ALDH1 expression predicts progression of premalignant lesions to cancer in Type I endometrial carcinomas

In type 1 endometrial cancer, unopposed estrogen stimulation is thought to lead to endometrial hyperplasia which precedes malignant progression. Recent data from our group and others suggest that ALDH activity mediates stemness in endometrial cancer, but while aldehyde dehydrogenase 1 (ALDH1) has been suggested as a putative cancer stem cell marker in several cancer types, its clinical and prognostic value in endometrial cancer remains debated. The aim of this study was to investigate the clinical value of ALDH1 expression in endometrial hyperplasia and to determine its ability to predict progression to endometrial cancer. Interrogation of the TCGA database revealed upregulation of several isoforms in endometrial cancer, of which the ALDH1 isoforms collectively constituted the largest group. To translate its expression, a tissue microarray was previously constructed which contained a wide sampling of benign and malignant endometrial samples. The array contained a metachronous cohort of samples from individuals who either developed or did not develop endometrial cancer. Immunohistochemical staining was used to determine the intensity and frequency of ALDH1 expression. While benign proliferative and secretory endometrium showed very low levels of ALDH1, slightly higher expression was observed within the stratum basalis. In disease progression, cytoplasmic ALDH1 expression showed a step-wise increase between endometrial hyperplasia, atypical hyperplasia, and endometrial cancer. ALDH1 was also shown to be an early predictor of EC development, suggesting that it can serve as an independent prognostic indicator of patients with endometrial hyperplasia with or without atypia who would progress to cancer (p = 0.012).

Association between cancer stem cell marker ALDH1 and clinical and morphological factors of colorectal cancer prognosis.

3522 Background: Cancer stem cells (CSCs) capable of self-sustaining and multipotent differentiation are considered among the most important factors limiting treatment effectiveness. ALDH1 is a marker of colorectal cancer (CRC) CSCs; it is involved in cell differentiation and proliferation, determines resistance to alkylating chemotherapeutic agents, and also induces epithelial-mesenchymal transition (EMT), which increases the invasive and metastatic potential of tumors. The purpose of the study was to assess the association between the expression of the ALDH1 CSC marker in tissues of CRC of different stages and clinical and morphological factors of the disease prognosis. Methods: The study included 299 patients (aged 42-86 years, mean age 64.2±1.7) with stage II-IV CRC T1-4N0-2M0-1; histologically verified G1-G3 adenocarcinoma in all patients. Tissues of surgically removed tumors were studied with IHC analysis using mouse monoclonal anti-ALDH1 antibodies (clone B-5, Santa Cruz Biotechnology) diluted 1:1800 and the Reveal Polyvalent HRP-DAB Detection System. The percentage of cells positively stained for ALDH1 among all tumor cells was assessed. Statistical analysis was performed using the STATISTICA 13.0 program (StatSoftInc., USA). Results: Positive ALDH1+ expression was registered in 52.5% of all patients, negative expression – in 47.5%. Statistically significant association was observed between the ALDH1 expression and the CRC stage, since the ALDH1+ expression increased from stage II to stage IV (p = 0.003). The ALDH1 expression was statistically significantly associated with the depth of tumor invasion (p = 0.018) and the presence of distant metastases (p < 0.001). No significant relationship was observed between the ALDH1 expression and regional lymph node metastasis (p = 0.788). Statistically significant association was registered between the ALDH1 expression and the tumor grade (p < 0.001), perineural invasion (p = 0.010) and lymphocytic infiltration (p < 0.001). No significant relationship was observed between the tumor histological structure (p = 0.979), lymphovascular invasion (p = 0.772) and ALDH1 expression. Tumor site was not statistically significantly associated with ALDH1 expression (p = 0.349). Conclusions: The study demonstrated statistically significant association between the ALDH1 expression and clinical and morphological characteristics of CRC, determining invasive and metastatic potential of the tumor, and ALDH1 may be an independent prognostic factor and a new therapeutic target for the regulation of the progression process.

ALDH1 expression in primary and recurrent vulvar carcinoma.

e17573 Background: Our aim was to evaluate ALDH1 expression as a stem cell marker in primary and recurrence vulvar squamous cell carcinoma. Methods: 27 patients aged 27-80 years with vulvar squamous cell carcinomas, together with their verified histopathological and clinical data were included in the study. All of them underwent treatment the first stage of which was surgery in National Research Centre for Oncology in 2015-2020. In 10 of them I stage of vulvar squamous cell carcinomas was diagnosed, in 13 – II stage, 4 had relapses after complex treatment and 2-10 years` remission. All paraffin-embedded samples of the primary and recurrent vulvar carcinoma were recruited. The presence of ALDH1 was detected by immunohistochemistry using murine monoclonal antibodies to ALDH1 (clone B-5 Santa Cruz Biotechnology) diluted 1:800 and Reveal Polyvalent HRP-DB Detection System. Positively stained cells were counted among all the tumor cells as well as a part of tumors positive for ALDH1 ( > 10% of cells). Statistics analysis was performed by program STATISTICA 13.0 (StatSoftInc., the USА). Results: By immunohistochemical staining, the expression of ALDH1 was observed in 5 patients with I stage of vulvar squamous cell carcinomas, in 5 patients with II stage and in all the patients with recurrence. Mean values of ALDH1 expression in patients with I stage were 14.1±4.3%, in patients with II stage – 16.0±3.6%, in patients with recurrence – 29.6±3.6%. Maximal ALDH1 accumulation was observed in tumor cells of patients with recurrence, it was 1.9 times higher than in the II stage (p = 0.03) and 2.1 times exceeded ones of the I stage (p = 0.027). All the differences were statistically significant according to Student`s t-test (p < 0.05). The was no statistically significant difference between values of the patients with I and II stages (p = 0.738), but it is noteworthy that ALDH1-expressing cells prevailed in tumor stroma especially in II stage. Conclusions: There is some discrepancy in the assessment of ALDH1 prognostic value in vulvar carcinoma patients as well as in understanding if it is related predominantly to cancer stem cells or to normal stem cells either. Our results though obtained on a few cases of vulvar squamous cell carcinomas show that ALDH1 expression in recurrent carcinomas markedly exceeds that in primary tumors and is related to tumor cells while in primary tumors is related rather to stromal cells.

FUBP1 Promotes Colorectal Cancer Stemness and Metastasis via DVL1-Mediated Activation of Wnt/β-Catenin Signaling

Background: Most colorectal cancer (CRC) patients die from distant metastasis. Approximately 50% of CRC patients develop liver metastases, while 10% to 30% of patients appear pulmonary metastases. The occurrence of metastasis is considered to be almost exclusively driven by cancer stem cells (CSCs) formation. However, the key molecules that confer the stem-transformation of CRC and subsequent metastasis remain unclear.Methods: FUBP1 was screened in CRC CSCs by mass spectrometry and was examined by immunohistochemistry in a cohort of CRC tissues. The stemness induction and mechanism of FUBP1 were elucidated both in vivo and in vitro.Results: FUBP1 was upregulated in 85% of KRAS mutation and 25% of wildtype CRC patients which correspondence to the metastasis rate. Further, elevated FUBP1 was positively correlated with CRC lymph node metastasis and clinical stages and negatively associated with overall survival, whatever KRAS mutation or wildtype. Overexpression of FUBP1 significantly enhanced migration, invasion, tumor sphere formation and CD133/ALDH1 expression of CRC cells in vitro and the tumorigenicity in vivo. Mechanistically, FUBP1 promoted the initiation of CSCs by activating Wnt/β-catenin signaling via directly binding to the promoter of DVL1, a vigoroso activator of β-catenin. The knockdown of DVL1 tremendously blockaded the stem-transformation and tumorigenicity of CRC. Activation of Wnt/β-catenin signaling by DVL1 increased pluripotent transcription factors, including c-Myc, NANOG, SOX2 rather c-Myc alone. Moreover, FUBP1 was upregulated at the post-transcriptional level. Elevated FUBP1 in KRAS wildtype CRC patients is due to the decrease of Smurf2, which promotes ubiquitin-mediated degradation of FUBP1. Whereas FUBP1 was upregulated in mutated KRAS patients through both inhibition of caspase-3-dependent cleavage and decreased Smurf2.Conclusions: Our results demonstrate for the first time that FUBP1 is a novel oncogene for the initiation of CSCs as a new endogenous Wnt signaling powerful agonist and may provide an important prognostic factor and therapeutic target for metastasis in both KRAS mutation and wildtype CRC.

TRPM7 Induces Tumorigenesis and Stemness Through Notch Activation in Glioma

We have reported that transient receptor potential melastatin-related 7 (TRPM7) regulates glioma stem cells (GSC) growth and proliferation through Notch, STAT3-ALDH1, and CD133 signaling pathways. In this study, we determined the major contributor(s) to TRPM7 mediated glioma stemness by further deciphering each individual Notch signaling. We first determined whether TRPM7 is an oncotarget in glioblastoma multiforme (GBM) using the Oncomine database. Next, we determined whether TRPM7 silencing by siRNA TRPM7 (siTRPM7) induces cell growth arrest or apoptosis to reduce glioma cell proliferation using cell cycle analysis and annexin V staining assay. We then examined the correlations between the expression of TRPM7 and Notch signaling activity as well as the expression of GSC markers CD133 and ALDH1 in GBM by downregulating TRPM7 through siTRPM7 or upregulating TRPM7 through overexpression of human TRPM7 (M7-wt). To distinguish the different function of channel and kinase domain of TRPM7, we further determined how the α-kinase-dead mutants of TRPM7 (α-kinase domain deleted/M7-DK and K1648R point mutation/M7-KR) affect Notch activities and CD133 and ALDH1 expression. Lastly, we determined the changes in TRPM7-mediated regulation of glioma cell growth/proliferation, cell cycle, and apoptosis by targeting Notch1. The Oncomine data revealed a significant increase in TRPM7 mRNA expression in anaplastic astrocytoma, diffuse astrocytoma, and GBM patients compared to that in normal brain tissues. TRPM7 silencing reduced glioma cell growth by inhibiting cell entry into S and G2/M phases and promoting cell apoptosis. TRPM7 expression in GBM cells was found to be positively correlated with Notch1 signaling activity and CD133 and ALDH1 expression; briefly, downregulation of TRPM7 by siTRPM7 decreased Notch1 signaling whereas upregulation of TRPM7 increased Notch1 signaling. Interestingly, kinase-inactive mutants (M7-DK and M7-KR) resulted in reduced activation of Notch1 signaling and decreased expression of CD133 and ALDH1 compared to that of wtTRPM7. Finally, targeting Notch1 effectively suppressed TRPM7-induced growth and proliferation of glioma cells through cell G1/S arrest and apoptotic induction. TRPM7 is responsible for sustained Notch1 signaling activation, enhanced expression of GSC markers CD133 and ALDH1, and regulation of glioma stemness, which contributes to malignant glioma cell growth and invasion.

Expression pattern of ALDH1, E-cadherin, Vimentin and Twist in early and late onset sporadic colorectal cancer

Aim: To evaluate the expression pattern of ALDH1 (aldehyde dehydrogenase 1), E-cadherin, Vimentin and Twist in early and late onset sporadic colorectal cancer (CRC) and to study association of their expression with the occurrence of CRC at a young age. Materials & methods: Immunohistochemistry of ALDH1, E-cadherin, Vimentin and Twist was performed on 103 pretreated CRC biopsy samples. Results: ALDH1 expression was found to have strong correlation with early onset CRC (p < 0.0001). Conclusion: High ALDH1 expression correlates with the early onset of CRC. ALDH1 over-expression correlates with poor overall survival in colon cancer.

HSPA1L Enhances Cancer Stem Cell-Like Properties by Activating IGF1Rβ and Regulating β-Catenin Transcription

Studies have shown that cancer stem cells (CSCs) are involved in resistance and metastasis of cancer; thus, therapies targeting CSCs have been proposed. Here, we report that heat shock 70-kDa protein 1-like (HSPA1L) is partly involved in enhancing epithelial–mesenchymal transition (EMT) and CSC-like properties in non-small cell lung cancer (NSCLC) cells. Aldehyde dehydrogenase 1 (ALDH1) is considered a CSC marker in some lung cancers. Here, we analyzed transcriptional changes in genes between ALDH1high and ALDH1low cells sorted from A549 NSCLC cells and found that HSPA1L was highly expressed in ALDH1high cells. HSPA1L played two important roles in enhancing CSC-like properties. First, HSPA1L interacts directly with IGF1Rβ and integrin αV to form a triple complex that is involved in IGF1Rβ activation. HSPA1L/integrin αV complex-associated IGF1Rβ activation intensified the EMT-associated cancer stemness and γ-radiation resistance through its downstream AKT/NF-κB or AKT/GSK3β/β-catenin activation pathway. Secondly, HSPA1L was also present in the nucleus and could bind directly to the promoter region of β-catenin to function as a transcription activator of β-catenin, an important signaling protein characterizing CSCs by regulating ALDH1 expression. HSPA1L may be a novel potential target for cancer treatment because it both enhances IGF1Rβ activation and regulates γβ-catenin transcription, accumulating CSC-like properties.

ALDH1 Cancer Stem Cell Marker as a Prognostic Factor in Triple-Negative Breast Cancer

Breast cancer is the most common cancer with an increasing incidence in Asia. About 20% of all breast cancers are triple-negative breast cancers (TNBCs). BCSC is a subset of tumor cells that has stem cell-like characteristics, such as a high capacity for self-renewal and tumor initiation, which implies that BCSC may cause aggressiveness of TNBC. ALDH1 has a role in early stem cell differentiation through its function in the oxidation of retinol to retinoic acid, proposed to be a strong candidate for breast cancer stem cells. Various studies have shown that ALDH1 is one of the markers of CSC that can be used as a prognosis indicator because it can be a biological marker for poor prognostic factors in TNBC. This study assessed the prognostic survival rate with a retrospective cohort method in TNBC patients. A total of 54 of 55 patients treated at RSCM were tested for the expression of ALDH1 through an immunohistochemistry assay of breast cancer tissue using ALDH1 staining. Survival analysis was done to obtain the prognostic data of ALDH1. Positive ALDH1 expression was obtained at 38.89% in TNBC patients. One-year survival and three years of survival in TNBC patients with positive ALDH1 expression were 42.9% and 33.3%, respectively. In this study, ALDH1 can be used as a poor survival prognostic factor with HR 2.636 and p value 0.013. The conclusion of this study is that ALDH1 can be used as a poor prognostic factor in TNBC patients although it cannot be an independent prognostic factor.