| Cancer is the biggest threat to human life. In2008, there were12.7million new cancercases and7.6million deaths according to statistics. A total of1.3million new breast cancercases and0.45million death cases are reported in the world each year, and breast cancer isthe commonest malignancy in women. Studies have confirmed that breast cancer progressesthrough pathological stages starting with mammary epithelial hyperplasia, progressing intoatypical hyperplasia, carcinoma in situ and invasive carcinoma. During the malignanttransformation of breast cancer, a series of complex changes in cellular biology andbiological behavior occur, and how to regulate this series of changes is unclear. Studies onthe malignant transformation and the mechanism that mammary epithelial cells progressesto breast cancer help to understand the pathogenesis of breast cancer, furthermore, it isexpected to explore a new therapeutic target.It has been confirmed that a variety of phospholipid metabolites are involved inregulating cellular signal transduction. A sphingosine metabolite, sphingosine l-phosphate（S1P） not only regulates the cell cycle as the second intracellular messenger, but alsoregulates various biological processes through the interaction with specific cell surfacereceptors, including cell proliferation, apoptosis, invasion, migration and adhesion moleculeexpression. Sphingosine kinase （Sphk） plays a key rate-limiting role in sphingolipidmetabolism during the S1P biosynthetic process. Two different SphK isoforms （SphK1andSphK2） have been identified in human and mouse tissues. The human SphK1and SphK2genes are located on chromosome17and chromosome19, respectively. Although SPHK2and SPHK1have a high degree of homology in the conserved region and comprise the samefour conserved （C1–C4） regions and carboxy terminus, both their tissue distribution andcatalytic activity are not the same. Sphk1lacks transmembrane domains, but Sphk2containsnumerous transmembrane domains. Moreover, both exhibit different expressions. Theresults mentioned above suggest that Sphk2and Sphk1have different biological functions and regulatory mechanisms. Sphk1is mainly distributed in brain, heart, lungs, liver, spleen,and hematopoietic immune system, which is the main regulator of S1P production, and isalso associated with the over-proliferation of a variety of tumor cells.SphK1is oncogenic, since it enhances colony formation in soft agar. After transfectingSphK1into NIH313fibroblasts, the growth of these cells is enhanced in vitro, and caninduce tumor formation after being transplanted into NOD/SCID mice. Sphk1isover-expressed in a variety of tumor tissues, such as lung cancer, stomach cancer,endometrial cancer, prostate cancer, head and neck squamous cell carcinoma, thyroid cancer,brain cancer, colon cancer, kidney cancer and non-Huo Hodgkin lymphoma, etc., S1P wasreported to stimulate proliferation of hepatic stellate cells and fibroblasts, resulting in liverfibrosis and even deteriorating state. However,inhibiting Sphk1can induce tumor celldifferentiation in human liver and gradually lose their malignant phenotype, indicating thatSphk1/S1P pathway may play a key role in the tumor formation of the liver cells. Kohno Met al. through establishing models of Apc Min/+mouse intestinal polyposis, increasedexpression of Sphk1was found during the malignant transformation process of intestinalpolyposis, and played an important role in the adenoma epithelial cell proliferation; while inApc Min/+SPHK （-/-） model, the adenoma epithelial cell proliferation was inhibited.Also, studies on the samples of breast cancer observed that Sphk1was not expressed innormal breast tissues, weakly expressed in atypical hyperplasia tissues, whereas stronglyexpressed in breast cancer tissues, suggesting that Sphk1may play an important role inbreast cancer formation. The above studies suggest that, Sphk1may play a key role in themalignant tumor transformation process of either stromal cells or epithelial cells.Also, Sphk1may play an important role in cancer progression. Sphk1rearranges actininto membrane ruffles according to the migration need. Up-regulating Sphk1inducesendothelial cell migration and promotes migration. Sphk1is also essential for thenucleotide-induced migration of mesangial cells. Sphk1is a complex loci of a variety ofcells surface receptors, wherein LPA, S1P, EGF receptors have been shown to be involvedin the regulation of tumor cell migration and invasion. Shida D et al. reported that theinteraction between LPA1（Lysophosphatidic acid） and EGRF receptors would increaseSphk1expression, thereby promoting cancer cell migration and invasion. Anti-metastaticmolecule KAI1can down-regulate Sphk1activity, thereby inhibiting the metastasis of pancreatic cancer cells. Also, it can down-regulate hepatocyte growth factor-mediatedSphk1activation, thus preventing liver cancer cells metastasis. Ruckhaberle E, et al. foundthat breast cancer patients with low expression of Sphk1had a lower risk of metastasis.These results suggest that, Sphk1may be a key regulator of cell migration and can promotetumor cell invasion and metastasis. However, some researchers also stated that comparedwith normal subjects, patients with prostate cancer had lower S1P levels and lower Sphk1activity in the circulating erythrocytes; moreover, in the hormone-resistant prostate cancer,decreased circulating S1P was a sign of tumor progression, and was associated withprostate-specific antigen （PSA） levels, lymph node metastasis and mortality. The studiesmentioned above suggest that Sphk1may inhibit the progression and metastasis of prostatecancer. Thus, Sphk1may promote tumor cell invasion and metastasis, but it is stillcontroversial. Sphk1may play a different role in different tumors or different tumorpatients, and its mechanism of promoting or inhibiting metastasis remains unclear,especially in breast cancer study.Through systematically reviewing the studies about Sphk1in breast cancer, we foundthat most studies focused on breast cancer cell lines, but few studies on its role in themalignant transformation on the normal breast epithelium. Meanwhile, the correlationbetween Sphk1expression and clinicopathological features and prognosis in breast cancerpatients has not been clearly defined. Further researches need to be conducted to study therole and mechanism of Sphk1in breast cancer and to investigate whether it is involved inthe breast cancer metastasis process.With respect to the above problems, we used immunohistochemistry （IHC） method todetect Sphk1protein expression in breast cancer tissues, and analyzed its relationship withboth the clinicopathological features and the expression of epithelial-mesenchymaltransition （EMT） key protein E-cadherin （E-cad）. In our study, we used chemical carcinogen7,12-demet hylbenz [a] anthrancene （DMBA） to induce the malignant transformation ofnormal human mammary epithelial cells （MCF-10A）, used RT-PCR, Western-blottingmethods to detect mRNA and protein expression of Sphk1and E-cad and analyzed therelationship between Sphk1and cell proliferation and invasion. Also, we used lentivirustransfection to achieve over-expression of Sphk1in MCF-10A cells, and used Sphk1agonists tumor necrosis factor （TNF-α）, Sphk1inhibitor agent dimethyl sphingosine （DMS） to treat the MCF-10A cells with Sphk1overexpression. Also, we used siRNA to interfereexpression of Sphk1in human breast cancer cell lines （MCF-7）, applied RT-PCR andWestern-blotting assay to detect mRNA and protein expression of Sphk1and E-cad, appliedCCK-8method to detect cell proliferation, Transwell method to detect cell invasion ability.Meanwhile, we analyzed the correlation between Sphk1and cell proliferation and invasionability, and explored the role of TNF-α/Sphk1/E-cad pathway in breast epithelial malignanttransformation and progression of breast cancer. With the above studies, we aim toprimarily investigate the role of Sphk1in breast cancer occurrence and progress and itsmolecular mechanism, so as to provide an experimental basis for further revealing thebiological function of Sphk1in breast cancer, and also to provide new ideas for thedevelopment of breast tumor molecular targets.Test methods and main results:1. Relationship between Sphk1and clinicopathological features of breast cancer andE-cad expressionMethods: IHC method was used to detect Sphk1protein expression in breast tissues,and its relationship with clinicopathological features of breast cancer and E-cad expressionwas also analyzed.Results: Higher Sphk1expression was observed in breast cancer tissues than that inbreast fibroadenoma tissues (P=1.69×10-11); Sphk1expression was associated with clinicalT stage （P=3.11×10-5）, pN stage (P=9.22×10-10), ER expression （P=0.034） and HER-2expression （P=0.013）, and had no correlation with age, menopausal status, or PR expression;E-cad and Sphk1expressions were determined to be negatively correlated （P=1×10-13）;E-cad （–）/Sphk1（+） carriers showed significantly higher ratio of axillary lymph nodemetastasis compared with E-cad （+）/Sphk1（–） carriers (P=1.3592×10-10).2. Effect of Sphk1on MCF10A cells malignant transformationMethods:（1） DMBA was used to induce MCF-10A cells, RT-PCR andWestern-blotting assay were applied to detect mRNA and protein expression levels ofSphk1and E-cad after induction, CCK-8method was used to detect cell proliferation,Transwell assay to detect the invasion level, soft agar colony formation assay to detectnon-anchorage-dependent cell growth capacity（;2）lentivirus was transfected into MCF-10Acells to achieve overexpression of Sphk1, RT-PCR and Western-blotting assay was used to detect mRNA and protein expression level of Sphk1and E-cad, CCK-8assay to detect cellproliferation, Transwell assay to detect cell invasion ability.（3）Sphk1agonist TNF-α andSphk1inhibitor DMS were added in MCF-10A cells with overexpression of transfectedSphk1, RT-PCR and Western-blotting assay was used to detect mRNA and protein levels ofSphk1and E-cad, CCK-8method to detect cell proliferation and Transwell method to detectcell invasion.Results:（1） after DMBA induction, Sphk1mRNA and protein levels in MCF-10Acells of the thirtieth generation were significantly higher than those of the non-induced cells,but E-cad expression was significantly lower; cell proliferation and invasion ability wassignificantly improved; cells formed colonies in soft agar, but failed in control cells.（2）after transfection, Sphk1mRNA and protein levels in MCF-10A-Sphk1cells wereconfirmed to be significantly higher than that of the controls （MCF-10A andMCF-10A-empty plasmid cells）.（3） after transfection, E-cad mRNA and protein expressionlevels were obviously decreased, TNF-α could enhance the ability of Sphk1in regulatingE-cad expression and DMS could inhibit this effect.3. Effect of Sphk1on MCF-7cell proliferation and invasionMethods: siRNA was used to interfere the Sphk1expression in MCF-7cells, RT-PCRand Western-blotting assay to detect mRNA and protein expression levels of Sphk1andE-cad, CCK-8cell assay to detect proliferation ability and Transwell assay to detect cellinvasion capability.Results: After siRNA transfection, Sphk1mRNA and protein expression levels ofMCF-7cells were significantly decreased, and E-cad mRNA and protein expression levelswere significantly increased. Proliferation and invasion of MCF-7cell were significantlydecreased.Conclusions:1. Sphk1is correlated with the high malignancy of breast cancer and lymph nodemetastasis.2. DMBA can induce the malignant transformation of MCF-10A cells. After themalignant transformation, Sphk1expression levels are significantly increased, E-cadexpression levels are decreased and cells proliferation and invasion capacity are enhanced;3. Transfecting the over-expressed Sphk1can enable occurrence of malignant phenotype in MCF-10A cells, down-regulate E-cad expression and increase the invasioncapacity of cell proliferation; Sphk1siRNA can up-regulate the E-cad expression of MCF-7cells d and decrease the invasion capacity of cell proliferation4. TNF-α/Sphk1/E-cad pathway may play an important role in the process of breastcancer invasion. |
PDF Full Text Request