The Roles Of ALDH1A1 In Glioma Invasion And Colorectal Carcinoma Metastasis And Their Mechanisms

Invasion and metastasis are the important properties of malignant tumor. They also are main factors of recurrence of cancer. Glioma is the most aggressive brain tumor with high invasiveness and poor prognosis. One of the most lethal characteristics of gliomas is their capability of invading the side normal brain tissues of glioma. At the same time, colorectal cancer(CRC) is one of the most common causes of cancer-related deaths in China. Lymph lode and liver metastasis are more common in the stage III of colorectal cancer. So we choose the glioma disease to study invasion of magligant tumor and the CRC as a model to study cancer metastasis.Recently, cancer stem cell(CSC) hypothesis gives us a new thinking insight into the mechanism underlining glioma and CRC initiation and progression. Aldehyde dehydrogenase 1A1(ALDH1A1) protein has been confirmed as a cancer stem cell’s marker and a candidate biomarker for colorectal carcinoma. But the mechanisms how to regulate malignant tumor biology behavior are not clear in glioma and CRC. We found that ALDH1A1 positive cells contribute to the progression of glioma including invasion,proliferation and poor prognosis in glioma. ADLH1A1 was frequently overexpressed in the high-grade glioma(WHO grade III-IV) as compared to the low-grade glioma(WHO grade I-II) patients. Multivariate Cox regression analysis revealed that ALDH1A1 was an independent marker for glioma patients’ outcome. ALDH1A1 positive cells can increase the levels of m RNA and protein for matrix metalloproteinase 2, 7 and 9(MMP2, MMP7 and MMP9). Then, roles of ALDH1A1 in invasion and metastasis of colorectal carcinoma and glioma were explored.To further explore the influence of ALDH1A1 on the diagnostic and prognostic value and the invasive property of colorectal carcinoma and glioma and the possible mechanisms,this study first confirmed that the expression patterns of ALDH1A1 were heterogeneous in the CRC and corresponding adjacent tissues. We defined the ratio of ALDH1A1 level inadjacent mucosa to that in tumor tissues as RA/C and found that the capabilities of tumor invasion and metastasis in the tumors with RA/C<1 were significantly higher than those with RA/C≥1. At the same time, the localization of β-catenin expressed in cell nuclei was correlation to the expression type of ALDH1A1. In addition, the heterogeneous of ALDH1A1 expression can recruit the infiltration of M2 type macrophages, and the number of M2 type macrophage can predicte the overall survival in RA/C<1 of CRC which had shorter overall survival time.This article included two parts as follows:Part I: The roles and correlated molecular mechanisms of ALDH1A1 in invasion of glioma.Glioma is one of the most common malignancies in brain with atrocious patient survival. The patients with glioma have poor prognosises, the median survival ranging from12-15 months. Many genetic factors alterations have been correlated with poor or better outcomes in glioma. Although much more molecules are demonstrated for their correlation to the malignancy and prognosis of glioma, more sensitive, reliable and practical indicators to reveal high invasiveness remain explored not only for pathological diagnosis but also for prognostic prediction and targeted therapy.The main experiment methods, datas and conclusions are listed as follows:1. ALDH1A1 expression is correlated with glioma histological grade and predicts worse prognosis.We quantitatively evaluated the surgical specimens of 117 low grade glioma patients(LGG)(WHO I-II) and 120 high grade glioma(HGG) patients(WHO III-IV). ALDH1A1 overexpression with IHC staining score over 5 was found respectively in 10(8.5%) of 117 LGG patients and 41(34.2%) of 120(HGG) patients(WHO III-IV). Furthermore, the specimens with high expression of ALDH1A1 also exhibited high level of proliferation protein Ki67. ALDH1A1 level was statistically correlated with Ki67. The estimated OS rate of low score group of glioma patients(n = 95) was significantly better than that of high score group of glioma patients(n = 19)(P =0.000). DFS rate was remarkably different between the patients with low and ones with high expression of ALDH1A1(P = 0.001). So,ALDH1A1 can be used as a biomarker for patients’ outcome, and its detection in the glioma specimens can predict patients prognosis.2. High expression of ALDH1A1 is correlated with molecular classification of GBM.We performed IHC staining to analyze the individual expression of TP53, PDGFRA and EGFR in our series of specimens from 72 GBM patient in our research. Overall, 50%samples were defined as “Pronural-Like” subtype as characterized by PDGFRA- and/or TP53- positive staining, which included 12.5% samples with ALDH1A1 over-expression.Other 18.1% samples belonged to “Classical-Like” subtype with EGFR-positive but PDGFRA- and TP53- negative staining. The remaining 23(31.9%) samples that did not fit any of these criteria were classified as ‘‘Other” subtype. Statistical analysis revealed that the expression of ALDH1A1 was significantly different among theses subtypes(P=0.0001).These results indicate that ALDH1A1 expression is correlated with the malignance and molecular classification of GBM.3. The expression of ALDH1A1 in glioma is associated with the status of IDH1.Previous studies showed that the radiation and chemotherapy sensitivity, prognosis are associated with the status of IDH1. We have finished research for 56 cases of gliomas specimens to detecte the state IDH1 mutations and found that the rate of IDH1 mutation was 35.8%(20/56). ALDH1A1 high expression is 40%(8/20) in IDH1 mutations cases relatived to 72.2%(26/36)in IDH1 wild ones(P<0.01). The results showed that the expression of ALDH1A1 was correlationed with IDH1 mutations state.4. ALDH1A1 positive glioma cells possess higher capabilities of colony formation,proliferation and invasion.We first examined the proportion of ALDH1A1 highcells in the glioma cell line U251,and found that ALDH1A1 high cells accounted for 6.9% ± 2.1% of the whole-cell population.Similar results were also obtained that ALDH1A1 highcells accounted for 4.2% ± 1.9% of the whole-cell population from primary glioblastoma cells. We then examined the bio-behaviors of the sorted ALDH1A1+ cells and found that these cells possessed higher capabilities of colony formation as compared with ALDH1A1- ones. The percentage of colonies formed by ALDH1A1+ cells were higher than those formed by ALDH1A1- cells in U251 and primary glioblastoma cells. Proliferation assay revealed that ALDH1A1+ cells sorted from either U251 and primary glioma cells were highly proliferated(P < 0.05). Furthermore,orthotopical xenotransplantation murine model studies demonstrated that SCID mice transplanted with ALDH1A1+ cells were alive longer period than the other one. These data suggest that ALDH1A1 positive cells play an important role for glioma progression.Pathological studies of orthotopically transplanted tumors demonstrated that ALDH1A1 positive tumor cells exhibited stronger invasiveness and the boundary of transplanted tumor formed from ALDH1A1 positive tumor cells was less clear than that formed from ALDH1A1 negative ones. To explore the corresponding mechanism(s), we next examined the invasion capabilities of FCAS-sorted ALDH1A1 positive cells. As compared to ALDH1A1 negative ones, ALDH1A1 positive cells respectively sorted from U251 and primary GBM cells had a greater invasion capability. Gene expression analysis further revealed that ALDH1A1 positive tumor cells sorted from either U251 or the primary GBM expressed higher levels of MMP9, MMP7 and MMP2. Meanwhile, ALDH1A1 positive cells had more abundances of MMP9, MMP7 and MMP2 as compared to ALDH1A1 negative cells. These results demonstrate that the highly invasive capabilities of ALDH1A1 positive cells are associated with the increased levels of MMPs expression.Part II: The roles and correlated molecular mechanisms of ALDH1A1 in invasion and prognosis of colorectal carcinoma.CRC is one of the most common malignancies with annual increases by approximately4.2%. Although studies have showed several molecules related to metastasis and prognosis of this cancer, sensitive, reliable and practical indicators are still unavailable for surgical pathology. Recently, several reports showed that ALDH1A1 may be a biomarker of CSCs and can be used as a prognostic predictor of many cancers, including CRC. However, the conclusions for the prognostic significance of ALDH1A1 expression in CRC tissues and corresponding adjacent mucosa remain controversial.The main experiment methods, datas and conclusions are listed as follows:1. The expression levels of ALDH1A1 in CRC and adjacent tissues and the significance of clinicopathology.(1) We analyzed the expression of ALDH1A1 by immunohistochemistry(IHC) in CRC samples containing both the tumor tissue and the corresponding adjacent mucosa. A significantly heterogeneity in staining patterns was also observed. One hundred and ninety-eight out of 406(48.8 %) adjacent mucosa samples were positively stained for ALDH1A1. The staining intensity was fairly variable, ranging from low(1.0 ± 0.0) to high(2.7 ± 0.5). The overall expression score was 4.0 ± 2.8. In tumor tissues, 55.4% samples(225/406) were ALDH1A1 positive. These results derived from IHC staining were further verified in the fresh CRC samples from 12 patients by real time PCR and western-blotting.(2)We studied the influence on prognosis of ALDH1A1 by a univariate Cox proportional hazard regression model indicated that RA/C correlated with the outcome of221 CRC patients. Patients in the RA/C ≥1 group had a median survival of 57.1 months,whereas patients in the RA/C<1 group had a median survival of 35.4 months. The RA/C ratio,together with other prognostic clinicopathologic factors, was also studied in a multivariate model(221 patients). These results indicated the favorable prognostic values for RA/C in terms of prolonged overall survival(OS)(P=0.000).2. The expression levels of ALDH1A1 in CRC is correlation to the expression ofβ-catenin and localization in colorectal carcinoma.The aberrant activation of β-catenin signaling has been reported to be involved in a number of tumors, most notably CRC. The nuclear translocation of β-catenin profoundly regulates the invasion, metastasis and differentiation of CRC. In addition, β-catenin is used as a supplemental marker in the analysis to stratify the expression profiles of investigated genes in CRC patients. Therefore, we investigated the relationship between RA/C and the nuclear translocation of β-catenin. By immunohistochemistry, we observed that in the tumor tissue of patients from the RA/C≥1 group, β-catenin was predominantly located at the cell membrane, relatived to the the RA/C<1 group located at nuclear translocation. The nuclear translocation rates in two groups were 36.0%(45/125, RA/C<1 group) and 16.9%(21/124,RA/C≥1 group), respectively. These results indicate that there are significant differences in the nuclear translocation of β-catenin between the two groups of CRC patients as divided by RA/C.3. The expression levels of ALDH1A1 is correlation to the expression of CD68 and CD163 in colorectal carcinoma.We next studied whether there was association of TAMs of M2 type macrophages with the expression of ALDH1A1 in the primary CRC. CD68 and CD163 staining are found in the cytoplasm. CRC specimens were analyzed to study the correlation of CD68 positive and CD163 positive macrophages with the two groups. IHC staining of these sections showed more CD68 positive and CD163 positive macrophages located at the nearby of ALDH1A1 positive cells of RA/C<1 group than RA/C≥1 one of CRC. Our results demonstrate the number of macrophages in the tumor microenvironment have a wide variability among RA/C≥1 and RA/C<1 group of CRCs.Conclusion: In summary, we found in the study of ALDH1A1 in glioma, the ALDH1A1 positive cells possesses higly invasive, colony formation, proliferation capability. Our study reveal that ALDH1A1 positive glioma cells are highly invasive due to high levels of MMP2, MMP7 and MMP9. MMPs are well known to be capable of degrading extracellular matrix proteins to promote tumor cell invasion.ALDH1A1 positive cells possesses highly invasive, metastasis properties in colorectal carcinoma. Our results indicate for the first time that the two distinct expression patterns of ALDH1A1 in CRC and its adjacent tissue are associated with different clinicopathological features and prognosis in CRC patients. Additional prospective studies are warranted to explore whether the nuclear translocation of β-catenin provides a molecular mechanism for these observations. On the other hand, ALDH1A1 positive cells had a correlation with the distribution of TAMs in CRC. Maybe M2 polarized TAMs could promote the invasion.