MMP12 Knockout Prevent Weight and Muscle Loss Induced by Cancer Cachexia

Weight loss and muscle wasting can have devastating impacts on survival and quality of life of patients with cancer cachexia. Here, we have established a hybrid mouse of ApcMin/+ mice and MMP12 knockout mice (ApcMin/+; MMP12-/-) and found that knockout MMP12 can suppress the weight and muscle loss of ApcMin/+ mice. In detail, we found that interleukin 6 was highly upregulated in the serum of cancer patients and MMP12 was increased in muscle of tumor-bearing mice. Interestingly, the interleukin 6 secreted by tumor cells led to MMP12 overexpression in the macrophages, which further resulted in degradation of insulin and insulin-like growth factor 1 and interruption of glycolipid metabolism. Notably, depletion of MMP12 prevented weight loss of ApcMin/+ mice. Our study uncovers the critical role of MMP12 in controlling weight and highlights the great potential of MMP12 in the treatment of cancer cachexia.


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The IL-6 level has been proposed to be high in patients with skeletal muscle loss (Peixoto da Silva et al., suppressing insulin receptor substrate-1 and downstream targets (Puppa et al., 2012). In short, various 106 studies have proven that IL-6 can induce insulin resistance, thereby indirectly exacerbating muscle loss 107 in CAC. In our study, we found that IL-6 secreted by tumors would target muscle macrophages, and then 108 MMP12 in these activated macrophages will be upregulated, degrading insulin and insulin-like growth 109 factor 1. So, we thought one molecular crosstalk may exist in bearing mice. Increased expression of IL-110 6 derived from cancer cells up-regulates MMP12 in macrophages, which affects skeletal glycolipid me-111 tabolism over a long period of time, may resulting in loss of skeletal muscle for a long time.

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To date, for patients with muscle and weight loss induced by CAC, multimodal interventions including 113 drugs, nutritional support and physical exercise may be a reasonable approach for future research to 114 better understand and prevent loss of muscle (Fonseca et al., 2020). Although several drugs have had 115 positive clinical effects in increasing lean body mass, their effects on body function are limited. There 116 are no effective medical interventions or approved drug therapies that can completely reverse muscle 117 loss caused by CAC, which brings difficulties to the treatment of chemotherapy drugs (Daou, 2020) 118 (Fonseca et al., 2020). Taken together, the combination of MMP12 inhibitors and chemotherapy drugs 119 may bring new challenges and ideas for the treatment of cancer cachexia to improve the quality of life.

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Cells incubated with fresh media were used as the untreated (-IL-6) negative controls. Finally, western 259 blotting was used to quantify MMP12 in RAW264.7 cells under different conditions. 260 2. 18 Isolation of primary peritoneal macrophages 261 24-week-old WT and Apc Min/+ mice were sterilized with 75% ethanol after cervical dislocation. The 262 mouse abdomen was opened from the peritoneum, and 5 mL fatal bovine serum was injected with a 263 syringe, which was allowed to remain inside the abdomen for 5 minutes, with gentle massaging for 30 s.

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The fluorescent peptide sequence:

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The two experiments are as follows: (1) Fluorescence intensity: After mixed incubation of MMP12 and 280 peptide (37℃, 2hours), the fluorescence intensity was measured with a fluorescence microplate reader.

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(2) MS Analysis Report: After the MMP12 and peptide were mixed and incubated, the lower liquid af-   To investigate the weight dynamics of the mice, we determined the mouse body weight by retroactive 317 examination of the body weight from 5-week to 24-weeks old. The current weight curves have shown 318 that compared with the weight gain in wild type (WT) mice over time, the weight of Apc Min/+ mice 319 reached its maximum peak at 12-week-old and then declined until to die at approximately 24-week-old 320 ( Figure 1A). Surprisingly, in comparison to the Apc Min/+ mice control, the body weight of Apc Min/+ ; 321 MMP12 -/mice increased by approximately 70% at the same age ( Figure 1B). While, there were no sig-322 nificant differences between WT mice and MMP12 -/mice in body weight ( Figure 1C). As well known,

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MMP12 -/mice tended to increase compared with that of Apc Min/+ mice but the increase was not statisti-330 cally significant ( Figure 1D). However, a significant increase of approximately 4.5% in the muscle-to-331 body weight ratio was observed in Apc Min/+ ; MMP12 -/mice compared with Apc Min/+ mice ( Figure 1E).

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To further confirm the histological changes of WAT ( Figure 1F) and muscle area ( Figure 1H) in the 333 four mice group at 24-week-old, we performed hematoxylin and eosin staining to assess the histologi-334 cal area by the ImageJ software ( Figure 1F, H). We observed the fat area is larger in MMP12 -/mice 335 17 17 compared with WT mice, but there has no difference between Apc Min/+ mice and Apc Min/+ ; MMP12 -/-336 mice ( Figure 1G). The H&E staining of muscle to show that the area of Apc Min/+ ; MMP12 -/mice is es-337 timated to be approximately 1-2-fold larger than Apc Min/+ mice ( Figure 1I). Meanwhile, no difference in    among many CAC-muscle loss patients who lost weight and were close to death, IL-6 was almost the 368 only increased cytokine among many factors. Therefore, we mainly focus on whether IL-6, which is 369 related to muscle loss, is caused by tumors (Carson and Baltgalvis, 2010b). We observed that the clinical 370 colorectal cancer patients had significantly higher serum IL-6 levels than the normal healthy group (Fig-371 ure 3A). In vivo, a similar trend was found in Apc Min/+ mice, and serum IL-6 levels in Apc Min/+ mice were 372 significantly increased compared with WT mice at 15-24-week-old ( Figure 3B). We demonstrated that 373 the IL-6 mRNA levels were higher in intestinal tumors of Apc Min/+ mice than in normal intestinal epithe-374 lium of WT mice by qPCR ( Figure 3C). Previous study reported MC38 cells and CT26 cells all can secret  and treated with increasing doses of IL-6 (0, 2, 5 10,30ng/ mL) for 72h. Next, RAW264.7 cells were 393 treated continuously with IL-6 (30 ng/ml) for 0, 3, 6, and 9hours. Cells incubated with fresh media were 394 used as the untreated negative controls ( Figure 4F). We found that within a certain concentration range 395 (<30ng/ml), as the IL-6 dose increased, the expression of MMP 12 in RAW264.7 cells also increased 396 when treated with IL-6 ( Figure 4G, H). Next, the expression of MMP12 in RAW264.7 increased as the  Meanwhile, immune gene data proved that IL-6 receptor (IL-6R) is highly expressed on myeloid cells, 399 including F480 + macrophages ( Figure 4K). The previous studies proved that IL-6 can be derived from 400 MC38 and CT26 tumor cells . Taken together, these findings suggest that tumor-derived 401 IL-6 can stimulate macrophages and up-regulate MMP12 in macrophages.

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Notably, MMP12 levels increase in muscle tissues and peritoneal macrophages not in serum.

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25 IL-6 is mainly secreted by a variety of immune cells and is also highly expressed in a variety of cancer 503 cells (Mauer et al., 2015). Our in vitro studies proved that MC38 cell lines can secrete IL-6. Our animal 504 experiments in vivo confirmed that the serum IL-6 of Apc Min/+ mice was also higher than that of WT 505 mice at 15 weeks and 24 weeks, which is consistent with the previous study (Baltgalvis et al., 2008). IL-

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In summary, we suspected that there may be a possibility that MCP1 and KC can also cooperate with 517 IL-6 to recruit macrophages, thereby activating alternative macrophages to polarize M2 macrophages, 518 resulting in MMP12 secretion. The above results suggested that in the period of CAC, the key is that 519 IL-6 secreted by tumor cells plays a major role (Baltgalvis et al., 2008). In Table 2.  demonstrate that macrophage MMP12 increase treatment with IL-6. We speculated that this is caused by 526 the IL-6 secreted by tumor cells, and we firstly proved that IL-6 can directly upregulate macrophage 527 MMP12. However, we did not determine whether the IL-6 produced by macrophages acts on macro-528 phages themselves. Similarly, we have not explored whether other cytokines secreted by tumors or mac-529 rophages themselves play a synergistic or indirect role along with IL-6.

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In present study, we used fluorescence intensity measurement and ionization mass spectrometry meth-531 ods to prove that insulin and IGF-1 can indeed be degraded by MMP12, but the specific sites and

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MMP12, as a macrophage matrix metalloproteinase, has been repeatedly reported to degrade insulin 536 and affect insulin sensitivity. We verified MMP12 can degrade insulin or IGF-1 in vitro, which is con-537 sistent with the previous study (Kettner et al.). As the two key hormones in tumor microenvironment, 538 insulin resistance is correlated with in insufficient insulin, lack of insulin receptor, or decreased insulin 539 sensitivity, which will reduce the uptake of glucose in organs, which suggests that MMP12 is closely

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In summary, our results identified that knocking out MMP12 in Apc Min/+ mice significantly reduced mus-

Acknowledgments 586
We thank technician Dr. Hao Chen for their help in clinical sample collection. We appreciate that Prof.

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Ming Li gave us advices for this study. We would also like to thank Jingzhou Xie, Lixun Huang, Yongjia