Role of Selenium-containing Proteins in Cancer and Development

许多硒领域的研究人员提出，硒化合物对硒的许多健康益处负有责任，而另一些人则认为硒蛋白可能起作用。这些健康益处包括预防癌症、心脏病和其他心血管和肌肉疾病，抑制病毒表达，延缓艾滋病毒阳性患者的艾滋病进程，减缓衰老过程，并在哺乳动物发育、男性生殖和免疫功能方面发挥作用。我们实验室首次提出，这些健康益处很大程度上是由于硒蛋白中的硒作为氨基酸--硒半胱氨酸(SEC)的存在。为了阐明硒蛋白在癌症预防和发展中的作用，我们对几种硒蛋白的功能进行了表征。在过去的一年里，我们专注于更详细地研究两种硒蛋白，硫氧还蛋白过氧化物酶1(Tr1)和谷胱甘肽过氧化物酶4(Gpx4)的作用。我们之前已经证明，利用RNA干扰技术在肺癌细胞系中敲除TR1会导致大多数恶性表型更接近于正常细胞，这表明TR1缺乏是抗肿瘤的。在过去的一年里，我们专注于TR1在癌症发展中作用的分子基础。我们观察到，在硒蛋白中，TR1在许多癌细胞系中唯一过表达，并且它在由k-ras驱动的小鼠恶性细胞系DT细胞中被敲除，导致了亲代(正常)细胞特有的形态变化。当这些细胞在缺乏血清的培养液中生长时，Tr1基因缺陷的恶性肿瘤细胞失去了自给自足的生长，出现了S期的缺陷进展，并降低了DNA聚合酶α的表达。这些研究提供的证据表明，TR1对于恶性细胞生长信号的自给自足至关重要，TR1在很大程度上维持了恶性细胞的癌症特征，并进一步证实了TR1是癌症治疗的主要靶点。随着我们对TR1基因敲除的研究扩展到包括乳腺癌细胞系和肺癌细胞系，我们发现TR1基因缺失的癌细胞比对照细胞对肿瘤坏死因子-α诱导的细胞凋亡更敏感，TR1基因缺失可能与ERK活性降低有关，侵袭和趋化能力显著降低，其中一种MAP激酶JNK2可能参与了乳腺癌细胞系的侵袭和趋化作用。作为我们关于TR1在癌症中的作用的项目的一个副业，我们挑战了癌症干细胞假说，该假说认为肿瘤的生长是由一种罕见的亚群细胞驱动的，称为癌症干细胞。支持这一理论的研究在很大程度上是基于异种移植实验，在异种移植实验中，人类癌细胞在免疫受损的小鼠中生长，只有肿瘤干细胞产生肿瘤，肿瘤干细胞通常占恶性肿瘤的不到1%。我们使用我们的小鼠肺癌和乳腺癌细胞系来表明，在这两个恶性细胞系中随机选择的单个细胞产生的所有克隆在同种异体组织相容小鼠移植后形成肿瘤。我们的研究表明，大多数恶性细胞而不是癌症干细胞可以维持肿瘤，必须重新评估癌症干细胞理论。我们还研究了Gpx4在细胞内的作用。Gpx4是一种含硒的抗氧化酶，其细胞内功能尚未确定。为了阐明Gpx4在细胞内的功能，我们在小鼠成纤维细胞系(NIH3T3细胞)中敲除了Gpx4，当该酶的水平降至正常水平的80%以下时，该酶会对细胞造成严重的损伤。其表达的部分下调导致了与膜功能相关的形态变化。出乎意料的是，Gpx4基因敲除细胞的活性氧水平没有变化，但这些细胞表现出氧化脂质副产物水平的高度增加。到目前为止，这些数据已经确定了Gpx4在膜脂过氧化代谢中的重要作用，但令人惊讶的是，作为一般抗氧化酶的作用是有限的。

Many investigators in the selenium field have proposed that selenocompounds are responsible for the many health benefits attributed to selenium, while others have suggested that selenoproteins are likely responsible. These health benefits include preventing cancer, heart disease and other cardiovascular and muscle disorders, inhibiting viral expression, delaying the progression of AIDS in HIV positive patients, slowing the aging process and having roles in mammalian development, male reproduction and immune function. Our laboratory was the first to propose that these health benefits are due largely to the presence of selenium in selenoproteins as the amino acid, selenocysteine (Sec). To elucidate the role of selenoproteins in cancer prevention and development, we are characterizing the function of several selenoproteins. During the past year, we have focused our attention on studying in greater detail the role of two selenoproteins, thioredoxin peroxidase 1 (TR1) and glutathione peroxidase 4 (GPx4). We had previously shown that the knockdown of TR1 using RNA interference technology in a lung cancer cell line resulted in most of the malignant phenotypes being reversed more towards those of normal cells suggesting that TR1 deficiency is antitumorigenic. This past year, we have focused on the molecular basis of TR1s role in cancer development. We observed that among selenoproteins TR1 is uniquely over-expressed in a number of cancer cell lines and its knockdown in DT cells, a malignant mouse cell line that is driven by k-ras, resulted in morphological changes characteristic of the parental (normal) cells. When these cells were grown in serum-deficient medium, TR1 deficient malignant cells lose self-sufficiency in growth, show defective progression in their S phase and decreased expression in DNA polymerase alpha. These studies provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely to maintain the cancer characteristics of malignant cells and further substantiate that TR1 is a primary target in cancer therapy. In expanding our studies on TR1 knockdown to include a breast cancer cell line as well as the lung cancer cell line, we have found that TR1 deficient cancer cells are more sensitive to TNF-alpha induced apoptosis than control cells and TR1 deficiency is possibly involved in decreased activation of ERK, that invasion and chemotaxis are significantly reduced and that one of the MAP kinases, JNK2, may possibly be involved in invasion and chemotaxis in the breast cancer cell line. As a sidelight to our project on the role of TR1 in cancer, we challenged the cancer stem cell hypothesis which posits that tumor growth is driven by a rare subpopulation of cells, designated cancer stem cells. Studies supporting this theory are based in large part on xenotransplantation experiments wherein human cancer cells are grown in immunocompromised mice and only cancer stem cells, often constituting less than 1% of the malignancy, generate tumors. We used our mouse lung and breast cancer cell lines to show that all colonies derived from randomly chosen single cells in these two malignant cell lines form tumors following allografting histocompatible mice. Our study suggested that the majority of malignant cells rather than cancer stem cells can sustain tumors and that the cancer stem cell theory must be reevaluated. We also examined the intracellular role of GPx4. GPx4 is a selenium-containing, antioxidant enzyme whose intracellular function has not been established. To elucidate its function intracellularly, we knocked down GPx4 in a mouse fibroblast cell line (NIH3T3 cells) that resulted in severe damage to the cells when the level of this enzyme was reduced below 80% of its normal level. Partial reduction in its expression led to morphological changes associated with membrane function. Unexpectedly, GPx4 knockdown cells showed unchanged levels of reactive oxygen species, but these cells manifested highly increased levels of oxidized lipid byproducts. The data thus far have established an essential role of GPx4 in the metabolism of membrane lipid hydroperoxides, and surprisingly, a limited role as a general antioxidant enzyme.