ERK2調控G-CSF在侵略性癌細胞中的表現

Abstract

顆粒性白血球群落刺激性因子 (granulocyte colony-stimulating factor, G-CSF)為醣蛋白的一種，可由單核球細胞、巨噬細胞、內皮細胞分泌，其主要功能為促進嗜中性白血球的分化與增生。G-CSF也可作用在其他細胞，例如穩定骨髓中單核球細胞、幹細胞的分化與增生，促進輔助性T細胞的活化與抑制發炎反應等，也因此目前G-CSF在治療疾病的臨床應用上相當廣泛。譬如在癌症治療方面，重組G-CSF目前被當作是化療佐劑的一種。由於接受化療的病人通常會有嗜中性白血球減少症，藉由G-CSF的作用來提高病人血液中的嗜中性白血球數量，以降低病人在接受治療過程中受到感染的風險。此外，在白血病病患接受化療之前，先給予重組G-CSF也被認為能提高癌細胞對治療藥劑的敏感性，藉此提高癌症治療的效率。然而當G-CSF表現過多時，也會造成免疫相關疾病。例如類風濕性關節炎常因為過多的G-CSF促使過量嗜中性白血球增生，最後導致嚴重的發炎反應產生。所以了解內生性G-CSF表現調控相當重要，但目前對此G-CSF在細胞中的調控尚未完全清楚。近年來有許多病例報告指出，在某些患有惡性腫瘤的病人檢體G-CSF有大量表現情形，隨著癌細胞侵略性增加，其G-CSF表現也相對提高。後續也有文獻提出，癌細胞表現的G-CSF會增加其轉移能力與侵略性。通常癌細胞的轉移與侵入對於癌症的致死率有高度相關性。因此了解G-CSF在癌細胞的表現與在癌細胞中是如何被調控對於癌症的治療具有一定的幫助。所以本研究的目的是想藉由不同侵入性的癌細胞來了解G-CSF的表現與G-CSF在癌細胞中的調控。 首先我們藉由RT-PCR與ELISA的實驗結果得知，在具較高轉移能力與侵入性的肺癌細胞CL1-5、乳癌細胞MDA-MB-231 IV1、口腔癌細胞SCC-15與HSC-3皆會表現較高量的G-CSF。接著以MEK1/2抑制劑PD98059和U0126處理細胞，發現在抑制磷酸化ERK1/2表現下，各癌細胞中的G-CSF表現量都明顯減少。此外，藉由shRNA knockdown MDA-MB-231 IV1細胞的ERK2蛋白表現，細胞中的G-CSF表現也明顯減少。我們也利用細胞激素TNF-α刺激MDA-MB-231 Parental細胞，發現MDA-MB-231 Parental細胞中G-CSF的表現量有明顯提高，且誘發的G-CSF表現會因U0126的處理而被抑制。在TNF-α的刺激之下，MDA-MB-231 Parental細胞中的磷酸化ERK1/2會提高表現，當U0126存在時，磷酸化ERK1/2的表現也隨即被抑制了。由此可知，癌細胞表現G-CSF會透過MAPK/ERK訊息傳遞路徑來調控。此外，我們藉由Western blot的方法也發現，在MDA-MB-231 IV1細胞中的磷酸化C/EBPβ表現量比Parental細胞表現的還要高，在MEK1/2抑制劑的處理之下，磷酸化的C/EBPβ表現量也被抑制了。由以上結果了解，MAPK/ERK訊息傳遞路徑去調控G-CSF表現會透過磷酸化的C/EBPβ。我們也用其他訊息傳遞路徑的蛋白激酶抑制劑去檢測癌細胞表現G-CSF是否會透過其他調控，由實驗結果顯示PI3K抑制劑，Ly294002會抑制SCC-15細胞的G-CSF表現。但對於其他細胞，PI3K、JNK和mTOR抑制劑都不影響G-CSF表現。由此可知在這些癌細胞中，對於G-CSF的調控主要是經由MAPK/ERK訊息傳遞路徑。最後我們檢測了G-CSF受器在細胞中的表現，發現這些癌細胞並不表現G-CSF受器。 由以上實驗結果，我們可以確定在具高度轉移與侵入性的癌細胞中會表現較高量的G-CSF。在這些癌細胞中，G-CSF的表現主要是透過MAPK/ERK與C/EBPβ訊息傳遞路徑的調控。但在癌細胞中，ERK是如何影響C/EBPβ對G-CSF的調控、G-CSF如何影響癌細胞轉移與侵入性的詳細機制仍須進一步的探討。

Granulocyte colony-stimulating factor (G-CSF) is one of the glycoprotein that is secreted by monocytes, macrophages, fibroblasts and endothelia cells. The major function of G-CSF is to support the maturation, differentiation and proliferation of neutrophils. G-CSF mobilizes monocyte and stem cell from bone marrow into the blood and improves Type 2 T-helper cell response. G-CSF also induces the expression of anti-inflammatory cytokines in monocytes. In clinical, recombinant G-CSF (rG-CSF) has been used as an adjuvant for the prevention and amelioration of neutropenia following cancer chemotherapy. Case reports demonstrated that patients have received the injection of rG-CSF have a reduced risk of infection. Administrating rG-CSF before chemotherapy increases the sensitivity of cancer cells to drugs which then increases the efficacy of therapy. However, G-CSF over-expression is linked to inflammatory diseases, such as Rheumatoid Arthritis. G-CSF increases the number of neutrophils in bone marrow which leads to chronic inflammation-response in specific tissues. Understanding the regulation of G-CSF in immune and cancer cells is very important for disease therapy. In recent years, many case reports showed that G-CSF producing tumors are malignant and highly invasive; and the tumors grow rapidly and prognosis is poor. Therefore, G-CSF has been considered as a therapeutic condidate; and understanding how G-CSF is regulated in cancer cells is equally important. In this study, we investigated the expression levels of G-CSF in various cancer cells, and the involvement of ERK in the expression of G-CSF in these cancer cells. We found that expression level of G-CSF is higher in cancer cells with higher migration and invasion ability, including CL1-5 lung cancer cell, MDA-MB-231 IV1 breast cancer cell, SCC-15 and HSC-3 oral cancer cells. When treating these cells with MEK1/2 inhibitor PD98059 and U0126, the cells resulted in inhibition of G-CSF expression in these cancer cells. Knockdown ERK2 in MDA-MB-231 IV1 breast cancer cell resulted in decreased G-CSF mRNA expression; while knockdown ERK1 resulted in higher G-CSF mRNA expression. Suggesting that activation of ERK2 is essential for G-CSF expression, but not ERK1. Treatment with TNF-α led to phosphorylation of ERK1/2 and up-regulation of G-CSF in MDA-MB-231 Parental cells. Pre-treatment with U0126 prevented ERK1/2 phosphorylation and G-CSF expression in these cells. According to these studies, we confirm that activation of ERK is essential for G-CSF expression in invasive cancer cells. Western blot showed that MDA-MB-231 IV1 cells express more phospho-C/EBPβ than MDA-MB-231 Parental cells. Treatment with U0126 inhibited C/EBPβ phosphorylation in MDA-MB-231 IV1 cells. It is possible that ERK regulates G-CSF, at least in part, via phospho-C/EBPβ. G-CSF expression in SCC-15 cells can be inhibited by LY294002, a PI3K inhibitor. However, other inhibitors, such as SP600125 and Rapamycin, have no effect on G-CSF expression in cancer cells used in this study. The results demonstrated that ERK2 is the major regulator of G-CSF expression in these cancer cells. Finally, we showed that the cancer cells we studied do not express G-CSF receptor. In summary, we demonstrate that G-CSF is up-regulated through an ERK2 dependent pathway in cancer cells with high level of migration and invasion. However, it is not clear how ERK regulates G-CSF, the mechanism underlie requires further studies.