The Effect Of PAK1and HIF-1α In The Invasion And Metastasis Of The Colorectal Carcinoma

[Bacground]Colorectal cancer(CRC) is one of the most common types of malignant tumors worldwide.In China,CRC is the fourth leading cause of cancer-related mortality. The incidence rate of CRC in china is increasing fast during the past decades. Although great advances have been made in the diagnosis and treatment of this cancer, the survival rate of CRC patients is not improved profoundly. Metastasis is the main cause affecting the therapeutic efficacy and leading to the death of cancer patients. Like other malignancies, the occurrence and development of CRC maybe a multi-step complex process involving multiple factors. From the initial benign adenomatous polyps to the later metastasis, the entire process could take several years or even decades. Since its development showed significant stage specificied characteristics, the CRC has been regarded as the "model of tumor" among various malignancies.The past studies showed that the incidence of CRC is closely related to cell cycle, apoptosis, signal transduction, invasion and metastasis. To find genes associated with metastasis and elucidate their functions in CRC and to search for the ideal markers for the early diagnosis and prognosis will be helpful for clinical diagnosis, prognosis analysis and possible target treatment of CRC. In recent years, researchers has paid close attention to PAK and HIF-1α, which has abnormal expression in many tumors and may contribute to the growth infiltration and metastasis of tumor cells. The expression of PAK1and HIF-1α is related to the diagnosis or prognosis in some tumors. However, It has not been exactly identified about the molecular mechanism and the clinical value of the high expressions of PAK and HIF-1α.PAKs(p21-activated kinase) family has six members in humans including PAK1-3and PAK4-6, PAK1is the first identified and best characterized member of the PAK family of serine/threonine protein kinases. The gene of PAK1locate between11q13.5and q14area in human chromosome, which plays important roles in cells forming, movement, survival and proliferation regulation. PAK1is also involved in other fundamental cellular processes, including cell division, apoptosis, and gene transcription. In molecular biology,such a crucial role in cellular life explains the fact that this kinase is subjected to a complex and exquisite regulation capable of integrating a variety of signals according to specific physiological needs. The molecular mechanisms of PAK1regulation in human cancers remain to be further clarified.CRC develops through multiple processes involving progressive disruption of cell proliferation, differentiation,and survival. Mutations in KRas and BRaf have been found in40%and10%of CRC, respectively. KRas and BRaf can activate the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). The overexpressions and activations of the MAPK and ERK play important roles in the progression of CRC. Alternatively, KRas stimulates the phosphatidylinositol3-kinase (PI3K)/AKT-dependent pathway. Mutations in PI3K occur in30%of epithelial cancers, and22%of CRC mutations in both KRas and PI3K. we have investigated the importance of PAK1in the molecular biology of the CRC cells. PAK1is the downstream effector of KRas and BRaf.Through activating KRas and BRaf, PAK1can promote transformation through facilitating the ERK/MAPK pathway and enhance cell migration and survival by stimulating AKT. PAK1expression increases with the progression of CRC. In this study, we have investigated the importance of PAK1in the biology of colon cancer cells. Reduction of PAK1expression decreased the activities of ERK and AKT leading to decreased cell proliferation, migration/invasion, and survival. Dual inhibition of ERK and AKT suppressed these cellular proce sses to levels comparable to those achieved by reduction of PAK1expression, whereas inactivation of either the ERK or AKT pathway alone partially inhibited cell migration/invasion and survival and had no effect on proliferation. Since KRas activates PAK1via PI3K-dependent pathways, and mutations in KRas and PI3K play important roles in CRC, it is possible that PAK1functions as a convergence point receiving signals from Ras and PI3K and stimulates CRC to grow and migrate by activation of AKT and ERK. In conclusion PAK1stimulates CRC cell proliferation, migration, invasion, and survival via ERK-and AKT-dependent pathways. These findings establish the central importance of PAK1in CRC signal transduction and clarify the mechanism by which PAK1regulates CRC growth and migration.HIF-1(hypoxia inducible factor-1) is the heterodimeric transcription factor composing by the subunits of a and β,its expression and activity is tightly regulated by the cellular oxygen concentration, the activity of HIF-1is mainly regulated by the subunit of α. HIF-1α (A nuclear transcription factor) is commonly found in the hypoxic conditions of the human and mammalian cells, it plays a central linking role in tumorigenesis and promote the progression of tumor.In the tiny solid tumors of angiogenesis stage, HIF-1α can provide energy to the tumor cells by regulating the glycolysis and enhancing the body’s tolerance to the hypoxia,when the tumors expand to few millimeters, HIF-1α can promote the proliferation, invasion and metastasis of the tumors by controling the angiogenesis and adjusting the oxygen transporting capacity.HIF-1α can regulate the expressions of more than40kinds of genes (eg, of Snail, The Slug, Twist, the SIP-1, etc.) and it palys important role in maintaining the energy metabolism of tumors, promoting angiogenesis, enhancing the invasion and metastasisan of the tumors.The molecular mechanisms of HIF-la regulation in human cancers remain to be further clarified.The EMT(epithelia-mesenchymal transition) refer to the progression of epithelial cells(tightly junction and adhesion between cells) changing into the mesenchymal cells(activity and move freely in the cell matrix).EMT plays an important role in the ability obtaining for the invasion and metastasis of the squamous cell carcinoma. Important transcription factor involved in the regulation of the EMT include Twist, Snail, Slug and SIP-1, all of which can inhibit the expression of E-cadherin (an important cell inhibitor for the invasion and metastasis of the tumor cells) and downregulate the E-cadherin, ultimatly inducing the progression of the EMT.It has been found that there are synergies between the expressions of HIF-1α and EMT-related transcription factors inside the tumor, HIF-la can regulate and induce the expressions of the EMT-related transcription factors(Twist，, of Snail, The Slug, SIP-1) directly or indirectly and then promote the progression of the EMT by coordinating the interaction of the various transcription factors. HIF-1α and Twist are part of the bHLH(basic helix-loop-helix) family members and widely involved in the regulation of organ development, tumorigenesis, cell proliferation and differentiation processes. Hypoxia response element (HRE) has been confirmed in the neighboring promoter of Twist, Yang have found that HIF-la could upregulate the expression of Twist and promote the formation of EMT and ultimately to facilitate the invasion and metastasis of the tumor by combining with the HRE.HIF-la closely linked with the occurrence of the EMT which can promote the overexpression of Snail and SIP1. Under hypoxic conditions, HIF-1α can reduce the expression of E-cadherin and promote the progression of the EMT by activate the transcription factor SIP1and prevent the convergence of PO1II and E-cadherin.lysyl oxidase like2(LOXL2) and lysyl oxidase (LOX) have been shown to be HIF-1targets which could regulate Snail during hypoxia induced metastasis by the progess of EMT. HIF-1α can promote the transcription of the MT4-MMP and the progression of EMT by activating the Slug and combining with the E-box(in the neighboring promoter of MT4-MMP).In conclusion, the activation of HIF-1a is a critical microenvironmental factor during tumor progression which can regulate multiple genes participating in the tumor growth,angiogenesis,progression of the EMT, invasion and metastasis of the tumor.[Objective]We hypothesized that PAK1and HIF-la may play important roles in the invasion and metastasis of the CRC by discussing the molecular mechanisms that PAK1and HIF-la promote the invasion and metastasis of tumor.We absence the related research reports by combinly detecting the expressions of PAK1and HIF-1α in the colorectal cancer currently,so we will combinly detect the expressions of PAK1and HIF-la in the level of protein and the level of mRNA in the CRC respectively and then research the effects of PAK1and HIF-1a in the invasion and metastasis of the CRC as well as analyzing the relationship between them.[Methods]1, We collected60cases of CRC from Shunde First People’s Hospital Southern Medical University in March2011to October2011.36males and24females, aged51-82(median66).34cases of colon cancer,26cases of rectal cancer.10cases of well differentiated,31cases of moderately differentiated,19cases of poorly differentiated about histological grade.10cases of A,19cases of B,19cases of C,12cases of D about Dukes staging.2,We used the methods of fluorescence in situ hybridization and immunohistochemistry to detect the expressions of PAK1and HIF-1a in60colorectal carcinoma specimens and normal colorectal mucosa tissue respectively and then analyzed the expression significance and correlation of PAK1and HIF-1a in the colorectal carcinoma.3, We analyzed the expression of PAK1and HIF-1a in the level of mRNA and protein with McNemar(P<0.05, results have statistically significant).We analyzed the connection between the expression of PAK1and HIF-1a and the clinical pathological parameters of CRC with pearsion x2(P<0.05, results have statistically significant).We analyzed the correlation between PAK1and HIF-1a in the level of protein with Spearman(P<0.05, results have statistically significant).[Results]1, The positive expression rate of PAK1-mRNA in60cases of normal colorectal rectal mucosa tissue was8.33%(5/60),in60cases of CRC was70.00%(42/60), the positive expression rate of PAK1-mRNA in CRC was significantly higher than PAK1-mRNA in normal colorectal rectal mucosa tissue(x2=47.881P<0.001). The positive expression rate of PAK1-protein in60cases of normal colorectal rectal mucosa tissue was11.67%(7/60), in60cases of CRC was76.67%(46/60),the positive expression rate of PAK1-protein in CRC was significantly higher than PAK1-protein in normal colorectal rectal mucosa tissue(x2=51.400P<0.001). The positive expression rate of PAK1-mRNA and PAK1-protein in CRC was void of statistically significance(x2=0.682P=0.268)2, The positive expression rate of HIF-1α-mRNA in60cases of normal colorectal rectal mucosa tissue was20.00%(12/60),in60cases of CRC was73.33(%) (43/60), the positive expression rate of HIF-1α-mRNA in CRC was significantly higher than HIF-1α-mRNA in normal colorectal rectal mucosa tissue(x2=32.257P<0.001). The positive expression rate of HIF-1α-protein in60cases of normal colorectal rectal mucosa tissue was16.67%(10/60), in60cases of CRC was78.33%(47/60),the positive expression rate of PAK1-protein in CRC was significantly higher than PAK1-protein in normal colorectal rectal mucosa tissue(x2=45.750P<0.001). The positive expression rate of HIF-1α-mRNA and HIF-1α-protein in CRC was void of statistically significance(x2=0.711P=0.264).3, The positive expression rate of PAK1-mRNA in CRC and the normal colorectal rectal mucosa tissue was void of statistically significance(x2=1.564P=0.211), The positive expression rate of HIF-1α-mRNA in CRC and the normal colorectal rectal mucosa tissue was void of statistically significance(x2=0.045P=0.832). The expressions of PAK1-mRNA and HIF-1α-mRNA have statistically significant in dukes stage of the CRC(P<0.05). The expressions of PAK1-mRNA and HIF-1α-mRNA with lymph node metastasis were significantly higher than those without lymph node metastasis in CRC (P<0.05). The expressions of PAK1-mRNA is not connected with the grade of differentiation of the CRC (x2=2.286P=0.130), The expressions of HIF-1α-mRNA is connected with the grade of differentiation of the colorectal cancer cell (x2=10.260P=0.003).4, The expressions of PAK1and HIF-1a in the level of protein in CRC were positively correlated(r=0.293,P=0.023).[Conclusions]1, PAK1-mRNA has overexpression in the CRC, The expressions of PAK1-mRNA and PAK1-protein have no significant differences in the CRC.2, HIF-1α has overexpression in the CRC, The expressions of HIF-1α-mRNA and HIF-1α-protein have no significant differences in the CRC. 3, The expressions of PAK1-mRNA has no significant difference in the Colon cancer and Rectal cancer respectively.Overexpressions of PAK1-mRNA has associated with the invasion of tumor and metastasis of the lymph node in the CRC. The expressions of HIF-1α-mRNA has no significant difference in the Colon cancer and Rectal cancer respectively.Overexpression of HIF-1α-mRNA has associated with the invasion of tumor and degree of tumor differentiation and metastasis of the lymph node in the CRC.4, PAK1and HIF-1α may have a mutually reinforcing effects in the invasion and metastasis of the colorectal carcinoma.