Extracellular Vesicles Derived From Esophageal Squamous Cell Carcinoma Containing P4HB Regulates Cancer Cachexia And Muscle Wasting

Esophageal cancer(EC)is one of the most malignant tumors in the world.EC has become the eighth most common cause of cancer incidence and the sixth leading cause of cancer mortality worldwide.According to histopathological subtypes,EC is classified into esophageal adenocarcinoma(EAC)and esophageal squamous cell carcinoma(ESCC),constituting greater than 90%of esophageal cancer cases.Patients with ESCC are accompanied with metabolism abnormality,change in energy consumption,and ongoing weight loss,which is called as cachexia.Muscle wasting is commonly acknowledged as the hallmark characteristic of cachexia.At present,no drug has been approved to relieve ESCC-associated cachexia because of under-recognized mechanisms and lack of appropriate ESCC-induced animal model.Extracellular vesicles(EVs)are microvesicles secreted from cells.EVs containing some cargos,such as functional proteins,are released to the extracellular space via fusion with plasma membrane and participate in intercellular communication.EVs can be internalized by recipient cells and influence their biological functions.Previous studies demonstrated that certain proteins and miRNAs in EVs could work as tumor-derived signaling factors for triggering muscle degradation,suggesting the pivotal role of EVs in cancer-associated cachexia.However,whether EVs derived from ESCC can confer certain molecules to trigger cancer-induced muscle wasting remains to be elucidated.Our previous whole-genome and whole-exome sequencing studies identified an amplification region encompassing prolyl 4-hydroxylase subunit beta(P4HB),with extremely high occurrence frequency(9.09%)in 154 ESCC cases.Furthermore,epigenome-wide association study revealed the strong association(1.7×10-16)of P4HB with body mass index,and functional study unveiled P4HB mediated mechanical stretch stress and/or advanced glycosylation end products-triggered proliferation and apoptosis of vascular smooth muscle cells.Thus,we focused on P4HB investigating the function and mechanism of which induces ESCC-associated muscle wasting.The main achievements of this study are summarized as the following:1.P4HB played an important role in the progression of esophageal squamous cell carcinoma.P4HB overexpression could predict poor survival of patients with ESCC utilizing TCGA dataset,indicating that the expression of P4HB could be an independent prognosis factor for ESCC.By analyzing human ESCC tissue microarray,it indicated the clinical significance of P4HB.We found that P4HB was upregulated in YES2 cells.2.We established and identified an ESCC-induced cachexia mouse model.We first developed an ESCC model of cachexia through subcutaneous implantation of four human ESCC cell lines.We successfully generated an ESCC xenograft mouse model of cachexia using YES2 cell line.3.P4HB had association with muscle wasting and cachexia.Performing subcutaneous implantation in nude mice,we verified that P4HB overexpression shorten the lifespan of mice and expedited the development of muscle wasting.Next,we found that P4HB was also upregulated in EVs derived from YES2 cells.Treatment with EVs with stable P4HB overexpression induced murine myoblast apoptosis and suppressed murine myoblast proliferation.4.In the mechanism research,utlizing co-immunoprecipitation assay,mass spectrometry,western blot and confocal microscopy assay,we found that ESCC-EVs with high levels of P4HB evoked myoblast apoptosis and muscle wasting by activating the PHGDH/Bcl-2/caspase-3 pathway,eventually leading to ESCC-associated cachexia.5.In the drug treatment investigation,we conducted drug treatment investigation in vitro and in vivo assays with a small molecule inhibitor of P4HB,CCF642.As a result,we verified that treatment with CCF642 inhibited murine and rat myoblast apoptosis in a dose-dependent manner,and prevented cachectic wasting in nude mice.Taken together,we establish,for the first time,an ESCC-induced cachexia mouse model in this study.We found that ESCC-derived EVs containing P4HB induced murine apoptosis and regulated PHGDH/Bcl-2/caspase-3 pathway,resulting in muscle wasting.This study unveils that P4HB might be served as a potential therapeutic target for cachexia treatment in ESCC and provides a valuable resource for future etiology and drug screening research for this prevalent disorder.