Chemotherapy-Induced Molecular Changes in Skeletal Muscle

Paraneoplastic conditions such as cancer cachexia are often exacerbated by chemotherapy, which affects the patient’s quality of life as well as the response to therapy. The aim of this narrative review was to overview the body-composition-related changes and molecular effects of different chemotherapy agents used in cancer treatment on skeletal-muscle remodeling. A literature search was performed using the Web of Science, Scopus, and Science Direct databases and a total of 77 papers was retrieved. In general, the literature survey showed that the molecular changes induced by chemotherapy in skeletal muscle have been studied mainly in animal models and mostly in non-tumor-bearing rodents, whereas clinical studies have essentially assessed changes in body composition by computerized tomography. Data from preclinical studies showed that chemotherapy modulates several molecular pathways in skeletal muscle, including the ubiquitin–proteasome pathway, autophagy, IGF-1/PI3K/Akt/mTOR, IL-6/JAK/STAT, and NF-κB pathway; however, the newest chemotherapy agents are underexplored. In conclusion, chemotherapy exacerbates skeletal-muscle wasting in cancer patients; however, the incomplete characterization of the chemotherapy-related molecular effects on skeletal muscle makes the development of new preventive anti-wasting strategies difficult. Therefore, further investigation on molecular mechanisms and clinical studies are necessary.


Introduction
According to the International Agency for Research on Cancer, there were 19.3 million new cancer cases worldwide in 2020 [1]. Cancer incidence is expected to increase by 56.7% over the next two decades, with 30.2 million cases estimated in 2040 [2]. Cancer has been associated with muscle wasting, which is often exacerbated by chemotherapy, one of the most common treatments [3]. Despite the plethora of benefits presented by chemotherapy regimens, there are still significant side effects to consider, which affect people differently. Nausea, loss of appetite, tiredness, fatigue, and weight loss are some of the side effects often experienced by patients undergoing chemotherapy treatments [4][5][6]. Some of these side effects are explained by the impact of chemotherapy on skeletal muscle (SkM) [7,8]. For instance, cancer patients receiving chemotherapy often experience body-weight loss, which can be attributed to SkM wasting. Such SkM loss may further exacerbate cancer-associated cachexia, a multifactorial syndrome accompanied by systemic inflammation, metabolic disarrangement, anorexia, and insulin resistance. This syndrome affects up to 80% of cancer patients [9,10]. ↓ skeletal muscle, CSA, IRS-1, and GSK3-b mRNA, GLUT4, AMPK α (pT172) levels, activity of mitochondrial complex 3, IL-10 levels ↑ AST, uric acid, corticosterone/testosterone ratio, insulin, glucose, FFA, activity of mitochondrial complex 1 = IL-6 and TNF-α levels [17] 12 mg/kg ( ↓ absolute respiratory sensitivity, membrane potential, specific force, fatigue resistance, rate of sarcoplasmic reticulum Ca 2+ uptake ↑ soleus half-relaxation time [19]

Overview of Selected Studies
In vitro studies (Table A1) [58,[87][88][89] on the effect of chemotherapy in cell lines or in ex vivo muscle tested different chemotherapeutic agents and doses, making it difficult to establish a response pattern to chemotherapy. Regarding preclinical studies [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32], the majority used male mice. From the 20 articles listed in Table 1, one [14] reported the effects of chemotherapy on knockout mice for the tumor-necrosis-factor receptor 1 (TNFR1) and only five focused on the effects of chemotherapy in animals with cancer (commonly C26 adenocarcinoma-cell inoculation). Indeed, most of the studies (n = 20) herein reviewed evaluated the effects of chemotherapy in healthy animals, making it difficult to translate the molecular findings to the clinical-oncology setting. The most studied chemotherapeutic agent was doxorubicin (7 papers), a potent anticancer drug known for its dose-dependent toxicity in many organs, including the heart and SkM [90]. The most studied SkM was the extensor digitorum longus (EDL) muscle, a fast-twitch muscle; however, other muscles, such as the slow-twitch soleus, the mixed-muscle gastrocnemius, and the fast-twitch quadriceps were also analyzed. Differences in both morphological and molecular changes induced by chemotherapy reported among studies (Table 1) can be related to the different chemotherapeutic agent, dosages, animal model and age, and SkM analyzed.
Among clinical studies, 38 focused on the effect of neoadjuvant chemotherapy or chemoradiation, 4 on adjuvant therapy, 4 involved palliative care, and 8 studies did not mention the moment of chemotherapy. Esophageal cancer was the most common cancer studied, followed by colorectal and gastric, pancreatic and lung, ovarian, breast and esophagogastric, and lastly, nasopharyngeal, cervical, endometrial, muscle-invasive bladder, and biliary-tract cancer. Differences between and within studies were found for other factors such as stage of cancer, number of chemotherapy cycles, duration of treatment, and type of treatment, as observed in Table 2, which made data interpretation and integration difficult.

Effects of Chemotherapy on Skeletal-Muscle Mass and Function
Loss of muscle mass and function negatively impacts the quality of life of cancer patients and is a marker of poor prognosis and survival [11,12,91]. In addition, SkM loss is also associated with reduced tolerance to anticancer treatments and exercise intolerance (fatigue) [11,12,91]. In most clinical studies (Table 2 [ ), SkM measurements rely on multifrequency bioelectrical impedance and mostly on computerized tomography (CT), a technique widely used for clinical purposes because it allows for the visualization of muscle cross-sections [92]. These studies showed that SkM index (SMI) and psoas muscle index (PMI) decreased in patients submitted to chemotherapy, independently of the drugs used. However, the SkM area (SMA) was reported to increase in one study published by Heus et al. [42]. This result was unexpected; however, chemoradiation was used in this study. Although the muscles measured were not in the direct field of radiation, chemoradiation may have decreased the inflammatory state, preventing SkM mass loss [42]. In addition, the lumbar SkM index (LSMI) seems to vary with the chemotherapeutic regimen used; it seems to be higher when patients are treated with paclitaxel [64] and lower in patients treated with different chemotherapy drugs [37,62,64]. All these bodycomposition-related indexes, such as SMI, can be useful as prognostic factors for overall survival [93]. Nonetheless, the application of CT scans and other imaging approaches in the analysis of body composition of cancer patients for the evaluation of SkM wasting is unrealistic, not only due to difficulty of data interpretation in a busy clinical setting but also due to its high costs. In clinical studies, SkM function has been assessed by hand-grip strength, which was diminished in patients with esophageal cancer and non-small-cell lung cancer treated with multiple chemotherapy therapies [48,49]. Additionally, a cross-sectional area of vastus lateralis was reported to be diminished with a lower cross-sectional area of type II fibers in breast-cancer patients [58].
In preclinical studies, EDL, soleus, gastrocnemius, and quadriceps muscles were analyzed, and loss of mass was reported for all these muscles following chemotherapy, which resulted, in most of the studies, in a lower body weight, and in some cases, diminished food intake was reported [21,26]. However, when sorafenib [25] and FOLFIRI [32] were administered, the mass of gastrocnemius and tibialis anterior increased [25,32]. The specific force of soleus [24] and maximal specific force of EDL were decreased [14], as well as average speed (which was measured using a software that processed beam breaks quantified by using locomotion and rearing records) [23]. Diminished SkM function assessed by the forelimb-grip strength was reported in healthy and tumor-bearing animals treated with FOLFIRI [32]. SkM-phenotype remodeling was reported since the ratio of glycolytic to oxidative fibers in quadriceps muscles increased [31], suggesting an enhanced susceptibility to fatigue following treatment with FOLFIRI or FOLFOX, and a decrease in quadriceps myofiber diameter after cisplatin treatment was also stated [21]. Glycolytic fibers are particularly susceptible to cancer-induced atrophy, as observed in muscle biopsies from human cancer patients [94]. Consequently, it is expected that chemotherapy will worsen the atrophy of type II fibers in these patients. Nevertheless, to the best of our knowledge, no preclinical studies have examined the effects of chemotherapy on cancer-induced muscle-fiber atrophy.

Molecular Impact of Chemotherapy on Skeletal Muscle
During and following chemotherapy, catabolic pathways seem to overcome the anabolic ones, potentiating the muscle wasting often observed in cancer patients. The following subsections overview the signaling pathways modulated in SkM by different anticancer agents from an integrated perspective. In overall, SkM mass is regulated by distinct pathways, namely, the catabolic ubiquitin-proteasome pathway (UPP), the autophagylysosome pathway (ALP), the myostatin/high-affinity type-2 activin receptor (ActRIIB) pathway, and the anabolic insulin-like growth factor 1 (IGF-1)/phosphatidylinositol-3kinase (PI3K)/Akt (or protein kinase B, PKB)/mammalian target of rapamycin (mTOR) pathway [10,95]. Both muscle wasting and growth result from the balance between these pathways in favor of catabolic or anabolic processes, respectively [10,[95][96][97]. Other sig-naling pathways contribute to chemotherapy-induced SkM remodeling like metabolism reprogramming, satellite-cell activation, and inflammation-associated pathways, such as interleukin 6 (IL-6)/Janus kinase-signal transducer and the activator of transcription protein (JAK/STAT) and nuclear-factor kappa-light-chain enhancer of activated B cells (NF-κB).

Metabolic Reprogramming
Following chemotherapy, the metabolism becomes less reliant on mitochondria for energy generation [98], reflected in the decreased density (given the citrate synthase (CS) activity) and functionality of this organelle and a more glycolytic phenotype. This metabolic switching was reported in quadriceps muscle from male mice treated with FOLFIRI and FOLFOX [31]. In vastus lateralis of breast-cancer patients, a lower average of mitochondrial area, particularly of the intermyofibrillar mitochondria subpopulation, was reported; however, the number of mitochondria evaluated by fluorometric dyes was not affected by chemotherapy [58]. Mitochondrial density reflects the balance between biogenesis and clearance by mitophagy. The expression of peroxisome proliferator-activated receptorgamma coactivator 1 alpha (PGC1α), a master player of mitochondrial biogenesis, was negatively impacted in the quadriceps of male mice following FOLFIRI and FOLFOX [31]. Still, unchanged levels of this coactivator were seen in soleus and EDL muscles from male mice treated with 5-fluorouracil (5-FU) [27]. PGC1α transcriptionally regulates the mitochondrial transcription factor A (TFAM), which is necessary for mtDNA maintenance [99]. Yet, TFAM levels were also found to be unchanged in soleus and EDL muscles [27]. Regarding mitophagy, decreased levels of parkin, an E3 ligase that synergistically acts with PTEN-induced kinase 1 (PINK1) for mitochondria engulfment by autophagosomes, was reported in lower-limb muscles treated with a combination of doxorubicin and dexamethasone [28]. Nonetheless, no changes in the levels of the mitochondria-fusion protein OPA-1 and -fission protein DRP-1 were observed [27]. Down-regulation of mitophagy may lead to the accumulation of dysfunctional mitochondria once no changes in biogenesis seem to occur and the density of this organelle is maintained. In fact, a significant decrease in ATP production was reported in the tibialis anterior of both non-tumor-bearing and tumor-bearing male mice treated with FOLFIRI [32]. Nevertheless, when the activity of mitochondrial oxidative phosphorylation (OXPHOS) complexes was measured in EDL of male rats, complex I activity was increased whereas complex III was decreased following treatment with doxorubicin [17]. Succinate dehydrogenase (SDH) activity and cytochrome c content were also diminished [31], suggesting a decreased oxidative capacity of SkM.
SkM is highly reliant on free fatty acids for energetic purposes. Fatty acids are oxidized in mitochondria and, thus, diminished fatty-acid oxidation (FAO) can be expected given the lower content of functional mitochondria in treated SkM. On the other hand, some proteins related to fatty-acid metabolism were reported to be upregulated by chemotherapy. The content of fatty-acid-binding protein (FABP) 1 increased in the quadriceps of male mice treated with FOLFIRI [31]. This protein belongs to the FABPs family, whose main function is to facilitate the transport of long-chain free fatty acids into the cell [100]. Moreover, the expression of stearoyl-CoA desaturase-2 (SCD2), one of the enzymes responsible for monounsaturated-fatty-acid synthesis [101], was also found to be increased in the quadriceps [31], suggesting increased fatty-acid production in wasted muscle. Additionally, the expression of acyl-CoA thioesterase (Acot) 2, which hydrolyzes coenzyme A (CoA) esters into free fatty acid and CoA, was diminished in the quadriceps after FOLFIRI treatment [31]. If not balanced by FAO, increased accumulation of free fatty acids and, eventually, of triglycerides can be presumed in wasted SkM. In fact, impaired FAO due to altered mitochondrial function was already associated with the accumulation of intramyocellular lipid droplets in muscle fibers of cachectic-cancer patients [102]. Other proteins associated with lipid metabolism were reported to be modulated by chemotherapy, including the expression of the apolipoproteins A and B (Apoa1, Apoa2, and Apob), essential in cholesterol metabolism, which were observed to be upregulated in the quadriceps muscles [31].
Amino acids derived from SkM proteolysis can also support energy generation [103]. Enhanced oxidation of branched-chain amino acids (BCAA; meaning Leu, Ile, and Val) in SkM was associated with the development of muscle wasting in cachectic subjects [104]. Still, BCAAs play a role in SkM that goes beyond energetic metabolism. BCAAs, particularly Leu, may activate mTORC1 signaling, boosting protein synthesis in SkM [105]. However, no changes in the levels of BCCAs induced by FOLFIRI were observed [32].
The accumulation of dysfunctional mitochondrial makes SkM more reliant on glucose oxidation for ATP generation. Thus, the glucose-uptake and -glycolysis rate should increase. Insulin regulates glucose uptake in SkM by promoting, among other cellular processes, glucose transporter type 4 (GLUT4) translocation from intracellular vesicles to the sarcolemma. However, GLUT4 content was found to be decreased in male rats' EDL when treated with doxorubicin [17], which may indicate impaired insulin signaling, as supported by the observed diminished content of IGF-1 tyrosine-receptor insulin-receptor substrate 1 (IRS-1) [17]. In this same study, the levels of phosphorylated 5 AMP-activated protein kinase (AMPK), another regulator of GLUT4 translocation, were found to be downregulated [17]. Still, the expression of mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), known to be activated by insulin, increased in male mice's quadriceps after FOLFIRI and FOLFOX [31]. Moreover, the levels of lactate were unchanged, whereas the activity of pyruvate dehydrogenase (PDH) complex, which converts pyruvate into acetyl-CoA, decreased in the tibialis anterior from healthy and tumor-bearing male mice treated with FOLFIRI [32]. The activity of the glycolytic enzyme hexokinase was unchanged in the tibialis anterior from healthy mice but decreased in the same muscle from mice with C26 adenocarcinoma [32]. Therefore, these results do not support an increased reliance on glycolysis in wasted SkM, despite lower density of functional mitochondria. Taken together, data suggest that anticancer drugs promote the accumulation of dysfunctional mitochondria in SkM and an overall decrease in its metabolic rate characterized by decreased oxidative capacity, glycolysis, and lipid accumulation.

IGF-1/PI3K/Akt/mTOR Pathway
The IGF-1/PI3K/Akt/mTOR pathway is known to stimulate protein synthesis in SkM [10,106,107]. This signaling mechanism is downregulated by most of the cytotoxic agents used in chemotherapy. Cisplatin plus 5-FU plus leucovorin was shown to reduce the levels of the downstream target of the IRS-1 [88] and doxorubicin downregulated IRS-1 expression [17], whereas IGF-1, the triggering player of this pathway, was reported to be diminished by cisplatin [21]. The downregulation of the downstream player Akt was observed in some animal studies using different anticancer drugs [21,27,31] and in in vitro studies [88,89], resulting in reduced phosphorylation of mTOR and, eventually, of p70S6 kinase (p70S6K) and eukaryotic translation-initiation factor 4E binding protein 1 (4E-BP1). Indeed, decreased phosphorylation of these proteins was observed in L6 skeletal myoblasts [88]. mTOR may regulate SkM mass through two complexes, the mTOR complex 1 and complex 2 (mTORC1 and mTORC2) [10]. mTORC1 inhibits 4E-BP1 and activates p70S6K, enhancing protein synthesis, whereas mTORC2 phosphorylates Akt at serine 473, which was reported to be diminished in mice soleus and EDL [27]. In addition, mTORC2 induces autophagy through FOXO3 activation [10,106]. Overall, such data highlight the downregulation of IGF-1/PI3K/Akt/mTOR pathway in SkM following treatment with anticancer drugs.

Regulation of Satellite-Cell Activation
Satellite cells are a heterogeneous group of stem cells [108] that mediate the life-long maintenance of SkM tissue [109]. These cells are responsible for postnatal muscle growth, repair, and regeneration [110], and are fundamental for SkM function. In atrophic conditions, a reduction of satellite-cell number and differentiation capacity was reported, thus affecting SkM regeneration [111]. Both MYF5 and Pax7 satellite cells are fundamental players in SkM regeneration [111]. MYF5 is a myogenic factor essential to muscle devel-opment and regeneration because it plays an important role in myogenic differentiation and maintenance [112]. Similarly, Pax7 is a key transcriptional regulator in SkM that, when activated, induces proliferating myogenic precursor-cell differentiation [113]. Curiously, an increase in MYF5 content was reported in mouse soleus, whereas in EDL, the Pax7 + was decreased following treatment with doxorubicin [18]. Therefore, the role of anticancer drugs on the regulation of satellite-cell activation needs to be further explored.
There are several pathways that modulate the satellite-cell maturation process. In fact, a JAK/STAT signaling cascade has been suggested to be required for the regeneration of muscle fibers [97], but upregulation of JAK/STAT signaling was reported to inhibit satellitecell function [114]. IGF-1/PI3K/Akt/mTOR pathway may also stimulate satellite-cell differentiation acting through many steps of the process. For example, IGF-1 may stimulate satellite-cell proliferation and differentiation [115], whereas protein synthesis induced by the IGF-1/PI3K/Akt/mTOR pathway contributes to the maturation of myotubes, thus leading to muscle-fiber regeneration [111]. However, as previously described, cisplatin diminished IGF-1 expression [21] and the IGF-1/PI3K/Akt/mTOR pathway was reported to be downregulated [21].

Myostatin/ActRIIB Pathway
The growth-differentiation factor 8 (GDF-8), also known as myostatin, is an autocrine/paracrine cytokine and a member of the TGF-β family. This cytokine is highly expressed in SkM compared with cardiac muscle or adipose tissue and it negatively regulates SkM mass and growth [10,116,117]. Myostatin signaling is modulated by anticancer drugs. Indeed, myostatin mRNA was raised in quadriceps treated with cisplatin [21] and gastrocnemius and soleus muscles treated with gemcitabine [29], whereas the expression of activin A, another member of the TGF-β superfamily, was increased in gastrocnemius and soleus muscles after gemcitabine therapy [29]. When activated, myostatin binds to the ActRIIB, which was reported to be increased in female-mouse SkM after treatment with gemcitabine [29]. The myostatin/ActRIIB complex leads to the activation of receptor-like kinase 4 or 5 (ALK4 and ALK5) [10,116,117]. The complex formed activates transcription factors SMAD2 and SMAD3 through phosphorylation [10,116,117]. Cisplatin increases SMAD2 phosphorylation [21]. These two members of the SMAD family form a trimeric complex with SMAD4 that can translocate into the nucleus and activate or inhibit the transcription of genes, such as the one for myoblast-determination protein 1 (MyoD), inhibiting the myogenic program ( Figure 1) [10,116,117]. In general, the data imply that anticancer drugs contribute to diminished muscle growth and differentiation by raising myostatin expression.

IL-6/JAK/STAT Pathway
The JAK/STAT pathway mediates the effect of diverse cytokines, such as IL-6. Although the levels of IL-6 were reported to be unchanged in different muscles [17,26], this cytokine binds to its receptor complex, IL-6R-gp130, activating the tyrosine kinase JAK [97]. Activated JAK undergoes a conformational change characterized by dimerization and phosphorylation and then activates the signal transducer and activator of transcription proteins [97]. Indeed, oxaliplatin seems to activate STAT3 phosphorylation [23], thus promoting its translocation to the nucleus and binding to specific regulators, enhancing the protein-coding genes in the promoter region, such as the E3 ligases muscle atrophy F-Box protein 32 (MAFbx/atrogin-1) and muscle RING-finger 1 (MuRF-1) [97]. The data indicate that anticancer drugs promote the activation of JAK/STAT pathways, boosting proteolysis by enhancing atrogin-1 and MuRF-1.
Taken together, anticancer drugs contribute to the activation of several pathways, such as NF-κB, IL-6/JAK/STAT, as well as directly increasing expression of the two muscle-specific E3 ubiquitin ligases, thus leading to enlarged UPP activity.

NF-κB Pathway
NF-κB activation in SkM is triggered by diverse stimuli that are often related to inflammation, such as proinflammatory-cytokine interleukin 1 (IL-1) and tumor-necrosis-factor alpha (TNF-α). Even though the NF-κB pathway is responsible for regulating proinflammatorycytokine production, leukocyte recruitment, or cell survival, these functions can either protect against inflammation or enhance it. This transcription factor is dimeric and can form homoor heterodimers of distinct subunits, such as p65, p50, and p100, among others. These dimers are often activated in wasting conditions [10,118]. When stimulated, NF-κB can act through two different routes, the canonical and the non-canonical pathways, the first of which is responsible for survival, proliferation, inflammation, and immune regulation. This pathway involves the activation of p50/p65 dimers, which translocate into the nucleus, where it binds to the appropriate cognate DNA-binding sites, thus inducing diverse gene transcriptions such as those belonging to UPP and ALP ( Figure 1) [10,118]. Indeed, 5-FU treatment increased phosphorylation of the p65 subunit in soleus and EDL [27]. When TNF-α binds to its receptor, it activates several intermediary steps that result in the activation of a complex composed of two catalytic subunits, IKK-α and IKKβ, and a regulatory subunit, IKK-γ/NF-κB essential modulator (NEMO) [10,118]. Curiously, unchanged TNF-α mRNA was reported after doxorubicin [17] and 5-FU [26] treatment, whereas decreased interleukin 1 beta (IL-1β) mRNA was observed in male mice treated with 5-FU [26].

Autophagy-Lysosome Pathway
The ALP refers to a fundamental process for the removal of misfolded proteins and damaged organelles. The process prevents the accumulation of protein aggregates, thus maintaining protein homeostasis in SkM [10,95]. Several players of this pathway have been evaluated in SkM to mechanistically explain chemotherapy-induced muscle wasting, namely, the lipidated microtubule-associated protein 1 light-chain 3 alpha (LC3), p62 [10,95], and BNIP3 [111]. Indeed, BNIP3 mRNA was increased following treatment with oxaliplatin in male mice [23], as well as in C2C12 mouse myoblasts in the presence of cisplatin [89]. Other in vitro studies (Table A1) also supported the activation of the ALP pathway induced by anticancer drugs since LC3CII mRNA [89] and beclin1 mRNA [88] were increased. Several components of other pathways, such as AMPK, mTOR, mitochondrial proteins (OPA1, DRP1), and NF-κB, can mediate autophagy, thus activating or inhibiting ALP [10,119]. In fact, AMPK phosphorylated at tyrosine 172 levels increased in EDL after doxorubicin [17], which may activate autophagy at different regulation levels by phosphorylating specific ALP-related complexes, including ULK1 and PIK3C3/VPS34 [120].

Ubiquitin-Proteasome Pathway
UPP is one of the main proteolytic systems responsible for the degradation of misfolded or defective proteins in SkM [10,95,121]. This pathway involves ubiquitin-activating enzymes (E1), ubiquitin-conjugating proteins (E2s), and ubiquitin-protein ligases (E3s), which act sequentially to polyubiquitinate the proteins that are then recognized by the 26S proteasome [10,121]. In SkM, MuRF-1 and atrogin-1 are two muscle-specific E3 ubiquitin ligases involved in UPP-mediated proteolysis. Both MuRF-1 and atrogin-1 are usually upregulated in atrophic muscle, as they are considered markers of muscle wasting [10,95,121]. Nevertheless, MuRF-1 and atrogin-1 mRNA were unchanged after FOLFIRI and FOLFOX treatment [31] and, unexpectedly, MuRF-1 mRNA decreased in male mice treated with cisplatin [20]. Still, MuRF-1 and atrogin-1 mRNA were increased by distinct chemotherapy agents in several animal studies ( [13,21,23,29]) and one in vitro study [89]. These inconsistent results about MuRF-1 and atrogin-1 expression among studies may be explained by the differences in the chemotherapy agents, drug doses, animal species, or health status.
Activation of FOXOs family members such as forkhead box O3 (FOXO3) can also lead to activation of atrogin-1 and MuRF-1, thus increasing UPP activity. FOXO3 mRNA was enhanced in animals submitted to gemcitabine plus cisplatin [29] and to oxaliplatin [23]. Similarly, cisplatin increased FOXO3 mRNA but decreased FOXO3a phosphorylation in quadriceps muscles [21], enhancing the activation of the UPP by increasing the expression of both atrogin-1 and MuRF-1. Another FOXO element, FOXO1, was evaluated following anticancer treatment. However, FOXO1 mRNA was unchanged [21,23].
Taken together, anticancer drugs contribute to the activation of several pathways, such as NF-κB, IL-6/JAK/STAT, as well as directly increasing expression of the two musclespecific E3 ubiquitin ligases, thus leading to enlarged UPP activity.

Conclusions
In this narrative review, we analyzed the molecular effects of chemotherapy agents on SkM based on the existing literature. The effects of cystemustine, gemcitabine, docetaxel, paclitaxel, and other chemotherapy agents on SkM have been studied; however, the number of studies is small when compared with those on doxorubicin, cisplatin, and 5-FU. Doxorubicin, one of the most studied chemotherapeutic agents, is very effective against cancer but its use is limited by dose-dependent toxic side effects in many organs, such as the heart and SkM. Therefore, studying the effects of other existing therapeutic regimens, including newer ones, such as the combination of 5-FU, leucovorin, oxaliplatin, and docetaxel (FLOT), is critical to better understanding the systemic changes induced by therapy and to explaining the associated side effects. One of the major limitations of clinical trials is the ethical constrains on the collection of SkM biopsies from cancer patients, although these samples are essential for a better understanding of the cellular and molecular changes induced by chemotherapy in SkM. Another limitation in the investigation of this issue is the low number of preclinical studies investigating this issue, with only three studies having been performed with C2C12 or L6 cell lines so far. To improve the translational application of preclinical research, human cell lines, such as the RCMH cell line, should be considered [122].
In addition, more preclinical studies using tumor-bearing animals and clinical trials with well-defined criteria are needed to support the development of novel pharmacological and nonpharmacological therapies. Such criteria should include the chemotherapy regimen, cancer stage, number of chemotherapy cycles, and duration of treatment. By integrating pharmacological therapies with exercise training, nutrition, and psychological support into tailored multimodal approaches, cancer patients at risk of muscle wasting can achieve better postoperative recovery. Therefore, it is crucial to prioritize research efforts in this area to improve the quality of life of cancer patients and enhance survival outcomes.  Acknowledgments: This work was supported by the research centers CIAFEL and ITR (UIDB/00617/2020 and LA/P/0064/2020, respectively), the Institute of Biomedicine (iBiMED; UIDB/04501/2020 and POCI-01-0145-FEDER-007628), LAQV-REQUIMTE (UIDB/50006/2020), and Instituto Português de Oncologia do Porto Francisco Gentil, EPE (CI-IPOP-134-2020). The figure was created with BioRender.com.