U0126. We further demonstrate that this axis mediates muscle atrophy induced by pancreatic cancer cell CM, as neutralization of IL-8 or treatment with SB225002 or U0126 significantly inhibit CM-induced myotube atrophy. Thus, these data support a key role of IL-8 released from human PC cells in initiating atrophy of muscle cells via CXCR2-ERK1/2.​.