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Extracellular heat shock protein 90 alpha (eHsp90α, also known as HSP90AA1) has been widely reported to promote tumor cell motility and tumor metastasis in various types of cancer. Several extracellular proteins and membrane receptors have been identified as interacting proteins of eHsp90α and mediate its pro-metastasis function. However, the regulatory mechanism of eHsp90α activity remains largely unknown. Here, we report that clusterin, a protein newly demonstrated to interact with eHsp90α, modulates eHsp90α signaling. We found that clusterin potentiated the effects of eHsp90α on activation of the AKT, ERK and NF-κB protein families, epithelial-tomesenchymal transition (EMT) and migration in breast cancer cells. Furthermore, in vivo investigations demonstrated similar synergistic effects of eHsp90α and clusterin on tumor metastasis. Notably, the effects of eHsp90α and clusterin were mediated by low-density lipoprotein receptor-related protein 1 (LRP1). Proximity ligation assay and co-immunoprecipitation experiments demonstrated that clusterin participated in eHsp90α-LRP1 complex formation, which enhanced the binding affinity of eHsp90α to LRP1. Collectively, our data establish a role of clusterin as a newly discovered modulator of eHsp90α, and unravel detailed molecular mechanisms underlying the synergistic metastasis-promoting effects of clusterin and eHsp90α.*Score represents for the protein score of each individual matched protein by the mass spectrometry analysis, and was calculated through the following formula: score=(sum of all cross-correlation factors of 0.8 or above) + (peptide charge×peptide relevance factor). # Coverage represents for the peptides coverage of each individual matched protein by the mass spectrometry analysis, and was calculated by the following formula: coverage = the number of matched amino acids in mass spectrometry peptides/the total number of amino acids in the protein sequence.

Graphical Abstract Highlights d Secreted PKM2 is a potential serum biomarker for diagnosing lung cancer d Secreted PKM2 promotes lung cancer progression d Integrin b1 functions as a receptor for secreted PKM2 d Targeting secreted PKM2 and integrin b1 can attenuate lung cancer metastasis SUMMARYCancer cell-derived secretomes have been documented to play critical roles in cancer progression. Intriguingly, alternative extracellular roles of intracellular proteins are involved in various steps of tumor progression, which can offer strategies to fight cancer. Herein, we identify lung cancer progression-associated secretome signatures using mass spectrometry analysis. Among them, PKM2 is verified to be highly expressed and secreted in lung cancer cells and clinical samples. Functional analyses demonstrates that secreted PKM2 facilitates tumor metastasis. Furthermore, mass spectrometry analysis and functional validation identify integrin b1 as a receptor of secreted PKM2. Mechanistically, secreted PKM2 directly bound to integrin b1 and subsequently activated the FAK/SRC/ERK axis to promote tumor metastasis. Collectively, our findings suggest that PKM2 is a potential serum biomarker for diagnosing lung cancer and that targeting the secreted PKM2-integrin b1 axis can inhibit lung cancer development, which provides evidence of a potential therapeutic strategy in lung cancer.

Chemotherapy damages the intestinal mucosa, causing adverse gastrointestinal reactions. Clostridium butyricum (C. butyricum) reduces the incidence of diarrhea in digestive diseases, including inflammatory bowel disease. Therefore, the aim of the present study was to investigate the role of C. butyricum in patients undergoing chemotherapy. A total of 41 participants with lung cancer were enrolled, and divided into the C. butyricum (CB) or placebo group using 1:1 randomization to obtain 20 CB and 21 placebo participants. On the first and last day of the 3-week intervention, blood and stool samples were collected and analyzed. To analyze stool flora, 16S ribosomal RNA sequencing was performed. The incidence of chemotherapy-induced diarrhea was lower in the CB group compared with the placebo group. The lymphocyte count and platelet/lymphocyte ratio (PLR) was markedly altered between the two groups. Neutrophil/lymphocyte ratio (NLR) and PLR decreased within the CB group. At week 3, the lymphocyte/monocyte ratio (LMR) was higher in the CB group compared with the placebo group. Alterations in lymphocyte subsets and immunoglobulin levels were not significantly different. Albumin (ALB) level and weight did not differ significantly between the two groups. At 3 weeks the total flora diversity did not decrease in either group. Phyla in the CB group varied slightly, while the proportion of Firmicutes in the placebo group decreased significantly. No statistically significant difference was observed between the two groups, though the genera producing short-chain fatty acids tended to increase, and the pathogenic genera tended to decrease in the CB group, which was almost the opposite of the observation in the placebo group. Operational taxonomy unit analysis revealed a notable increase in beneficial flora, including the Clostridium and Lactobacillus genera of the CB group, compared with the placebo group. The present study highlighted that C. butyricum reduced chemotherapy-induced diarrhea in patients with lung cancer, reduced the systemic inflammatory response system and encouraged homeostatic maintenance.