Gastric cancer (GC) is the second cause of cancer death and almost a million people suffer from it every year in worldwide. Given that GC high-risk people can be prevented from GC development through controlling the environmental factors, it is undoubtedly im-portant to find out the genetic factors for discrimination of GC high-risk people. However, the number of already known genetic factors is still fewer in GC than in other main cancers.
Maspin, also called as SERPINB5 (SERine Proteinase INhibitor B family), was firstly known as a tumor suppressor in breast cancer. Its biological functions discovered so far in-clude the inhibition of metastasis and angiogenesis, and the promotion of cell adhesion and apoptosis. According to prior studies maspin has different roles in different types of tissues and even shows no function or expression in a few other cancers. Thus, it is interesting to study the function of maspin in an organ-specific manner.
Previous researches for cancer-related genetic factors mostly provided us information about gene names or risky loci but it is not sufficient to acquire real targets for treating GC patients. Here, we investigated the clinical relevance and the role of maspin in GC. It will give proper genetic markers and treatment planning for GC.
To estimate the genetic association of maspin with GC in Korea, we collected a total of 865 unrelated Korean participants including 255 diffuse type gastric cancer (DGC) cases, 179 intestinal type gastric cancer (IGC) cases and 431 unaffected controls with their written informed consent at Seoul National University Hospital, Hanyang University Guri Hospital, Chungnam National University Hospital, and Eulji University Hospital in Korea. 15 tagging SNPs (tSNP) were selected to cover from promoter to 3’ UTR of maspin (from 8000 bp upstream region to 3000 bp downstream region of maspin, tSNP r2 cut off P value=0.7). Among them, 1 tSNP was excluded at primer design step and genotyping result of 2 tSNPs was counted out because of low Hardy-Weinberg Equilibrium (HWE). Genotypic associa-tion with susceptibility to GC was statistically analyzed for all 12 tSNPs according to a co-dominant, dominant, and recessive genetic model using multivariate logistic regression analysis with adjustment for age and gender. All statistical analysis and experiments were separately proceeded in DGC and IGC. Two tSNPs, rs3744941 in promoter and rs8089104 in intron 1, showed significant association with susceptibility to DGC in dominant model (rs3744941 P=0.0038, rs8089104 P=0.002) but not to IGC. Based on the information of tSNPs we got more elaborated results using imputation. A total of 178 SNPs located from position 59,282,378 to 59,328,715 on chromosome 18q21.3 (1000 genomes data on June, 2011) were imputed by the 12 typed SNPs with the 1000 genomes data for the Chinese (CHB) and Japanese (JPT) populations using the MaCH v1.0.16 program. We found a sig-nificant association with DGC in a region spanning from distal promoter to intron 1 (domi-nant model p = < .01)
Maspin expression level has been known to be important in suppressing tumors. Pre-vious studies demonstrated that higher expression level of maspin was detected in normal gastric tissues than in GC tissues, but it was still not enough to conclude that maspin is a tumor suppressor in GC. So we firstly focused on significantly-associated tSNP in promo-ter, rs3744941 and found two haplotype constructs (non-risk haplotype and risk haplotype) including SNPs having more than 0.9 r2 with rs3744941. To investigate the effects of those haplotypes on promoter activity, luciferase activity was measured from the reporter vectors containing each promoter haplotypes followed by the luciferase gene. Risk haplotype-driven luciferase activity was lowered by 40 % as compared with non-risk haplotype-driven one in DGC cell line, MKN45, but each haplotype-driven luciferase activity was almost same in IGC cell line, MKN28 (cells were obtained from ATCC), which supported previous expression tests in GC tissues. Thus, it is likely that maspin expression is reduced in GC patients carrying the risk SNPs. To elucidate the role of maspin expression level in DGC tumorigenesis, maspin knockdown cell lines were generated using shRNA in MKN45. Maspin knockdown caused a 2-fold increase in growth rate as compared to control. In addi-tion, overexpression of maspin in MKN45 resulted in reduction of growth rate, demonstrat-ing that maspin plays a suppressive role in DGC growth.
In an attempt to explain how maspin affects on MKN45 proliferation, we examined both cell cycle and apoptosis. shRNA-mediated stable knockdown of maspin accelerated cell cycle progression in DGC cells. Peak of G2/M phase in control was shown at 12 hours after release from G1/S block, but in maspin knockdown cells, the peak appeared at 10 hours. However, neither up-regulation nor down-regulation of maspin led to changes in apoptotic cell death as compared to control. Therefore, maspin contributes to DGC cell pro-liferation via controlling the cell cycle speed.
So far, it has not been clearly identified in which signaling pathway maspin is involved in cancer. PI3K-, E2F1- and MEK-pathway were suggested to be related with maspin and thus, we firstly examined the proliferation rate of DGC after treatment of PI3K and MEK inhibitors. Both inhibitors did not cause any changes in DGC proliferation and maspin ex-pression level also was not changed. However, overexpression of E2F1 decreased the ex-pression level of maspin, which thereafter led to the invigoration of Cdc25c activity, whe-reas there was no alteration of E2F1 expression in maspin knockdown MKN45. In addition, an inactive, phosphorylated form of Cdc2 was increased in maspin down-regulated MKN45 and E2F1-overexpressed MKN45, so we concluded that lower level of maspin in DGC might accelerate G2/M transition via E2F1 pathway, especially by the activation of Cdc25c and cdc2.
We also tried to investigate other functions of maspin briefly, like angiogeneic or inva-sive properties, in DGC. Both the expression of VEGF (vascular endothelial growth factor) and the invasion capacity were not significantly altered when maspin expression was sup-pressed, but these results were not sufficient to conclude whether maspin suppressed angi-ogenesis and invasion, or not. Further functional study is required in the future to confirm more specific signaling pathway and other maspin functions in DGC cell.
In this study, we revealed the association of maspin with DGC and found it as a tumor suppressor in DGC. Of note is that the risk SNPs on maspin promoter are likely to reduce maspin expression and thereby accelerate cell cycle progression in DGC. These findings will contribute to give better guidelines for the assortment of DGC high-risk group and target selection for efficient treatment.
위암은 한국에서 가장 빈번하게 일어나는 암이며 전 세계적으로는 4번째로 많이 발생하는 암으로, 암으로 인한 사망자 수는 2위에 달한다. 알려져 있는 위험 요소로는 성별, 나이, nitrite류, 유전적 요인 등이 있으며 현재까지 다른 암들에 비해 유전적 요인이 많이 밝혀져 있지 않았다. 로렌 분류법에 따르면 위암은 장형위암과 미만형 위암으로 나뉘며 두 유형의 암은 유래와 발생과정이 다르기 때문에 확연히 구분되는 특성을 가진다. 미만형 위암의 경우 장형 위암에 비해 젊은 나이에 나타나며 침습적인 성장패턴을 보이고 특정 성별에 치우쳐 발생하는 경향을 보이지 않지만, 장형 위암은 여성에 비해 남성에게서 2배 정도 더 많이 나타나고, 확장형 성장 패턴을 보인다. 위암의 두 유형을 구별하여 감수성 유전변이들을 찾아내는 일은 위암의 예방과 진단, 치료에 중요한 역할을 할 것으로 예상되지만 아직 위암의 유형별로 감수성을 가지는 것으로 알려진 유전 변이들은 많지 않다.
본 실험에서는 암이 생성되는 기관에 따라 다른 암 억제 역할을 한다고 알려진 SERPINB5 (serpin peptidase inhibitor, clade B [ovalbumin], member 5) 라고도 불리는 maspin이 위암에서 하는 역할과 새로운 기능을 규명하였다. Maspin은 미만형 위암에 감수성을 가지고 있었으며 장형 위암과는 연관성을 보이지 않았다. 특히 maspin의 프로모터 지역에 있는 유전변이들이 미만형 위암 특이적 신호를 보였으며 (예를 들어, rs3744941에서, odds ratio = 1.8, 95% confidence interval = 1.2 to 2.7, P = .0038), 위암 감수성을 가지는 유전변이들을 포함한 maspin의 프로모터는 미만형 위암 세포에서 정상에 비해 40% 정도만 활성을 나타내고 있었다. 이렇게 maspin의 발현량이 줄어들 경우 세포의 증식 속도가 2배 정도 빨라졌으며, 반대로 maspin의 양을 증가시켰을 경우 증식 속도가 줄어드는 것이 관찰 되었다. Maspin의 발현량 변화는 세포사멸에 영향을 미치지 않았지만, maspin의 양이 감소할 경우 세포 주기가 빨라 졌기 때문에 미만형 위암 세포의 증식속도 조절은 maspin이 줄어들면서 세포 주기를 촉진시키기 때문으로 사료된다.
Maspin의 세포 주기 조절은 E2F1이 관여하는 경로를 거칠 것으로 추정된다. Maspin의 발현 감소는 E2F1의 발현량에 영향을 미치지 않았지만, E2F1의 과발현에 의해 maspin 발현량이 감소하였고, 감소한 maspin에 의해 E2F1이 G2/M기 전환이 일어날 때 조절한다고 알려져 있는 요소들 가운데 Cdc25c의 활성 증가와 비활성 cdc2 양의 감소가 관찰되었다. Cdc2의 활성이 Cdc25c에 의해 증가하는 것은 잘 알려진 사실이므로, E2F1이 maspin 발현을 억제시키면 Cdc25c의 활성이 증가하고, 이로 인해 cdc2와 cyclinB 합성체가 활성화 되면서 G2/M기 전환이 일어나는 것으로 여겨진다. 보다 세부적이고 명확한 maspin 관련 기작의 규명을 위해서는 더 많은 실험이 필요할 것이다.
Maspin은 다른 기관의 암에서 혈관형성과 조직에의 침입성을 억제하는 기능을 가진다고 알려져 있었기 때문에 미만형 위암에서도 그러한 기능을 가질 가능성이 있는지 VEGF (vascular endothelial growth factor) 발현량과 boyden chamber assay를 이용하여 간단히 살펴 보았다. Maspin이 감소된 미만형 위암 세포에서는 maspin이 감소되지 않은 경우와 비교했을 때, VEGF 발현량과 침습한 세포의 양이 유의한 차이를 보이지 않았다. 그러나 이러한 실험들을 통해서는 maspin이 미만형 위암에서 혈관형성 억제 기능과 조직에의 침입 억제 기능이 없다고 명확히 말할 수 없기 때문에 관련성을 명확히 하기 위해서는 앞으로 면밀한 연구가 요구된다.
본 연구에서는 maspin이 미만형 위암에 감수성을 가지고, 암 억제 기능을 가진다는 것을 밝혔다. 무엇보다도 maspin 프로모터의 위암 감수성 유전변이들이 maspin의 발현량을 낮추고, 이로 인해 세포 주기가 빨라지면서 미만형 위암 세포의 성장 속도가 증가한다는 사실을 알아낸 것이 가장 의미 있는 발견이었다. 우리는 이러한 연구 결과들이 미만형 위암에 걸릴 확률이 높은 사람들이 사전에 예방할 수 있게 도와주고, 이미 암에 걸린 사람들에게는 보다 효율적이 치료를 제공할 수 있는 기반을 제공하는 데에 공헌하기를 기대한다.
