1.

        在临床上，食管癌的发生发展和预后具有显著的性别差异。研究发现，70%的食管癌病例见于男性患者，且预后较女性差。食管癌患者体内雌雄激素及其对应受体表达水平与患者的预后有关，而具体分子机制尚不明确。本研究中，我们系统性探究了雄激素受体（Androgen Receptor, AR）, 雌激素受体α和β（Estrogen Receptor α/β， ERα/β）在食管癌中的功能和分子机制。我们推测，雄激素及其受体的功能和作用机制可能是导致食管癌男性高发的重要原因之一，  而雌激素信号通路的活化则一定程度上揭示女性食管癌患者的发病机理。

        首先， 我们检测AR, ERα/β在食管癌细胞系中的表达水平，发现男性患者来源的KYSE410和ZEC014细胞具有较高的AR表达，女性患者来源的KYSE510和 KYSE150细胞则分别表达ERα和ERβ。体内实验发现，KYSE410细胞在雄性小鼠中的生长速度显著快于雌鼠，雄鼠去势后细胞的生长显著减缓。另一方面，KYSE510细胞在雌鼠体内生长显著快于雄鼠，而KYSE150则相反。外源雌激素以受体依赖的方式促进KYSE510细胞生长，抑制KYSE150细胞生长。体外实验也一致表明雌雄激素受体通过结合对应激素配体，从而调控食管癌细胞生长。敲降激素受体表达显著削弱食管癌细胞对激素的应答反应。此外，激素受体调节剂（激动剂或拮抗剂）通过影响激素与受体的结合，从而调控受体阳性的食管癌细胞的体内外生长。

       本课题中，我们重点研究了AR调控食管癌细胞生长的分子机制。我们首先通过染色质免疫共沉淀联合二代测序（ChIP-seq），在KYSE410细胞中定义了AR在全基因组上的结合位点。基序富集分析（motif enrichment analysis）发现在激素诱导下增加的AR结合位点上有雄激素应答原件（full-length androgen receptor element, ARE）以及GATA原件富集，提示AR在激素诱导下直接结合到基因组DNA上，同时GATA转录因子家族成员可能作为AR的转录辅助因子共同调控基因转录。同时，RNA测序（RNA-seq）表明，雄激素处理显著抑制50%以上的基因表达，提示AR可能通过抑制基因转录来调控食管癌细胞的生长。ChIP实验发现激素处理下AR能够富集在两个下游基因DUSP4和FOSB的调控区域，而AR拮抗剂恩杂鲁胺（Enzalutamide）或者敲降AR表达则显著抑制了AR转录抑制活性。功能实验发现，DUSP4和FOSB在食管癌细胞中发挥抑癌基因功能。作为先导转录因子（pioneer factor）, GATA3通过募集转录抑制复合物（co-repressors）SMRT和HDAC3，介导AR的转录抑制功能。特异性敲降GATA3/SMRT/HDAC3，或者联合使用AR拮抗剂Enzalutamide和HDAC3抑制剂能够显著促进AR下游基因DUSP4和FOSB的表达，进而抑制食管癌细胞的生长。另一方面，我们也探究了激素处理下AR对上调基因UGT2B15的转录调控机制。结合Motif富集分析以及ChIP实验验证发现， AP-1家族成员c-Jun和FOSL2作为转录辅助因子（cofactors）与AR共同富集在UGT2B15的调控区域。特异性敲降c-Jun, FOSL2 或者使用AP-1抑制剂能够显著抑制AR的转录活性，从而抑制UGT2B15表达。体外实验表明，过表达UGT2B15能够显著促进食管癌细胞的运动，而不影响细胞的生长。同时，敲降AR/c-Jun/FOSL2能够显著抑制食管癌细胞的运动。GEO分析发现UGT2B15在食管癌组织中的表达水平显著高于癌旁，且UGT2B15与AR的表达存在显著正相关。

        综上所述，激素受体信号通路在食管癌细胞生长和运动中发挥着重要作用。本研究揭示了雄激素处理下AR在食管癌细胞全基因组上的结合位点，并且主要发挥转录抑制功能。GATA3通过募集SMRT/HDAC3转录抑制复合物介导AR的转录抑制活性，而c-Jun/FOSL2与AR形成转录复合物促进下游基因表达。AR通过抑制抑癌基因转录，上调癌基因表达促进食管癌细胞的生长和运动。联合使用AR拮抗剂恩杂鲁胺以及共调节分子（co-regulators）特异性抑制剂，显著抑制AR的转录程序，从而抑制食管癌细胞的恶性表型，为AR阳性的食管癌患者提供了新的临床治疗策略。此外，进一步揭示ERα/β的转录调控机制有助于完善激素受体信号通路在食管癌细胞中分子机制。针对食管癌患者不同受体的表达状态给予特异性治疗，同时研发新的受体调节剂，有助于为食管癌的临床治疗选择提供新的思路。

2.

       转移是导致肿瘤患者死亡的重要原因。在肿瘤转移过程中，多种分子组成信号传递网络对维持肿瘤细胞的转移潜能发挥着重要作用。实验室前期通过chemotaxis体外模型筛选，获得了运动能力存在显著差异的4种食管癌细胞亚型，即30-U/D和180-U/D。我们通过表达芯片进行差异基因的筛选鉴定，发现溶酶体相关膜蛋白3（LAMP3）在运动能力较强的食管癌细胞中显著高表达，提示LAMP3可能与食管癌细胞的运动相关。研究报道，LAMP3参与了不同类型肿瘤细胞的运动和浸润，然而具体调控机制尚不明确。本研究旨在探讨LAMP3在食管癌转移中的功能及分子机制。

       我们首先在多个食管癌GEO数据库中分析了LAMP3的表达情况。LAMP3在食管癌组织中的表达水平显著高于癌旁组织，且具有淋巴结转移的食管癌患者原发灶中LAMP3的表达水平显著高于非淋巴结转移的食管癌患者。我们也通过免疫组化实验进一步检测了LAMP3在食管癌以及淋巴结组织中的表达情况。体外transwell实验表明，稳定敲降LAMP3表达后，食管癌细胞的运动能力被显著削弱，而稳定过表达LAMP3的食管癌细胞，运动能力显著增强。体内实验表明，敲降LAMP3后，食管癌细胞在肺内定植的能力显著下降，淋巴结转移能力下降，转移阳性淋巴结的百分比也显著降低。机制研究发现，在chemotaxis状态下，LAMP3敲降后VASP 239位丝氨酸磷酸化显著升高，而过表达LAMP3后VASP 239位丝氨酸磷酸化水平显著降低。VASP敲降后，食管癌细胞的运动能力显著增强。当在LAMP3敲降的细胞中进一步敲降VASP表达，食管癌细胞的运动能力得到部分恢复。为了进一步确证VASP 239位丝氨酸磷酸化水平对LAMP3敲降的食管癌细胞的运动的影响，我们将VASP 239位丝氨酸突变为丙氨酸（S239A），在chemotaxis模型中，检测VASP 239位丝氨酸磷酸化情况。在两株食管癌细胞中，我们发现将VASP 239位丝氨酸突变后，chemotaxis状态下，LAMP3敲降的食管癌细胞中VASP在该位点的磷酸化水平被显著削弱。Transwell实验发现，LAMP3敲降后进一步突变VASP的食管癌细胞较LAMP3单独敲降相比，细胞的运动能力得到部分恢复。淋巴结转移实验中，LAMP3敲降组中，过表达突变型的VASP可以显著促进食管癌细胞的淋巴结转移。由于多种蛋白激酶参与VASP  239位丝氨酸的磷酸化，我们通过使用不同蛋白激酶抑制剂处理细胞，检测VASP的磷酸化水平和细胞的运动能力的改变。研究发现，PKA抑制剂处理细胞后，LAMP3敲降细胞内VASP的磷酸化水平没有明显改变，而对照组细胞的运动能力显著增强。我们推测PKA可能是影响VASP磷酸化的潜在激酶。我们在LAMP3敲降的食管癌细胞中进一步敲降PKA的两个调节亚基后，发现VASP的磷酸化水平显著下降而细胞的运动能力得到一定回复。表明PKA介导了LAMP3敲降后VASP的磷酸化过程。为了进一步研究LAMP3敲降后，PKA调控VASP磷酸化的分子机制，我们推测LAMP3敲降后可能影响溶酶体对目标蛋白的选择性降解过程。我们发现，在chemotaxis状态下，LAMP3敲降后，PKA的调节亚基PRKAR1A水平显著升高，同时，PKA锚定支架蛋白（AKAP）WAVE-1的表达显著升高。我们通过氯喹抑制溶酶体功能后，得到了同样的结果。Co-IP实验发现，WAVE-1与LAMP3存在相互作用。当我们敲降WAVE-1表达后，LAMP3敲降后诱导的VASP 239位丝氨酸磷酸化显著降低。因此我们推测，LAMP3敲降后，抑制了溶酶体对WAVE-1的选择性降解，WAVE-1作PKA的支架蛋白，为PKA选择性磷酸化VASP创造了条件。对于LAMP3如何选择WAVE-1进入溶酶体进行降解，我们还需要进一步研究。

        综上所述，本研究首次解释了LAMP3促进食管癌细胞运动的分子机制。通过chemotaxis模型，我们发现LAMP3敲降后，影响溶酶体对于PKA支架蛋白WAVE-1的选择性降解，从而促进了PKA对于VASP 239位丝氨酸的磷酸化，抑制了食管癌细胞的运动和体内转移。本研究也提示溶酶体通过对特定目标蛋白的选择性降解，进而调控食管癌细胞的运动和转移潜能。系统性鉴定溶酶体内特定蛋白底物以及蛋白降解过程，为揭示食管癌细胞转移的分子机制提供了新的思路。

1.

Esophageal squamous cell carcinoma (ESCC) is strongly characterized by a male-predominant propensity, as males account for more than 70% of ESCC patients. The levels of circulating hormone and hormone receptors (AR/ERα/ERβ) were associated with the prognosis of ESCC patients, however, the underlying mechanism remains unclear. In this study, we characterized the functions and molecular mechanisms of AR/ERα/ERβ in ESCC. We hypothesized that AR signaling pathway might be responsible for the male-dominated phenomenon in ESCC, and the activation of the ERα/ERβ signaling pathway may account for the development of ESCC in females.

Firstly, we analyzed the expression levels of AR, ERα and ERβ in ESCC cell lines and found that KYSE410 and ZEC014 cells derived from male patients exhibited higher AR expression, while ERα and ERβ were highly expressed in female patients-derived KYSE510 and KYSE150 cells respectively. The growth of KYSE410 in male mice was significantly faster than female mice, which was impaired after male mice were castrated. We also found that the growth of KYSE510 cells was significantly increased in female mice, while the growth of KYSE150 cells showed the opposite effect. Exogenous supplementation of estrogen promoted the growth of KYSE510 cells, while inhibited the growth of KYSE150 cells in a receptor-dependent manner. In vitro experiments further confirmed that estrogen and androgen receptors regulated the growth of ESCC cells by specifically binding to their ligands. Genetic depletion of hormone receptor significantly weakened their response to corresponding hormone ligands. In addition, hormone receptor modulators (agonists or antagonists) regulated the growth of ESCC cells in vivo and in vitro by modulating the binding of hormones to their receptors.

Here, we mainly focused on the molecular mechanism of AR in ESCC. We first defined the genomic binding sites of AR in KYSE410 cells through chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq). Motif enrichment analysis revealed that full-length androgen-responsive element (ARE) and GATA motif were enriched in the AR agonist R1881-induced AR binding sites, suggesting that AR may collaborate with members of the GATA transcription factor family to regulate gene transcription. Furthermore, R1881 treatment significantly downregulated more than 50% of gene expression, suggesting that androgen mainly represses gene expression in ESCC cells. Upon R1881 stimulation, AR was significantly enriched in the regulatory regions of the two R1881-repressed genes DUSP4 and FOSB. Additionally, pharmacological inhibition or genetic depletion of AR significantly repressed its transcriptional repression activity. Functional experiments found that DUSP4 and FOSB play tumor-suppressive roles in ESCC. As a pioneer factor, GATA3 mediated the transcriptional repression of AR by recruiting SMRT/ HDAC3 co-repressors to regulatory regions of target genes. Knockdown of GATA3/SMRT/HDAC3 or synergistic administration of enzalutamide and HDAC3 inhibitor significantly increased the expression of DUSP4 and FOSB, thereby inhibiting the proliferation of ESCC cells. Moreover, we also investigated the molecular mechanism of transcriptional activation of AR in regulating the R1881-induced UGT2B15 gene. We identified that AP-1 family member c-Jun/FOSL2 as transcription cofactors to facilitate AR binding to the regulatory region of UGT2B15. Genetic knockdown of c-Jun/FOSL2 or treatment of AP-1 inhibitor significantly repressed the transcriptional activation of AR, thereby inhibiting UGT2B15 expression. Overexpression of UGT2B15 significantly promoted the invasion of ESCC cells without affecting cell viability. Consistently, knockdown of AR/c-Jun/FOSL2 also significantly inhibited the invasion of ESCC cells. Finally, we found that the expression of UGT2B15 in ESCC was significantly higher than adjacent tissues, which was also positively correlated with AR expression in ESCC.

In summary, the hormone receptor signaling pathway plays an important role in ESCC. Our study, for the first time, reveals the genomic binding of AR in ESCC cells. AR functions as a transcriptional repressor upon androgen treatment. GATA3 mediates the transcriptional repression activity of AR by recruiting SMRT/HDAC3 corepressor complex to target gene loci. c-Jun/FOSL2 forms a transcriptional complex with AR to increase gene expression. AR promotes proliferation and invasion of ESCC cells by inhibiting the expression of tumor suppressors and increasing the expression of oncogenes. The synergistic administration of AR antagonist enzalutamide and co-regulators inhibitors significantly compromises the oncogenic AR transcription program, leading to growth inhibition of ESCC cells, and provides a therapeutic strategy for treating AR-expressing male ESCC patients. In addition, the mechanism underlying the transcriptional regulation of ERα/β will be further investigated to uncover estrogen-mediated signaling in female ESCC patients. Targeting hormone receptor pathway will be a potential therapeutic option for hormone-dependent ESCC.

2.

Metastasis is one of the major causes of cancer-related death. During metastasis, the maintenance of the metastatic potential of cancer cells involved the complex molecular network. Previously, our lab has established the chemotaxis-based in vitro screening model for highly invasive ESCC cell subtypes. We obtained four subtypes of ESCC cells with distinct motilities, namely 30-U/D and 180-U/D. Differential gene expression was identified by expression array and found that lysosomal-associated membrane protein 3 (LAMP3) was highly expressed in ESCC cell subtypes with higher motilities, suggesting that LAMP3 was associated with migrative abilities of ESCC cells. Several studies have reported that LAMP3 regulated migrative and infiltrative phenotypes in different types of cancer cells, however, the underlying mechanism remains elusive. The purpose of this study is to explore the function and molecular mechanism of LAMP3 in ESCC metastasis.

We first analyzed the mRNA expression of LAMP3 in multiple GEO databases of ESCC. The expression level of LAMP3 in ESCC patient tissues was significantly higher than that of adjacent tissues. Moreover, LAMP3 mRNA was significantly increased in primary tumors from LNM-positive ESCC patients compared with LNM-negative patients. These findings were also further validated in the ESCC tissues as well as metastatic lymph nodes by immunohistochemistry assay. In vitro study showed that knockdown of LAMP3 significantly inhibited the migration and invasion of highly invasive ESCC cells, whereas ectopic expression of LAMP3 in less invasive parental ESCC cells significantly enhanced their motilities. In vivo metastasis model showed that LAMP3 knockdown suppressed both the lung and lymph node metastasis of ESCC cells, and the ratio of positive lymph node metastasis also decreased significantly. Mechanistically, in chemotaxis state, the phosphorylation of VASP serine 239(S239) significantly increased after LAMP3 knockdown, which was attenuated in LAMP3-overexpressing ESCC cells. We further knocked down VASP expression in LAMP3-depleted cells and found that the motility of LAMP3-depleted ESCC cells was partially restored. Next, we mutated VASP S239 to alanine (S239A) to further investigate the role of VASP S239 in the motility of ESCC cells. We found VASP S239A significantly abolished VASP S239 phosphorylation in LAMP3-depleted ESCC cells in the chemotaxis state. LAMP3-depleted ESCC cells carrying VASPS S239 mutant exhibited increased invasive behaviors in vitro and in vivo. We next sought to identify potential protein kinases responsible for VASP S239 phosphorylation in LAMP3-depleted ESCC cells. By administration of different protein kinases inhibitors,  we found that the phosphorylation level of  VASP S239 remains constant in LAMP3-depleted cells after treatment of PKA inhibitor, which also increased the motility of ESCC cells in Ctrl group and impaired the inhibitory effects of LAMP3 depletion on ESCC motility. We further knocked down the two regulatory subunits of PKA in LAMP3-depleted ESCC cells and found that the phosphorylation level of VASP S239 was significantly decreased, leading to the restoration of ESCC cell motility. To further study the molecular mechanism of PKA-mediated VASP phosphorylation after LAMP3 knockdown, we hypothesized that LAMP3 knockdown might interfere with the selective degradation processes of protein by lysosomes. We found that in the chemotaxis state, after the knockdown of LAMP3, one of the PKA regulatory subunits PRKAR1A was significantly increased. Meanwhile, the expression of PKA anchored scaffold protein (AKAP) WAVE-1 was also significantly increased. Similar results were also obtained after LAMP3-depleted cells treated with chloroquine to impair lysosome functions. The interaction between WAVE-1 and LAMP3 was also confirmed by the Co-IP assay. When we knocked down WAVE-1 expression, the phosphorylation of VASP S239 was significantly reduced after WAVE-1 knockdown in LAMP3-depleted cells. Hence, we believed that LAMP3 knockdown may impair lysosomal degradation of WAVE-1, which served as a scaffold protein for PKA to phosphorylate VASP at Ser239, leading to blockade of ESCC metastasis.

In summary, this study reveals the molecular mechanism of LAMP3 in ESCC metastasis. We found that LAMP3 knockdown increased the level of PKA scaffold protein WAVE-1, which was actively involved in PKA-mediated VASP S239 phosphorylation, further inhibiting the motility and metastasis of ESCC cells. This study also implies that lysosomes may selectively degrade specific target proteins involved in the different biological processes. The perturbation of lysosomal degradation may impair cellular homeostasis, resulting in the rewiring of the signaling network. Therefore, systematic characterization of protein substrates and regulation of lysosomal degradation will provide new insights for revealing the molecular basis of ESCC metastasis.