Growth characteristics of early-stage (IA) lung adenocarcinoma and its value in predicting lymph node metastasis.

BACKGROUND: We aim to compare the differences in growth characteristics between part-solid and solid lung adenocarcinoma, and to investigate the value of volume doubling time (VDT) or mass doubling time (MDT) in predicting lymph node (LN) metastasis and preoperative evaluation in patients of early-stage (IA) non-small cell lung cancer (NSCLC). METHOD: We reviewed 8,653 cases of surgically resected stage IA lung adenocarcinoma between 2018 and 2022, with two follow-up visits at least 3 months apart, comparing diameter, volume, and mass growth of pSN and SN. VDT and MDT calculations for nodules with a volume change of at least 25%. Univariable or multivariable analysis was used to identify the risk factors. The area under the curve (AUC) for the receiver operating characteristic (ROC) curves was used to evaluate the diagnostic value. RESULTS: A total of 144 patients were included 114 with solid nodules (SN) and 25 with part-solid nodules (pSN). During the follow-up period, the mean VDTt and MDTt of SN were shorter than those of pSN, 337 vs. 541 days (p = 0.005), 298 vs. 458 days (p = 0.018), respectively. Without considering the ground-glass component, the mean VDTc and MDTc of SN were shorter than the solid component of pSN, 337 vs. 498 days (p = 0.004) and 298 vs. 453 days (p = 0.003), respectively. 27 nodules were clinically and pathologically diagnosed as N1/N2. Logistic regression identified initial diameter (p < 0.001), consolidation increase (p = 0.019), volume increase (p = 0.020), mass increase (p = 0.021), VDTt (p = 0.002), and MDTt (p = 0.004) were independent factors for LN metastasis. The ROC curves showed that the AUC for VDTt was 0.860 (95% CI, 0.778-0.943; p < 0.001) and for MDTt was 0.848 (95% CI, 0.759-0.936; p < 0.001). CONCLUSIONS: Our study showed significant differences in the growth characteristics of pSN and SN, and the application of VDT and MDT could be a valid predictor LN metastasis in patients with early-stage NSCLC.

Advances in diagnosis and prediction for aggression of pure solid T1 lung cancer.

A growing number of early-stage lung cancers presenting as malignant pulmonary nodules have been diagnosed because of the increased adoption of low-dose spiral computed tomography. But pure solid T1 lung cancer with ≤3 cm in the greatest dimension is not always at an early stage, despite its small size. This type of cancer can be highly aggressive and is associated with pathological involvement, metastasis, postoperative relapse, and even death. However, it is easily misdiagnosed or delay diagnosed in clinics and thus poses a serious threat to human health. The percentage of nodal or extrathoracic metastases has been reported to be >20% in T1 lung cancer. As such, understanding and identifying the aggressive characteristics of pure solid T1 lung cancer is crucial for prevention, diagnosis, and therapeutic strategies, and beneficial to improving the prognosis. With the widespread of lung cancer screening, these highly invasive pure solid T1 lung cancer will become the main advanced lung cancer in future. However, there is limited information regarding precision medicine on how to identify these "early-stage" aggressive lung cancers. To provide clinicians with new insights into early recognition and intervention of the highly invasive pure solid T1 lung cancer, this review summarizes its clinical characteristics, imaging, pathology, gene alterations, immune microenvironment, multi-omics, and current techniques for diagnosis and prediction.

Deep learning predicts malignancy and metastasis of solid pulmonary nodules from CT scans.

In the clinic, it is difficult to distinguish the malignancy and aggressiveness of solid pulmonary nodules (PNs). Incorrect assessments may lead to delayed diagnosis and an increased risk of complications. We developed and validated a deep learning-based model for the prediction of malignancy as well as local or distant metastasis in solid PNs based on CT images of primary lesions during initial diagnosis. In this study, we reviewed the data from multiple patients with solid PNs at our institution from 1 January 2019 to 30 April 2022. The patients were divided into three groups: benign, Ia-stage lung cancer, and T1-stage lung cancer with metastasis. Each cohort was further split into training and testing groups. The deep learning system predicted the malignancy and metastasis status of solid PNs based on CT images, and then we compared the malignancy prediction results among four different levels of clinicians. Experiments confirmed that human-computer collaboration can further enhance diagnostic accuracy. We made a held-out testing set of 134 cases, with 689 cases in total. Our convolutional neural network model reached an area under the ROC (AUC) of 80.37% for malignancy prediction and an AUC of 86.44% for metastasis prediction. In observer studies involving four clinicians, the proposed deep learning method outperformed a junior respiratory clinician and a 5-year respiratory clinician by considerable margins; it was on par with a senior respiratory clinician and was only slightly inferior to a senior radiologist. Our human-computer collaboration experiment showed that by simply adding binary human diagnosis into model prediction probabilities, model AUC scores improved to 81.80-88.70% when combined with three out of four clinicians. In summary, the deep learning method can accurately diagnose the malignancy of solid PNs, improve its performance when collaborating with human experts, predict local or distant metastasis in patients with T1-stage lung cancer, and facilitate the application of precision medicine.

Disruption of ZNF334 promotes triple-negative breast carcinoma malignancy through the SFRP1/ Wnt/ß-catenin signaling axis.

Zinc-finger proteins (ZNFs) constitute the largest transcription factor family in the human genome. The family functions in many important biological processes involved in tumorigenesis. In our research, we identified ZNF334 as a novel tumor suppressor of triple-negative breast cancer (TNBC). ZNF334 expression was usually reduced in breast cancerv (BrCa) tissues and TNBC cell lines MDA-MB-231 (MB231) and YCCB1. We observed that promoter hypermethylation of ZNF334 was common in BrCa cell lines and tissues, which was likely responsible for its reduced expression. Ectopic expression of ZNF334 in TNBC cell lines MB231 and YCCB1 could suppress their growth and metastatic capacity both in vitro and in vivo, and as well induce cell cycle arrest at S phase and cell apoptosis. Moreover, re-expression of ZNF334 in TNBC cell lines could rescue Epithelial-Mesenchymal Transition (EMT) process and restrain stemness, due to up-regulation of SFRP1, which is an antagonist of Wnt/ß-catenin signaling. In conclusion, we verified that ZNF334 had a suppressive function of TNBC cell lines by targeting the SFRP1/Wnt/ß-catenin signaling axis, which might have the potentials to become a new biomarker for diagnosis and treatment of TNBC patients.

ZBTB28 inhibits breast cancer by activating IFNAR and dual blocking CD24 and CD47 to enhance macrophages phagocytosis.

Breast cancer is the leading cause of cancer death in female. Until now, advanced breast cancer is still lack effective treatment strategies and reliable prognostic markers. In the present article, we introduced the physiologic and pathologic functions and regulation mechanisms of ZBTB28, a tumor suppressor gene, in breast cancer. ZBTB28 is frequently silenced in breast cancer due to promoter CpG methylation, and its expression is positively correlated with breast cancer patient survival. The antineoplastic effect of ZBTB28 in breast cancer was elucidated through a series of in vitro and in vivo measurements, including cell proliferation, apoptosis, cell cycle, epithelial mesenchymal transition (EMT), and growth of xenografts. Furthermore, ZBTB28 can directly regulate IFNAR to activate interferon-stimulated genes and potentiate macrophage activation. Ectopic ZBTB28 expression in breast cancer cells was sufficient to downregulate CD24 and CD47 to promote phagocytosis of macrophages, demonstrating that ZBTB28 was beneficial for the combination treatment of anti-CD24 and anti-CD47. Collectively, our results reveal a mode of action of ZBTB28 as a tumor suppressor gene and suggest that ZBTB28 is an important regulator of macrophage phagocytosis in breast cancer, holding promise for the development of novel therapy strategies for breast cancer patients.

Multiple targeted self-emulsifying compound RGO reveals obvious anti-tumor potential in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly vascularized, inflammatory, and abnormally proliferating tumor. Monotherapy is often unable to effectively and comprehensively inhibit the progress of HCC. In present study, we selected ginsenoside Rg3, ganoderma lucidum polysaccharide (GLP), and oridonin as the combined therapy. These three plant monomers play important roles in anti-angiogenesis, immunological activation, and apoptosis promotion, respectively. However, the low solubility and poor bioavailability seriously hinder their clinical application. To resolve these problems, we constructed a new drug, Rg3, GLP, and oridonin self-microemulsifying drug delivery system (RGO-SMEDDS). We found that this drug effectively inhibits the progression of HCC by simultaneously targeting multiple signaling pathways. RGO-SMEDDS restored immune function by suppressing the production of immunosuppressive cytokine and M2-polarized macrophages, reduced angiogenesis by downregulation of vascular endothelial growth factor and its receptor, and retarded proliferation by inhibiting the epidermal growth factor receptor EGFR/AKT/epidermal growth factor receptor/protein kinase B/glycogen synthase kinase-3 (GSK3) signaling pathway. In addition, RGO-SMEDDS showed considerable safety in acute toxicity tests. Results from this study show that RGO-SMEDDS is a promising therapy for the treatment of HCC.

Cancer cells escape p53's tumor suppression through ablation of ZDHHC1-mediated p53 palmitoylation.

The inactivation of tumor-suppressor genes contributes heavily to oncogenesis. The mutation of TP53 has been well-studied and recognized as a major factor in the development of tumors. Yet other means of p53 inactivation has not been well-elucidated. We previously identified a hypermethylated gene ZDHHC1 that suppresses tumor growth when the expression was restored, but the specific mechanism was yet to be found. The protein product of ZDHHC1 is an S-palmitoyltransferase and we have identified p53 as a substrate for ZDHHC1-mediated palmitoylation, specifically at the C135, C176, and C275 residues. The novel form of post-translational modification of p53 is required for the nuclear translocation of the tumor suppressor. p53 recruited DNMT3A to ZDHHC1 promoter and is responsible for the hypermethylation of ZDHHC1. The epigenetic feedback loop formed by ZDHHC1 and p53 sheds light on the inactivation of p53 without the presence of genetic mutations.

ZBTB28 induces autophagy by regulation of FIP200 and Bcl-XL facilitating cervical cancer cell apoptosis.

BACKGROUND: Among the common preventable cancers of women, cervical cancer has the highest morbidity. It is curable if detected at an early stage. However, reliable diagnostic and prognostic markers, which relate to physiologic and pathologic regulation of cervical cancer, are not available. In this study, one such potential marker, ZBTB28, was evaluated for its potential usefulness in cervical cancer assessment. METHODS: Public database analysis, reverse-transcription polymerase chain reaction (PCR), and methylation-specific PCR were employed to analyze ZBTB28 expression and promoter methylation. The importance of ZBTB28 in cervical cancer cells was assessed by cellular and molecular analysis in vitro and in vivo. RESULTS: This study assessed the anti-tumor effects of the transcription factor, ZBTB28, which is often silenced in cervical cancer due to CpG methylation of its promoter. We found ZBTB28 to directly affect cervical cancer cell proliferation, apoptosis, autophagy, and tumorigenesis. Also, it increased cancer cell chemosensitivity to Paclitaxel, Cisplatin, and 5-fluorouracil. Ectopic ZBTB28 expression inhibited the growth of cervical cancer xenografts in nude mice. Furthermore, electron microscopy demonstrated ZBTB28 to induce autophagosomes in cervical cancer cells. ZBTB28 induced cellular autophagy by the degradation of Bcl-XL, reduction of the Bcl-XL-BECN1 complex, and by interaction with the autophagy-related gene FIP200. ZBTB28-induced autophagy of cervical cancer cells was shown to mediate cellular apoptosis through the regulation of FIP200. CONCLUSION: These findings identify ZBTB28 as a tumor suppressor gene that can induce autophagy-related apoptosis in cervical cancer cells. As such, ZBTB28 may be a target for the treatment of uterine-cervical carcinoma. Further, ZBTB28 promoter methylation analysis may offer a new objective strategy for cervical cancer screening.

DNA methylation downregulated ZDHHC1 suppresses tumor growth by altering cellular metabolism and inducing oxidative/ER stress-mediated apoptosis and pyroptosis.

Cancer progression is an intricate biological process profiled by not only unscheduled proliferation, but also altered metabolism mechanisms. In this article, we introduced a novel tumor suppressor gene (TSG), Zinc Finger DHHC-Type Containing 1 (ZDHHC1, also known as ZNF377), frequently silenced due to epigenetic modification among various cancers, which exerts significant anti-tumor effects through metabolic regulation. Methods: Quantitative reversed-transcription PCR (qRT-PCR), reverse transcription PCR (RT-PCR) and Western blot were employed to demonstrate transcriptional and protein levels of targeted regulators. Methylation of ZDHHC1 promoter was detected by bisulfite genomic sequencing (BGS) and methylation specific PCR (MSP). Proteomics were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) and gas chromatography-mass spectrometry (GC-MS) were utilized for metabolomics analysis. Cellular functions were examined via corresponding approaches. Nude mice were used for xenograft tumor models. Indirect immunofluorescence staining was utilized to obtain precise location and expression of target proteins. Oxidative and ER stress indicators were detected using specific kits. Results: We found that ZDHHC1 expression was frequently silenced in multiple tumor cells and specimens due to methylation. Restoration of ZDHHC1 expression can curb cancer cell progression via stimulating apoptosis and cell cycle arrest, repressing metastasis, and reversing EMT transition and cell stemness. ZDHHC1's salient anti-tumor abilities were recognized in vivo as well. Metabolomic and proteomic analyses predicted inhibitory role of ZDHHC1 in glucose metabolism pathways in a CYGB-dependent manner, and in pentose phosphate pathway (PPP), which was validated by examining altered key factors. Moreover, we unraveled that ZDHHC1 dedicates to the increment of oxidative stress and endoplasmic reticulum (ER) stress to promote pyroptosis for anticancer purposes. Conclusion: Our study for the first time indicates ZDHHC1 is a potential tumor-suppressor frequently silenced due to promoter methylation, capable of negatively regulating metabolisms of tumor cells while stimulating oxidative stress and ER stress to expedite cell death through induction of pyroptosis and apoptosis, which can be exploited for development of new cancer prevention and therapies.

Epigenetic identification of ADCY4 as a biomarker for breast cancer: an integrated analysis of adenylate cyclases.

Aim: To explore the role of adenylyl cyclase isoforms and its epigenetics in cancer. Materials & methods: Adenylyl cyclase expression profiles, epigenetic alterations, prognostic value and molecular networks were assessed by use of public omics datasets. Results:ADCY4 was significantly downregulated in breast cancer. This downregulation was associated with promoter hypermethylation. High ADCY4 expression was correlated with better survival of patients with breast cancer and its different intrinsic subtypes and tumor stages. ADCY4 was shown to be strongly associated with G protein coupled receptors and the downstream cAMP signaling pathway, which was also significantly enriched in newly identified lysophosphatidic acid receptor 4 and glucagon-like peptide-1. Conclusion: ADCY4 may be used as an epigenetic biomarker for breast cancer, as well as a possible target for therapy.

The phosphoinositide hydrolase phospholipase C delta1 inhibits epithelial-mesenchymal transition and is silenced in colorectal cancer.

In this study, we found that the phospholipase C delta1 (PLCD1) protein expression is reduced in colorectal tumor tissues compared with paired surgical margin tissues. PLCD1-promoted CpG methylation was detected in 29/64 (45%) primary colorectal tumors, but not in nontumor tissues. The PLCD1 RNA expression was also reduced in three out of six cell lines, due to PLCD1 methylation. The ectopic expression of PLCD1 resulted in inhibited proliferation and attenuated migration of colorectal tumor cells, yet promoted colorectal tumor cell apoptosis in vitro. We also observed that PLCD1 suppressed proliferation and promoted apoptosis in vivo. In addition, PLCD1 induced G1/S phase cell cycle arrest. Furthermore, we found that PLCD1 led to the downregulation of several factors downstream of ß-catenin, including c-Myc and cyclin D1, which are generally known to be promoters of tumorigenesis. This downregulation was caused by an upregulation of E-cadherin in colorectal tumor cells. Our findings provide insights into the role of PLCD1 as a tumor suppressor gene in colorectal cancer (CRC), and demonstrate that it plays significant roles in proliferation, migration, invasion, cell cycle progression, and epithelial-mesenchymal transition. On the basis of these results, tumor-specific methylation of PLCD1 could be used as a novel biomarker for early detection and prognostic prediction in CRC.

Inactivation of ADAMTS18 by aberrant promoter hypermethylation contribute to lung cancer progression.

Lung cancer is the most frequently diagnosed cancer worldwide. Epigenetic regulation contributes to lung cancer pathogenesis. The ADAMTS18 tumor suppressor gene is inactivated in some cancers, but its involvement in lung cancer has not been shown. Immunohistochemistry, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and methylation-specific PCR were used to assay ADAMTS18 expression and promoter methylation in lung tumor tissues and adjacent tissues. Cell viability, transwell, and wound-healing assays, as well as flow cytometry were used to characterize the biological activity of ADAMTS18. The influence of ADAMTS18 on protein expression was assayed using western blots analysis, and its effect on chemosensitivity was assayed by the response to cisplatin. We found that ADAMTS18 was silenced in lung cancer cells by promoter methylation. Demethylation by the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, with or without the histone deacetylase inhibitor trichostatin A, restored ADAMTS18 expression. Compared with normal lung tissue, ADAMTS18 in lung tumors was frequently methylated. Overexpression of ADAMTS18 in lung cancer cells inhibited cell proliferation, migration, and invasiveness and induced G0/G1 cell cycle arrest. Furthermore, ADAMTS18 suppressed epidermal growth factor receptor/protein kinase B (EGFR/AKT) signaling, which sensitized lung cancer cells to cisplatin. Thus, our results demonstrated that the tumor suppressor gene ADAMTS18 was downregulated in lung cancer by promoter CpG methylation, and it promoted sensitivity to cisplatin via EGFR/AKT signaling. Our study suggests that ADAMTS18 promoter methylation is a potential epigenetic biomarker for early detection of lung cancer and warrants investigation as a therapeutic target for early-stage lung cancer.

The new 6q27 tumor suppressor DACT2, frequently silenced by CpG methylation, sensitizes nasopharyngeal cancer cells to paclitaxel and 5-FU toxicity via ß-catenin/Cdc25c signaling and G2/M arrest.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is prevalent in South China, including Hong Kong and Southeast Asia, constantly associated with Epstein-Barr virus (EBV) infection. Epigenetic etiology attributed to EBV plays a critical role in NPC pathogenesis. Through previous CpG methylome study, we identified Disheveled-associated binding antagonist of beta-catenin 2 (DACT2) as a methylated target in NPC. Although DACT2 was shown to regulate Wnt signaling in some carcinomas, its functions in NPC pathogenesis remain unclear. METHODS: RT-PCR, qPCR, MSP, and BGS were applied to measure expression levels and promoter methylation of DACT2 in NPC. Transwell, flow cytometric analysis, colony formation, and BrdU-ELISA assay were used to assess different biological functions affected by DACT2. Immunofluorescence, Western blot, and dual-luciferase reporter assay were used to explore the mechanisms of DACT2 functions. Chemosensitivity assay was used to measure the impact of DACT2 on chemotherapy drugs. RESULTS: We found that DACT2 is readily expressed in multiple normal adult tissues including upper respiratory tissues. However, it is frequently downregulated in NPC and correlated with promoter methylation. DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine restored its expression in NPC cells. DACT2 methylation was further detected in 29/32 (91%) NPC tumors but not in any (0/8) normal nasopharyngeal tissue samples. Ectopic expression of DACT2 in NPC cells suppressed their proliferation, migration, and invasion through downregulating matrix metalloproteinases. DACT2 expression also induced G2/M arrest in NPC cells through directly suppressing ß-catenin/Cdc25c signaling, which sensitized NPC cells to paclitaxel and 5-FU, but not cisplatin. CONCLUSION: Our results demonstrate that DACT2 is frequently inactivated epigenetically by CpG methylation in NPC, while it inhibits NPC cell proliferation and metastasis via suppressing ß-catenin/Cdc25c signaling. Our study suggests that DACT2 promoter methylation is a potential epigenetic biomarker for the detection and chemotherapy guidance of NPC.

TET1 exerts its anti-tumor functions via demethylating DACT2 and SFRP2 to antagonize Wnt/ß-catenin signaling pathway in nasopharyngeal carcinoma cells.

BACKGROUND: TET1 is a tumor suppressor gene (TSG) that codes for ten-eleven translocation methyl cytosine dioxygenase1 (TET1) catalyzing the conversion of 5-methylcytosine to 5-hydroxy methyl cytosine as a first step of TSG demethylation. Its hypermethylation has been associated with cancer pathogenesis. However, whether TET1 plays any role in nasopharyngeal carcinoma (NPC) remains unclear. This study investigated the expression and methylation of TET1 in NPC and confirmed its role and mechanism as a TSG. RESULTS: TET1 expression was downregulated in NPC tissues compared with nasal septum deviation tissues. Demethylation of TET1 in HONE1 and HNE1 cells restored its expression with downregulated methylation, implying that TET1 was silenced by promoter hypermethylation. Ectopic expression of TET1 suppressed the growth of NPC cells, induced apoptosis, arrested cell division in G0/G1 phase, and inhibited cell migration and invasion, confirming TET1 TSG activity. TET1 decreased the expression of nuclear ß-catenin and downstream target genes. Furthermore, TET1 could cause Wnt antagonists (DACT2, SFRP2) promoter demethylation and restore its expression in NPC cells. CONCLUSIONS: Collectively, we conclude that TET1 exerts its anti-tumor functions in NPC cells by suppressing Wnt/ß-catenin signaling via demethylation of Wnt antagonists (DACT2 and SFRP2).

Dickkopf-related protein 2 induces G0/G1 arrest and apoptosis through suppressing Wnt/ß-catenin signaling and is frequently methylated in breast cancer.

Dickkopf-related protein 2 (DKK2) is one of the antagonists of Wnt/ß-catenin signaling, with its downregulation reported in multiple cancers. However, how DKK2 contributes to breast tumorigenesis remains unclear. We examined its expression and promoter methylation in 10 breast tumor cell lines, 98 primary tumors, and 21 normal breast tissues. Compared with normal tissues, DKK2 was frequently silenced in breast cell lines (7/8). DKK2 promoter methylation was detected in 77.8% of cell lines and 86.7% of breast tumors; while rarely detected in normal breast tissues (19%), indicating common DKK2 methylation in breast cancer. Ectopic expression of DKK2 changed breast tumor cell morphology, inhibited cell proliferation and colony formation by inducing G0/G1 cell cycle arrest and apoptosis, and suppressed tumor cell migration by reversing epithelial-mesenchymal transition (EMT) and downregulating stem cell markers. Moreover, restored expression of DKK2 in MCF7 cells disrupted the microtube formation of human umbilical vein endothelial cells on Matrigel®. In vivo, the growth of MDA-MB-231 cells in nude mice was markedly decreased after stable expression of DKK2. DKK2 suppressed canonical Wnt/ß-catenin signaling by inhibiting ß-catenin activity with decreased active ß-catenin protein. Thus, our findings demonstrate that DKK2 functions as a tumor suppressor through inhibiting cell proliferation and inducing apoptosis via regulating Wnt signaling during breast tumorigenesis.

Dickkopf-Related Protein 2 is Epigenetically Inactivated and Suppresses Colorectal Cancer Growth and Tumor Metastasis by Antagonizing Wnt/ß-Catenin Signaling.

BACKGROUND/AIMS: Aberrant activation of the Wnt/ß-catenin signaling pathway plays a key role in the pathogenesis of multiple tumors including digestive cancers. Recent studies have reported that Dickkopf-related protein 2 (DKK2) is epigenetically inactivated in numerous types of cancers and that its gene products exhibit tumor-suppressive properties. However, the biological functions and underlying molecular mechanisms of DKK2 in colon carcinoma remains obscure. METHODS: We examined the expression of DKK2 in colon tumor cell lines by RT-PCR and its promoter methylation status in colon tumor cell lines and primary tumors by methylation-specific PCR (MSP). Ectopic expression of DKK2 was measured by RT-PCR prior to the other experiments. To investigate the function of DKK2, we assayed colony formation and cell proliferation, utilized flow cytometric analyses of the cell cycle and acridine orange/ethidium bromide (AO/EB) fluorescence staining for apoptosis, and examined wound healing, transwell migration and tumor growth in vivo. Western blots were used to explore the mechanisms of DKK2 in epithelial- mesenchymal transition and canonical Wnt/ß-catenin signaling. RESULTS: We show here that downregulation or silencing of DKK2 was closely associated with the hypermethylation status of its promoter and that DKK2 expression could be restored by demethylation treatment. Methylation of the DKK2 promoter was detected in nearly all tumors and tumor-adjacent tissues, but not in normal colon tissues. Ectopic expression of DKK2 in colon cell lines HCT116 and HT-29 inhibited colony formation and cell viability by inducing cell cycle G0/G1 arrest and apoptosis, and growth of stable DKK2-infected HCT116 cells in nude mice was decreased compared to controls. Furthermore, DKK2 restrained cell migration through partial reversal of epithelial-to- mesenchymal transition and also by downregulating several stem cell markers. Our data further showed that restoration of DKK2 expression resulted in downregulation of active ß-catenin and its downstream target genes. CONCLUSION: DKK2 appears to be a functional tumor suppressor regulating tumorigenesis of colorectal cancer by antagonizing Wnt/ß-catenin signaling.

DACT2 silencing by promoter CpG methylation disrupts its regulation of epithelial-to-mesenchymal transition and cytoskeleton reorganization in breast cancer cells.

Wnt signaling plays an important role in breast carcinogenesis. DAPPER2 (DACT2) functions as an inhibitor of canonical Wnt signaling and plays distinct roles in different cell contexts, with its role in breast tumorigenesis unclear. We investigated DACT2 expression in breast cancer cell lines and primary tumors, as well as its functions and molecular mechanisms. Results showed that DACT2 expression was silenced in 9/9 of cell lines. Promoter CpG methylation of DACT2 was detected in 89% (8/9) of cell lines, as well as in 73% (107/147) of primary tumors, but only in 20% (1/5) of surgical margin tissues and in none of normal breast tissues. Demethylation of BT549 and T47D cell lines with 5-aza-2'-deoxycytidine restored DACT2 expression along with promoter demethylation, suggesting that its downregulation in breast cancer is dependent on promoter methylation. Furthermore, ectopic expression of DACT2 induced breast cell apoptosis in vitro, and further inhibited breast tumor cell proliferation, migration and EMT, through antagonizing Wnt/ß-catenin and Akt/GSK-3 signaling. Thus, these results demonstrate that DACT2 functions as a tumor suppressor for breast cancer but was frequently disrupted epigenetically in this cancer.

Protocadherin 17 functions as a tumor suppressor suppressing Wnt/ß-catenin signaling and cell metastasis and is frequently methylated in breast cancer.

Protocadherins play important roles in the regulation of cell adhesion and signaling transduction. Aberrant expression of protocadherins has been shown to be associated with multiple tumorigenesis. We previously identified PCDH17, encoding protocadherin 17, as a frequently methylated and downregulated tumor suppressor gene (TSG) in gastric and colorectal cancers. Here, we examined the abnormalities and functions of PCDH17 in breast cancer pathogenesis. We used PCR and immunohistochemistry to check its expression pattern in breast tumor cell lines and primary tumors. Methylation-specific PCR (MSP) was applied to examine its promoter methylation status in breast tumor cell lines and primary tumors. The biological functions of PCDH17 in breast tumor cells were assessed using in vitro and in vivo assays. We found that PCDH17 was frequently downregulated or silenced in 78% (7/9) of breast tumor cell lines, as well as 89% (32/36) of primary tumors. Downregulation of PCDH17 in breast cancer was mainly due to the methylation of its promoter. Ectopic expression of PCDH17 in breast tumor cells inhibited cell proliferation and mobility through arresting cell cycle and inducing apoptosis. In breast tumor cells, PCDH17 significantly suppressed the active ß-catenin level and its downstream target gene expression. Thus, we found that PCDH17 functions as a tumor suppressor inhibiting Wnt/ß-catenin signaling and metastasis in breast cancer but is frequently methylated in primary tumors which could be a potential biomarker.