B7-H3在调控人乳腺癌细胞糖代谢中的作用及机制

Most cancer cells show increased glucose uptake and lactate production even in the presence of oxygen. This phenomenon, known as the Warburg effect (also termed as "aerobic glycolysis", is considered as one of the most fundamental metabolic alterations during malignant transformation. There is growing evidence to support the idea that the upregulation of glycolysis in cancer cells meet, partially if not all, the energetic requirement, as well as the metabolic requirement of tumor growth. In the reprogramming of metabolism in cancer cells, The transcription factor, hypoxia-inducible factor 1 (HIF-1), plays a crucial role by inducing the expression of nearly all the enzymes involved in glycolysis, as well as glucose transporters. HIF-1 consists of an O2-regulated HIF-1 alapha subunit and a constitutively expressed HIF-1 beta subunit. In the presence of oxygen, HIF-1 alapha undergoes a posttranslational modification and degraded quickly. Under hypoxic conditions, HIF-1 alapha is stabilized and activated. B7-H3, a newly indentified member of the B7 protein family, has been shown to play either immune stimulatory or inhibitory roles on T-cell proliferation, induction of CTL response, IFN-? production in T cells, and anti-tumor activity. Recently, B7-H3 protein has been found to be overexpressed in most human tumor types, including prostate cancer, non-small-cell lung cancer, breast cancer and et al, and the correlation between high expression of B7-H3 and poor outcome has been shown in almost all the tumor types. Although the majority of previous studies focused on the immunological function of B7-H3, this broad expression pattern suggests possible nonimmunological functions of B7-H3. Recently, a series of reports from Dr. Oystein Fodstad and Dr. Ming Tans' lab in U.S. demonstrated a critical role of this protein in tumor progression, metastasis and induction of drug resistance. However, there is no report about the role of B7-H3 in cancer metabolism so far. Our preliminary data elucidated that the B7-H3 knockdown human breast cancer cells showed a lower glycolytic rate than the control cells and, to a more extent, under hypoxic condition; we also found HIF-1α and its downstream targets LDHA and PDK1 were downregulated in B7-H3 knockdown cell. Based on the above results, we hypothesized that B7-H3 might play a crucial role in promoting glucose metabolism switch to glycolysis by HIF-1α, which eventually induce human cancer cell growth. To testify it, we aim to fully study the function of B7-H3 in regulating glucose metabolism in both B7-H3-knockdown and -overexpressing human breast cancer cells and the underlying mechanism which involved in HIF-1α. This study would elucidate a novel function of B7-H3 in regulating glucose metabolism in human cancer cells. These novel findings also have important implications for the design of new approaches to target B7-H3 expressing human breast cancers.

B7-H3为免疫调节蛋白B7家族的新成员，该分子在多种类型的肿瘤细胞包括乳腺癌细胞上高水平表达，并在肿瘤发展、转移及诱导肿瘤药物抗性中有重要作用，但迄今国内外尚无B7-H3与肿瘤代谢相关的报道。我们在前期研究中发现：B7-H3 沉默的人乳腺癌细胞糖酵解水平下调，在缺氧条件下这种下调更为明显；B7-H3沉默细胞中缺氧诱导因子HIF-1α及其下游靶基因LDHA及PDK1的蛋白水平也下调。基于上述结果，我们假设"B7-H3通过HIF-1α介导对人乳腺癌细胞糖代谢的调控并进而促进癌细胞增殖"。为验证上述假设，我们将从B7-H3沉默以及B7-H3过表达等多个角度探讨B7-H3对人乳腺癌细胞糖代谢及细胞增殖的影响；并围绕HIF-1α及其上下游调节通路进行信号通路研究。本研究的完成将在国内外首次揭示B7-H3在人乳腺癌细胞糖代谢中的调控作用和机制，并为人乳腺癌患者的靶向治疗提供新的思路。

B7-H3是免疫调节蛋白B7家族的一个新成员，以往的研究大多集中在该分子强大的免疫学功能方面。近来发现该分子在多种类型的肿瘤细胞包括乳腺癌细胞上高水平表达，而且这种高水平的蛋白表达往往与肿瘤患者愈后较差显著相关,提示该分子可能具备其他的非免疫学功能。我们和其他研究者还相继发现B7-H3在肿瘤发展、转移及诱导肿瘤药物抗性中的重要作用，但具体的机制尚未阐明。本研究探讨了B7-H3在肿瘤糖代谢变化过程中的调控作用及其相关机制。首先，我们分别在正常和缺氧条件下，从B7-H3 knockdown以及糖酵解抑制剂处理等多个角度考察了B7-H3对人乳腺癌细胞糖酵解及癌细胞增殖的影响。结果发现，在正常条件下，B7-H3 knockdown的人乳腺癌细胞乳酸产生率和葡萄糖摄取率显著降低，这种差异在缺氧条件下表现得更为显著；B7-H3 knockdown细胞增殖速度也显著降低，对糖酵解抑制剂oxamate和葡萄糖缺乏较不敏感，细胞生长的受抑制程度较低；在以半乳糖取代葡萄糖的培养基中，B7-H3 knockdown 细胞与对照组细胞（B7-H3高表达）的生长速度几乎一致；但B7-H3 knockdown对人乳腺癌细胞氧化磷酸化过程几乎无影响。上述结果表明，B7-H3可促进人乳腺癌细胞糖代谢由氧化磷酸化方向往糖酵解方向流动，促进细胞糖酵解，进而促进细胞的增殖。上述结果亦在人结直肠癌细胞系中得到证实，提示B7-H3对糖代谢的调节作用不仅仅限于人乳腺癌细胞中，而是在肿瘤糖代谢的调控过程中有普遍的作用。机制研究方面，我们发现B7-H3 knockdown细胞中HIF-1及其下游的直接靶蛋白LDHA和PDK1(两者均为糖酵解的关键酶)的蛋白水平均显著下调；瞬时转染B7-H3表达质粒使B7-H3低表达的HMEC细胞和231-shB7-H3细胞B7-H3 和HIF-1蛋白水平同时升高。上述结果表明B7-H3可通过HIF-1直接调节其下游靶蛋白LDHA和PDK1的蛋白表达水平，进而调控人肿瘤细胞的糖酵解过程；同时，我们还发现Stat3可能也参与了B7-H3对HIF-1α的调控过程。总之，本研究揭示了B7-H3在人肿瘤细胞糖代谢中的调控作用和机制，这些结果可为人乳腺癌患者的靶向治疗提供新的思路。