Supporting data for “Clusterin as a functional downstream target in Sox9-expressing hepatocellular carcinoma”.

Hepatocellular
carcinoma (HCC) is one of the deadliest cancers across the globe. Patients are
usually diagnosed at advanced stage. Presence of microvascular metastasis,
oversized tumor burden and insufficient liver remnant reduce the feasibility to
surgical and local ablative treatment modalities. Besides, high propensity of
tumor recurrence and metastasis increase the level of challenge in eliminating
HCC. As a matter of fact, targeted therapy confers survival benefits to HCC
patients in late phase. In this regard, elucidating the genetic events that
underlies HCC is of the utmost importance to supply potential biomarkers for
HCC detection and druggable targets for curative purpose.

As
a transcription factor, Sox9 masters embryogenesis and sexual development.
Emerging evidence illustrated that Sox9 overexpression facilitates tumor
initiation and progression in multiples solid cancers. Sox9 overexpression is
associated with aggressive behavior. Of note, Sox9 is a stem cell factor which
endows stemness properties in cancers. Based on our previous study, knocking
down Sox9 inhibited cell growth, cell migration and invasion, but enhanced
chemosensitivity. Apart from this, decline of Sox9 expression suppressed the
expression of stemness markers and tumorsphere formation ability. The oncogenic
potential of Sox9 was mediated by Wnt signaling pathway via transcription
regulation of Frizzled-7. To further dissect the molecular mechanism underlying
Sox9-expression HCC, we tried to identify the molecular target from cancer
secretome regulated by Sox9. Furthermore, its functional role and clinical
significance were evaluated.

To
this end, the HCC secretome upon Sox9 silencing in HCC cell lines was examined
by mass spectrometry. Our results revealed Clusterin (CLU) was a commonly
downregulated in the conditioned medium (CM) from Sox9 knockdown clones
compared with the control. A high serum CLU level was correlated with advanced
HCC stages. Moreover, CLU could better differentiate tumor stages and predict
clinical outcomes when used in combination with serum alpha-fetoprotein (AFP)
level. After investigating the clinical features of CLU, we further explored
the oncogenic potential of Sox9-CLU axis using in vitro assays. Through cell
migration and invasion assays, CM from Sox9 knockdown clones retarded cell
migration ability and invasiveness. Conversely, addition of CLU recombinant
proteins enhanced cell motility in the transwell assays. Enhanced cell
migration and invasion induced by Sox9 overexpression could be abrogated by the
use of neutralizing CLU antibody which reduced extracellular CLU expression. By
predictive analysis, Sox9 putative binding sites were identified in the
promoter region of CLU. The binding interaction was validation by chromatin
immunoprecipitation (ChIP) assay.

In
summary, findings from this study suggests that CLU is a downstream effector in
the secretome of Sox9-expressing HCC. Targeting secreted CLU may present a
novel therapeutic strategy to improve the disease outcome of Sox9-expressing
HCC.

肝细胞癌（HCC）是全球最为致命的恶性肿瘤之一。患者通常在晚期才被确诊。微血管转移的存在、肿瘤负荷过重以及残余肝功能不足，降低了手术治疗和局部消融治疗方法的可行性。此外，肿瘤复发和高转移倾向使得消除HCC的挑战级别大幅提升。实际上，针对晚期HCC患者的靶向治疗能够带来生存益处。鉴于此，阐明HCC背后的遗传事件对于提供HCC检测的潜在生物标志物和治愈目的的药物靶点至关重要。

作为转录因子，Sox9掌管胚胎发生和性发育。新近的证据表明，Sox9的过表达促进了多种实体瘤的肿瘤发生和进展。Sox9的过表达与肿瘤的侵袭性有关。值得注意的是，Sox9是一种干细胞因子，它赋予肿瘤干细胞特性。基于我们先前的研究，敲低Sox9可以抑制细胞生长、细胞迁移和侵袭，并增强化疗敏感性。除此之外，Sox9表达水平的降低还抑制了干细胞标志物的表达和肿瘤球体形成能力。Sox9的致癌潜力是通过Wnt信号通路介导的，该通路通过转录调控Frizzled-7的表达。为了进一步解析Sox9表达HCC背后的分子机制，我们试图从受Sox9调控的癌症分泌物中识别分子靶点，并评估其功能作用和临床意义。

为此，我们通过质谱分析了Sox9沉默后HCC细胞系的分泌组。我们的结果表明，与对照相比，Sox9敲低克隆的培养基（CM）中Clusterin（CLU）的表达普遍下调。高血清CLU水平与晚期HCC阶段相关。此外，当与血清甲胎蛋白（AFP）水平结合使用时，CLU可以更好地区分肿瘤阶段并预测临床结果。在研究CLU的临床特征之后，我们进一步通过体外实验探索了Sox9-CLU轴的致癌潜力。通过细胞迁移和侵袭实验，Sox9敲低克隆的CM减缓了细胞的迁移能力和侵袭性。相反，在Transwell实验中添加CLU重组蛋白增强了细胞的运动能力。通过使用中和CLU抗体，可以消除Sox9过表达引起的增强细胞迁移和侵袭，该抗体通过降低细胞外CLU表达来实现。通过预测分析，在CLU启动子区域识别了Sox9的潜在结合位点。通过染色质免疫沉淀（ChIP）实验验证了这种结合作用。

总之，本研究的结果表明CLU是Sox9表达HCC分泌组中的下游效应器。针对分泌的CLU可能提供一种新颖的治疗策略，以改善Sox9表达HCC的疾病预后。