Proteomics reveals the function reverse of MPSSS treated prostate cancer-associated fibroblasts to suppress PC-3 cell viability via the FoxO pathway

Prostate cancer (PCa) remains the malignancy with the highest morbidity in men (1). Although early-stage PCa usually causes no symptoms, cancer cells in prostate glands readily metastasize to bones, lymph nodes, lungs, and liver during PCa progression (2). Common strategies, including surgery, radiation therapy, and androgen-deprivation therapy, are promising treatments for early-stage PCa. However, advanced PCa still lacks effective therapeutic strategies (3-5). Thus, researchers are currently focused on inhibiting tumor progression to explore effective therapeutic strategies for PCa treatment (6, 7). Cancer-associated fibroblasts (CAFs), stromal cells derived from normal fibroblasts or epithelia, are one of the major cellular components in the tumor microenvironment (8, 9). Compared with normal fibroblasts, CAFs are activated with increased expression of key protein markers, such as α-SMA, FAP, vimentin and S100A4 protein (10, 11). In the tumor microenvironment, CAFs secrete various growth effectors or cytokines to the extracellular matrix and thereby promote tumor progression by directly enhancing cancer cell growth, angiogenic induction, the extracellular matrix modification, and tumor-promoting inflammatory mediation (11-14). Notably, CAFs could act as an immunosuppressive mediator for the formation of an immunosuppressive tumor environment by recruiting and activating multiple immune cells (15). As such, CAFs might be a potential target in anti-tumor immunotherapy (16, 17). Currently, increased research has focused on inhibiting the immunosuppressive function of prostate cancer-associated fibroblasts (prostate CAFs), which may suggest therapeutic strategies for PCa (18, 19). Polysaccharides from Lentinus edodes are known to exhibit potent anti-tumor and immunomodulatory functions. The anti-tumor mechanisms of L. edodes polysaccharides include regulating the innate immune system by mediating CAF function, preventing tumorigenesis, or directly killing tumor cells via cell cycle regulation or apoptotic induction (20-22). Our previous studies isolated a novel polysaccharide component (MPSSS) from L. edodes. We found the secretome of prostate CAFs treated with MPSSS could inhibit the proliferation of CD4+ and CD8+ T cells, which suggested that MPSSS could prevent the immunosuppressive function of prostate CAFs (22). However, although the secretome of MPSSS-treated prostate CAFs inhibit the proliferation of T cells, how the secretome of MPSSS-treated prostate CAFs affect PCa progression is still unclear. The secretome defines as all proteins secreted by the organism or living cells into the extracellular space, which consists of soluble proteins and extracellular vesicles (EVs) (23). The secretome of prostate CAFs pays vital roles in PCa tumor origination, progression (24, 25). Since EVs contains various molecules (proteins, RNA, DNA and lipid) (26), we speculate that soluble proteins and EVs of prostate CAFs might have the different influence on PCa cells. In this study, the secretome of prostate CAFs untreated/treated with MPSSS were separated into the high molecular weight secretome (>100 kD) and the low molecular weight secretome (3-100 kD) using 100 kD and 3kD MWCO memberane filtration to narrow down the range of active components. The high molecular weight secretome contained extracellular vesicles (EVs) and large soluble proteins, while the low molecular weight secretome contained the small soluble proteins. The secretome of prostate CAFs untreated/treated with MPSSS were used to explore the underlying function and molecular mechanism using quantitative proteomics and multiple biochemical approaches.

前列腺癌（prostate cancer, PCa）仍是全球男性发病率最高的恶性肿瘤(1)。尽管早期前列腺癌通常无明显临床症状，但在疾病进展过程中，前列腺腺体内的癌细胞极易向骨骼、淋巴结、肺脏及肝脏发生远处转移(2)。目前临床针对早期前列腺癌的常用治疗策略包括手术治疗、放射治疗与雄激素剥夺疗法，均具备较好的应用前景。然而，晚期前列腺癌仍缺乏有效的临床治疗手段(3-5)。因此，当前研究者正聚焦于抑制肿瘤进展，以探索前列腺癌治疗的有效方案(6,7)。

癌症相关成纤维细胞（cancer-associated fibroblasts, CAFs）是一类源自正常成纤维细胞或上皮细胞的基质细胞，是肿瘤微环境的核心细胞组成成分之一(8,9)。与正常成纤维细胞相比，活化的CAFs会高表达α-平滑肌肌动蛋白（α-SMA）、成纤维细胞活化蛋白（FAP）、波形蛋白（vimentin）及S100A4蛋白等关键蛋白标志物(10,11)。在肿瘤微环境中，CAFs可向细胞外基质分泌多种生长效应因子与细胞因子，通过直接促进癌细胞增殖、诱导血管生成、修饰细胞外基质以及介导促肿瘤炎症反应等多种途径加速肿瘤进展(11-14)。值得注意的是，CAFs还可通过招募并活化多种免疫细胞，参与构建免疫抑制性肿瘤微环境，发挥免疫抑制介导作用(15)。据此，CAFs或可成为抗肿瘤免疫治疗的潜在靶点(16,17)。

目前，越来越多的研究聚焦于抑制前列腺癌相关成纤维细胞（prostate cancer-associated fibroblasts, 简称前列腺CAFs）的免疫抑制功能，这或将为前列腺癌治疗提供全新的治疗思路(18,19)。香菇（Lentinus edodes）多糖已被证实具备显著的抗肿瘤与免疫调节活性。香菇多糖的抗肿瘤机制包括通过调控CAFs功能以调节先天免疫系统、抑制肿瘤发生，或通过调控细胞周期、诱导细胞凋亡直接杀伤肿瘤细胞(20-22)。

本团队前期研究从香菇中分离得到一种新型多糖组分（MPSSS），并发现经MPSSS处理后的前列腺CAFs分泌组可抑制CD4+及CD8+ T细胞的增殖，提示MPSSS能够逆转前列腺CAFs的免疫抑制功能(22)。然而，尽管经MPSSS处理的前列腺CAFs分泌组可抑制T细胞增殖，但其对前列腺癌进展的具体影响及分子机制仍未明确。

分泌组（secretome）指生物体或活细胞分泌至细胞外空间的全部蛋白质，涵盖可溶性蛋白与细胞外囊泡（extracellular vesicles, EVs）(23)。前列腺CAFs分泌组在前列腺癌的发生与进展过程中发挥关键调控作用(24,25)。由于细胞外囊泡携带有蛋白质、RNA、DNA及脂质等多种分子(26)，我们推测前列腺CAFs分泌的可溶性蛋白与细胞外囊泡或对前列腺癌细胞产生不同的调控效应。

本研究通过采用100 kD与3 kD分子量截留（molecular weight cutoff, MWCO）膜过滤法，将经MPSSS处理及未处理的前列腺CAFs分泌组分离为高分子量分泌组分（>100 kD）与低分子量分泌组分（3-100 kD），以缩小活性成分的筛选范围。其中，高分子量分泌组分包含细胞外囊泡与大型可溶性蛋白，低分子量分泌组分则包含小型可溶性蛋白。本研究将利用定量蛋白质组学与多种生化实验方法，探究上述前列腺CAFs分泌组的潜在功能与分子机制。