SMAD7 in keratinocytes promotes skin carcinogenesis by activating ATM-dependent DNA repair and an EGFR-mediated cell proliferation pathway.

SMA- and MAD-related protein 7 (SMAD7) is a general inhibitor of transforming growth factor-ß (TGF-ß) signaling that acts through interaction and degradation of TGF-ß receptors. SMAD7 has been demonstrated to be transcriptionally upregulated in chemical-induced skin tumors and TGF-ß-treated normal keratinocytes. To evaluate the function of SMAD7 in skin carcinogenesis in vivo, Smad7 transgenic mice that specifically express either wild-type (WT) SMAD7 (TG-Smad7-WT) or mutant SMAD7 (TG-Smad7-MT) in keratinocytes, as well as Smad7 keratinocyte-specific knockout (Smad72f/2f-K14Cre) mice, were subjected to chemical-induced skin carcinogenesis. WT-SMAD7-expressing transgenic mice showed significantly greater papilloma formation than did non-TG control and Smad7-MT mice. The expression of WT-SMAD7 attenuated DNA damage-induced apoptosis in epidermal keratinocytes by stimulating the ATM-dependent DNA repair pathway. Nonetheless, overexpression of WT-SMAD7 caused a susceptibility to 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation through activation of epidermal growth factor (EGF) signaling. In agreement with the transgenic mouse data, keratinocyte-specific deletion of SMAD7 markedly suppressed the tumor formation by inhibiting ATM and epidermal growth factor receptor (EGFR) signaling. Moreover, specific inhibition of EGFR signaling attenuated the hyperproliferation and tumor formation in TG-Smad7-WT mice. Taken together, these data support a novel role for SMAD7 as a tumor promoter in skin carcinogenesis where SMAD7 stimulates the DNA repair pathway and EGFR signaling activation.

MicroRNA-132 and microRNA-223 control positive feedback circuit by regulating FOXO3a in inflammatory bowel disease.

BACKGROUND AND AIM: Although many progresses have been achieved for inflammatory bowel disease (IBD), it is still remained as idiopathic disease to be completely controlled. MicroRNAs (miRNAs) have been identified as key players in many human diseases through degradation or translational inhibition of target genes. Because role of miRNAs in IBD is not completely understood yet, we need to identify miRNAs as novel targets for treatment of IBD. METHODS: Microarray analysis for miRNAs was performed using dextran sulfate sodium-induced colitis samples and selected differentially regulated miRNAs. Candidate genes were validated using in vitro system and IBD patient samples. Molecular mechanism for regulation of inflammatory signaling was identified using gene modulation system of miRNAs. RESULTS: We selected 14 upregulated and 15 downregulated miRNAs through microarray analysis. Among candidate miRNAs, significant upregulation of miR-132 and miR-223 was confirmed in inflamed mouse tissues as well as human IBD patient tissues. Through bioinformatics analysis, we identified FOXO3a as direct target of miRNAs and confirmed regulatory mechanism using luciferase assay. Expression of miRNAs clearly suppressed the level of IκBα through downregulation of FOXO3a, leading to enhanced NF-κB signaling to promote the production of pro-inflammatory cytokines. The downregulation of FOXO3a concurrent with upregulation of cytokines was significantly reversed by sequestration of miRNAs with miRNA sponges. CONCLUSIONS: Our findings provided the evidences that miR-132 and 223 are critical mediators in positive circuit for pathogenesis of IBD by negatively regulating FOXO3a to enhance the expression of inflammatory cytokines and can be a good therapeutic target for IBD treatment.

Evaluation of anti-tumorigenic activity of BP3B against colon cancer with patient-derived tumor xenograft model.

BACKGROUND: KIOM-CRC#BP3B (BP3B) is a novel herbal prescription that is composed of three plant extracts. Our preliminary study identified that BP3B exhibited potent anti-proliferative activity against various types of cancer cell lines in vitro. Because the in vivo anti-tumor effect of BP3B is not evaluated before clinical trial, we want to test it using patient's samples. METHODS: To confirm the in vivo anti-cancer effect of BP3B, we used genetically characterized patient-derived colon tumor xenograft (PDTX) mouse model. Anti-cancer activity was evaluated with apoptosis, proliferation, angiogenesis and histological analysis. RESULTS: Oral administration of BP3B significantly inhibited the tumor growth in two PDTX models. Furthermore, TUNEL assay showed that BP3B induced apoptosis of tumor tissues, which was associated with degradation of PARP and Caspase 8 and activation of Caspase 3. We also observed that BP3B inhibited cancer cell proliferation by down-regulation of Cyclin D1 and induction of p27 proteins. Inhibition of angiogenesis in BP3B-treated group was observed with immunofluorescence staining using CD31 and Tie-2 antibodies. CONCLUSION: These findings indicated that BP3B has a strong growth-inhibitory activity against colon cancer in in vivo model and will be a good therapeutic candidate for treatment of refractory colon cancer.

Transcriptional and post-translational regulation of Bim is essential for TGF-ß and TNF-α-induced apoptosis of gastric cancer cell.

BACKGROUND: Tumor necrosis factor-α (TNF-α) and transforming growth factor-ß (TGF-ß) are well known as central signaling molecules in natural antitumor mechanisms. However, some cancer cells are resistant to TNF-α or TGF-ß-induced death signaling. Herein, we investigated synergistic activities of TGF-ß and TNF-α and molecular mechanisms involved in apoptosis of gastric cancer cells. METHODS: SNU620, a human gastric carcinoma cell line was tested for cell viability by treatment of TGF-ß in combination with TNF-α. Cell apoptosis, proliferation, caspase activation and gene expression were tested using flow cytometry, Western blot, MTT assay, luciferase assay and real-time qRT-PCR analysis. Knockdown of target genes were performed using lentiviral shRNA system. RESULTS: TGF-ß sensitizes SNU620 cells undergoing TNF-α-induced caspase-dependent apoptosis. TNF-α and TGF-ß synergistically induced the degradation of poly(ADP-ribose) polymerase (PARP) and caspase cascade activation. We also confirmed that c-Jun NH2-terminal kinase (JNK) and Smad3 play critical roles in the apoptotic pathway. In addition, a pro-apoptotic protein Bim was critical for apoptosis and was regulated by TGF-ß and TNF-α at the transcriptional and post-translational levels. Expression of Bim was induced at the transcriptional level by Smad3 while Bim protein stability was maintained by a JNK-mediated pathway. CONCLUSION: By understanding the synergistic activation of TGF-ß and TNF-α in apoptosis, we may have a chance to identify good therapeutic approaches for the treatment of cancers that are resistant to death signals. GENERAL SIGNIFICANCE: Our results indicate that TGF-ß and TNF-α act in concert to activate apoptosis in gastric cancer cell through crosstalk between Smad and JNK signaling pathways.

Recent applications of RNA therapeutic in clinics.

Ribonucleic acid (RNA) therapy has been extensively researched for several decades and has garnered significant attention in recent years owing to its potential in treating a broad spectrum of diseases. It falls under the domain of gene therapy, leveraging RNA molecules as a therapeutic approach in medicine. RNA can be targeted using small-molecule drugs, or RNA molecules themselves can serve as drugs by interacting with proteins or other RNA molecules. While several RNA drugs have been granted clinical approval, numerous RNA-based therapeutics are presently undergoing clinical investigation or testing for various conditions, including genetic disorders, viral infections, and diverse forms of cancer. These therapies offer several advantages, such as high specificity, enabling precise targeting of disease-related genes or proteins, cost-effectiveness, and a relatively straightforward manufacturing process. Nevertheless, successful translation of RNA therapies into widespread clinical use necessitates addressing challenges related to delivery, stability, and potential off-target effects. This chapter provides a comprehensive overview of the general concepts of various classes of RNA-based therapeutics, the mechanistic basis of their function, as well as recent applications of RNA therapeutic in clinics.

Pellino 3 promotes the colitis-associated colorectal cancer through suppression of IRF4-mediated negative regulation of TLR4 signalling.

The incidence of colitis-associated colorectal cancer (CAC) has increased due to a high-nutrient diet, increased environmental stimuli and inherited gene mutations. To adequately treat CAC, drugs should be developed by identifying novel therapeutic targets. E3 ubiquitin-protein ligase pellino homolog 3 (pellino 3; Peli3) is a RING-type E3 ubiquitin ligase involved in inflammatory signalling; however, its role in the development and progression of CAC has not been elucidated. In this study, we studied Peli3-deficient mice in an azoxymethane/dextran sulphate sodium-induced CAC model. We observed that Peli3 promotes colorectal carcinogenesis with increased tumour burden and oncogenic signalling pathways. Ablation of Peli3 reduced inflammatory signalling activation at the early stage of carcinogenesis. Mechanistic studies indicate that Peli3 enhances toll-like receptor 4 (TLR4)-mediated inflammation through ubiquitination-dependent degradation of interferon regulatory factor 4, a negative regulator of TLR4 in macrophages. Our study suggests an important molecular link between Peli3 and colonic inflammation-mediated carcinogenesis. Furthermore, Peli3 can be a therapeutic target in the prevention and treatment of CAC.

Emerging roles of non­coding RNAs in the response of rectal cancer to radiotherapy (Review).

Radiotherapy (RT) followed by radical surgery is an effective standard treatment strategy for various types of cancer, including rectal cancer. The response to RT varies among patients, and the radiosensitivity of cancer cells determines the clinical outcome of patients. However, the application of RT to patients with radioresistant tumors may result in radiation­induced toxicity without clinical benefits. Currently, there are no effective methods to predict the response to RT. The limitations of the methods currently used to evaluate tumor radiosensitivity, which are mainly based on clinical and radiological features, are low sensitivity and specificity. Non­coding RNAs (ncRNAs) have emerged as a class of biomarkers for predicting radiosensitivity. In particular, the expression pattern of ncRNAs can predict the response to RT in patients with rectal cancer. Thus, ncRNAs may be used as potential biomarkers and therapeutic targets to improve the diagnosis and treatment outcome of patients with rectal cancer. In the present review, the current knowledge on the limitations of RT for rectal cancer and the association between ncRNA expression and sensitivity of rectal cancer to RT are presented. Additionally, the potential of ncRNAs as predictive biomarkers and therapeutic targets to mitigate resistance of rectal cancer to RT is discussed.

MicroRNA-130a modulates a radiosensitivity of rectal cancer by targeting SOX4.

Radioresistance poses a major challenge in the treatment of advanced rectal cancer. Therefore, understanding the detailed mechanisms of radioresistance may improve patient response to irradiation and the survival rate. To identify the novel targets that modulate the radiosensitivity of rectal cancer, we performed small RNA sequencing with human rectal cancer cell lines. Through bioinformatics analysis, we selected microRNA-310a (miR-130a) as a promising candidate to elucidate radioresistance. miR-130a was dramatically upregulated in radiosensitive rectal cancer cells and overexpression of miR-130a promotes rectal cancer cell radiosensitivity. Mechanically, miR-130a reversed the epithelial-mesenchymal transition phenotype of rectal cancer cells following inhibition of cell invasion upon irradiation. Moreover, miR-130a also inhibited the repair of irradiation-induced DNA damage followed by cell death. We identified that SOX4 was a direct target of miR-130a. Overexpression of SOX4 reversed the promotion activity of miR-130a on radiosensitivity. Together, our findings suggest that miR-130a functions as a radiosensitizer in rectal cancer and reveals a potential therapeutic target and preoperative prognostic marker for radiotherapy.

IMP2 and IMP3 cooperate to promote the metastasis of triple-negative breast cancer through destabilization of progesterone receptor.

Triple-negative breast cancer (TNBC) is one of the most aggressive malignancies and is associated with high mortality rates due to the lack of effective therapeutic targets. In this study, we demonstrated that insulin-like growth factor-II mRNA-binding protein 2 and 3 (IMP2 and IMP3) are specifically overexpressed in TNBC and cooperate to promote cell migration and invasion. Downregulation of both IMP2 and IMP3 in TNBC cells was found to produce a synergistic effect in suppressing cell invasion and invadopodia formation, whereas overexpression of IMP2 and IMP3 in luminal subtype cells enhanced epithelial-mesenchymal transition and metastasis. We also showed that IMP2 and IMP3 are direct targets of microRNA-200a (miR-200a), which is downregulated in TNBC. Conversely, IMP2 and IMP3 suppressed the transcription of miR-200a by destabilizing progesterone receptor (PR) mRNA through recruitment of the CCR4-NOT transcription complex subunit 1 (CNOT1) complex. Together, our findings suggest that IMP2 and IMP3 partially determine the characteristic phenotype and synergistically promote the metastasis of TNBC by downregulating PR. The identified IMP2/3-miR-200a-PR axis represents a novel double-negative feedback loop and serves as a new potential therapeutic target for the treatment of TNBC.

Protein Tyrosine Phosphatase N2 Is a Positive Regulator of Lipopolysaccharide Signaling in Raw264.7 Cell through Derepression of Src Tyrosine Kinase.

T cell protein tyrosine phosphatase N2 (PTPN2) is a phosphotyrosine-specific nonreceptor phosphatase and is ubiquitously expressed in tissues. Although PTPN2 functions as an important regulator in different signaling pathways, it is still unclear what is specific target protein of PTPN2 and how is regulated in lipopolysaccharide (LPS)-induced inflammatory signaling pathway. Here, we found that PTPN2 deficiency downregulated the expression of LPS-mediated pro-inflammtory cytokine genes. Conversely, overexpression of PTPN2 in Raw264.7 cells enhanced the expression and secretion of those cytokines. The activation of MAPK and NF-κB signaling pathways by LPS was reduced in PTPN2-knockdowned cells and ectopic expression of PTPN2 reversed these effects. Furthermore, we found that PTNP2 directly interacted with Src and removed the inhibitory Tyr527 phosphorylation of Src to enhance the activatory phosphorylation of Tyr416 residue. These results suggested that PTPN2 is a positive regulator of LPS-induced inflammatory response by enhancing the activity of Src through targeting the inhibitory phosphor-tyrosine527 of Src.

Smad7 Modulates Epidermal Growth Factor Receptor Turnover through Sequestration of c-Cbl.

Epidermal growth factor (EGF) regulates various cellular events, including proliferation, differentiation, migration, and tumorigenesis. For the maintenance of homeostasis, EGF signaling should be tightly regulated to prevent the aberrant activation. Smad7 has been known as inhibitory Smad that blocks the signal transduction of transforming growth factor ß. In the process of cell proliferation or transformation, Smad7 has been shown the opposite activities as a promoter or suppressor depending on cell types or microenvironments. We found that the overexpression of Smad7 in human HaCaT keratinocyte cells and mouse skin tissues elevated EGF receptor (EGFR) activity by impairing ligand-induced ubiquitination and degradation of activated receptor, which is induced by the E3 ubiquitin ligase c-Cbl. The C-terminal MH2 region but not MH1 region of Smad7 is critical for interaction with c-Cbl to inhibit the ubiquitination of EGFR. Interestingly, wild-type Smad7, but not Smad6 or mutant Smad7, destabilized the EGF-induced complex formation of c-Cbl and EGFR. These data suggest a novel role for Smad7 as a promoter for prolonging the EGFR signal in keratinocyte and skin tissue by reducing its ligand-induced ubiquitination and degradation.