BMP axis in cancer cachexia

BACKGROUND Cancer cachexia is a devastating metabolic syndrome characterized by systemic inflammation and massive muscle and adipose tissue wasting. Although cancer cachexia is responsible for about 25% of cancer deaths, no effective therapies are available, and the underlying mechanisms have not been fully elucidated. Its occurrence complicates patients’ management, reduces tolerance to treatments and negatively affects patient quality of life. Muscle wasting, mainly due to increased protein breakdown rates, is one of the most prominent features of cachexia. Blocking muscle loss in cachexia mouse models dramatically prolongs survival even of animals in which tumor growth is not inhibited. Recent observations showed that bone morphogenetic protein (BMP) signaling, acting through Smad1, Smad5 and Smad8 (Smad1/5/8), is a master regulator of muscle homeostasis. BMP-Smad1/5/8 axis negatively regulates a novel ubiquitin ligase (MUSA1) required for muscle loss induced by denervation. MATERIALS AND METHODS First aim of the present work was to test if alterations of the BMP signaling pathway occur in cancer-induced muscle wasting in patients. For this purpose we checked the state of activation of the BMP pathway in muscle of cachectic vs non–cachectic patients affected by colon, pancreatic and esophagus cancer and in control subjects. We checked by Western Blot the phosphorylation levels of Smad1/5/8 and of Smad3 and by quantitative Real-Time PCR (qRT-PCR) the expression levels of different atrophy-related genes The second aim was to evaluate the degree of muscle atrophy and distribution of muscle fibers in patients and control subjects using morphometric and immunohistochemical analyses. We also performed analysis on distribution of NCAM positive muscle fibers to assess the effect of denervation on muscle tropism. RESULTS From December 2014 we collected 95 rectus abdominis muscle biopsies of cancer patients and 11 from control subjects. In line with the results we obtained in C26 mice model (a well-established cancer cachexia experimental model) Smad1/5/8 phosphorylation, readout of the state of activation of the BMP pathway, was nearly completely abrogated in the muscles of cancer cachectic patients compared to cancer non-cachectic ones. Interestingly, the level of phosphorylation of Smad3 was not significantly affected suggesting specific effects of cancer growth on BMP pathway. The expression levels of different atrophy-related genes including MUSA1 were induced in the cachectic muscles. Interestingly, several BMP related genes are also changing the expression during cancer growth. We also found a correlation between suppression of BMP pathway, expression of atrophy related genes and Noggin, known to block BMP pathway. Morphometric analysis shown that patients with cancer cachexia have smaller myofiber diameter (in particular fast type fibers) in comparison to age-matched controls. In skeletal muscle from cancer patients (either cachectic or non-cachectic) we detected a prevalence of flat shaped, angulated and severely atrophic myofibers (i.e. morphological features of denervated myofibers), big fiber-type grouping (i.e. typical hallmark of denervation/reinnervation events) and numerous NCAM positive myofibers (i.e. specific marker of denervation). CONCLUSIONS These findings are consistent with the hypothesis that BMP inhibition is permissive to cachexia onset. Since the reactivation of the BMP-dependent signaling and MUSA1 suppression was sufficient to prevent tumor-induced muscle atrophy in our C26 mouse model (data not shown), the present data suggest that the BMP axis can be an effective target for therapeutic approaches to counteract cachexia also in cancer patients. The results of morphometric and immunohistochemical studies collected till now may suggest that denervation contributes to myofiber atrophy in cancer cachexia

Selected characteristics of male patients with bladder cancer and controls.

<p><i>N.S.</i> not significant, <i>OR</i>- odds ratio, <i>CI</i>-confidence interval,</p>a-<p><i>OR</i> adjusted by age and pack-years.</p

Combined effect of occupational exposure to pesticides and <i>GST</i> genotype on bladder cancer risk in male patients.

a<p>Active (present) if at least one active allele present.</p>b<p>Inactive (null) if no active alleles present. <i>OR</i>- odds ratio adjusted for age and pack years. <i>CI</i>- confidence interval.</p

<i>GSTA1, GSTM1, GSTT1</i> and <i>GSTP1</i> genotypes in relation to bladder cancer risk in male patients.

a<p>Active (present) if at least one active allele present.</p>b<p>Inactive (null) if no active alleles present. <i>OR</i>- odds ratio adjusted for age and pack-years. <i>CI</i>- confidence interval.</p

Glutathione S-transferase (GST) genotype distribution.

<p>Glutathione S-transferase (GST) genotype distribution.</p

<i>GSTT1, GSTP1, GSTO1, GSTO2, GSTM1</i> and <i>GSTA1</i> polymorphisms as the predictors for overall mortality among 101 patients with muscle invasive TCC after 5 yrs of follow-up by the Cox proportional hazards regression.

<p>Abbreviations: CI, Confidence Interval; HR, Hazard Ratio.</p>a<p>Adjusted for age and gender.</p>b<p>Adjusted for the covariates in Model 1 plus an additional adjustment for grade.</p

Survival analysis Kaplan–Meier curves according to <i>GSTO1</i> polymorphism for overall mortality (A), as well as mortality of TCC patients on chemotherapy (B).

<p>Survival analysis Kaplan–Meier curves according to <i>GSTO1</i> polymorphism for overall mortality (A), as well as mortality of TCC patients on chemotherapy (B).</p

Survival analysis Kaplan–Meier curves according to <i>GSTO2</i> polymorphism for overall mortality (A), as well as mortality of TCC patients on chemotherapy (B).

<p>Survival analysis Kaplan–Meier curves according to <i>GSTO2</i> polymorphism for overall mortality (A), as well as mortality of TCC patients on chemotherapy (B).</p

Patient characteristics at study entry.

<p>Patient characteristics at study entry.</p

<i>GST</i> genotypes in relation to the risk of cRCC.

<p><i>GST</i> genotypes in relation to the risk of cRCC.</p