Intravesical instillation-based mTOR-STAT3 dual targeting for bladder cancer treatment.

BACKGROUND: Recent intravesical administration of adenoviral vectors, either as a single injection or in combination with immune checkpoint inhibitors, exemplified by cretostimogene grenadenorepvec and nadofaragene firadenovec, has demonstrated remarkable efficacy in clinical trials for non-muscle invasive bladder cancer. Despite their ability to induce an enhanced immune reaction within the lesion, the intracellular survival signaling of cancer cells has not been thoroughly addressed. METHODS: An analysis of the prognostic data revealed a high probability of therapeutic efficacy with simultaneous inhibition of mTOR and STAT3. Considering the challenges of limited pharmaco-accessibility to the bladder due to its pathophysiological structure and the partially undruggable nature of target molecules, we designed a dual siRNA system targeting both mRNAs. Subsequently, this dual siRNA system was encoded into the adenovirus 5/3 (Ad 5/3) to enhance in vivo delivery efficiency. RESULTS: Gene-targeting efficacy was assessed using cells isolated from xenografted tumors using a single-cell analysis system. Our strategy demonstrated a balanced downregulation of mTOR and STAT3 at the single-cell resolution, both in vitro and in vivo. This approach reduced tumor growth in bladder cancer xenograft and orthotopic animal experiments. In addition, increased infiltration of CD8+ T cells was observed in a humanized mouse model. We provided helpful and safe tissue distribution data for intravesical therapy of siRNAs coding adenoviruses. CONCLUSIONS: The bi-specific siRNA strategy, encapsulated in an adenovirus, could be a promising tool to augment cancer treatment efficacy and overcome conventional therapy limitations associated with "undruggability." Hence, we propose that dual targeting of mTOR and STAT3 is an advantageous strategy for intravesical therapy using adenoviruses.

hsa-miR-CHA2, a novel microRNA, exhibits anticancer effects by suppressing cyclin E1 in human non-small cell lung cancer cells.

Despite considerable therapeutic advancements, the global survival rate for lung cancer patients remains poor, posing challenges in developing an effective treatment strategy. In many cases, microRNAs (miRNAs) exhibit abnormal expression levels in cancers, including lung cancer. Dysregulated miRNAs often play a crucial role in the development and progression of cancer. Therefore, understanding the mechanisms underlying aberrant miRNA expression during carcinogenesis may provide crucial clues to develop novel therapeutics. In this study, we identified and cloned a novel miRNA, hsa-miR-CHA2, which is abnormally downregulated in non-small cell lung cancer (NSCLC)-derived cell lines and tissues of patients with NSCLC. Furthermore, we found that hsa-miR-CHA2 regulates the post-transcriptional levels of Cyclin E1 (CCNE1) by binding to the 3'-UTR of CCNE1 mRNA. CCNE1, a cell cycle regulator involved in the G1/S transition, is often amplified in various cancers. Notably, hsa-miR-CHA2 overexpression led to the alteration of the Rb-E2F pathway, a significant signaling pathway in the cell cycle, by targeting CCNE1 in A549 and SK-LU-1 cells. Subsequently, we confirmed that hsa-miR-CHA2 induced G1-phase arrest and exhibited an anti-proliferative effect by targeting CCNE1. Moreover, in subcutaneous xenograft mouse models, intra-tumoral injection of polyplexed hsa-miR-CHA2 mimic suppressed tumor growth and development. In conclusion, hsa-miR-CHA2 exhibited an anticancer effect by targeting CCNE1 both in vitro and in vivo. These findings suggest the potential role of hsa-miR-CHA2 as an important regulator of cell proliferation in molecular-targeted therapy for NSCLC.

Isolation and Genomic Analysis of Single Circulating Tumor Cell Using Human Telomerase Reverse Transcriptase and Desmoglein-2.

As epithelial cells in the circulation are considered to originate from the tumor, the epithelial cell adhesion molecule has been commonly used as a standard marker for circulating tumor cells (CTCs) isolation. However, it seems to disappear after the epithelial-mesenchymal transition that most cancer cells undergo for intravasation. Thus, more advanced techniques for CTC detection are needed to better understand the clinical significance of CTCs. A cancer cell-specifically-infecting or replicating virus that codes a fluorescent monitor gene can be a solution to efficiently detect CTCs. Thus, the authors designed an adenovirus to bind to desmoglein-2, which is highly expressed in most cancer cells. A cancer-specific human telomerase reverse transcriptase promoter is inserted to control a viral E1 region. The adenovirus is utilized to compare the number of CTCs from renal cell carcinoma and prostate cancer patients before and after surgery. The isolated two or three CTCs are eligible for whole genome sequencing. The genomic analysis proves the difference of variants between primary tumors and CTCs. Taken together, it is a fast and exact serial method for CTC isolation and the enriched genome sequencing may be used to determine the prognosis and as a point-of-care system for patients with cancer.

The novel microRNA hsa-miR-CHA1 regulates cell proliferation and apoptosis in human lung cancer by targeting XIAP.

OBJECTIVES: MicroRNAs have critical roles in cancer development by regulating the expression of oncogenes or tumor suppressor genes. We identified and characterized a novel miRNA, miR-CHA1, in human lung cancer cells. The aim of this study was to investigate its novel function in human lung cancer by targeting XIAP. MATERIAL AND METHODS: Novel miRNA cloning, Real-time qRT-PCR, western blotting, dual luciferase assay, miRNA transfection, proliferation and apoptosis assay were carried on human lung cancer cell line A549. Fifteen paired NSCLC tissues and noncancerous lung tissues were collected. In vivo xenograft assay was performed. RESULTS: Expression of miR-CHA1 was downregulated in human lung cancer cell lines and tissues compared with normal cells and tissues. We identified a putative target gene, XIAP, whose expression was regulated by miR-CHA1 overexpression. XIAP is an inhibitor of apoptosis that represses the activation of caspase 3 and 9. XIAP mRNA and protein levels were directly suppressed by miR-CHA1. XIAP has an important role in carcinogenesis, and previous studies suggest that it may regulate cell survival and proliferation by its anti-apoptotic ability. CONCLUSION: Taken together, miR-CHA1 inhibited cell proliferation and induced apoptosis in vitro and in vivo by targeting XIAP. These data can be applied to identify novel therapeutic targets for lung cancer therapy.

Correction to: Selective killing of circulating tumor cells prevents metastasis and extends survival.

The original article [1] contains an inadvertent omission in the Funding declaration.

Anti-psoriatic effect of myeloid-derived suppressor cells on imiquimod-induced skin inflammation in mice.

Myeloid-derived suppressor cells (MDSCs) play an important role in controlling the immune response against cancer and in suppression of autoimmunity and allergic inflammation. However, the beneficial effects of MDSCs on the experimental mouse model of psoriasis have not been reported. Therefore, we investigated the anti-psoriatic effect of MDSCs on IMQ-induced skin inflammation in mice and explored the mechanisms involved. Our results showed that administration of MDSCs (1 × 106 or 2 × 106  cells) suppressed the development of IMQ-induced skin inflammation in mice as exemplified by a significant reduction in clinical severity scores and was associated with a reduction of histopathological changes, including inflammatory infiltration, epidermal hyperplasia and hyperkeratosis. The immunosuppressive effect of MDSCs (1 × 106 or 2 × 106  cells) corresponded to the production of Th1 cytokines (TNF-α, IFN-γ) and Th17 cytokines (IL-17A and IL-23) in the serum and dorsal skin. Administration of MDSCs (1 × 106 or 2 × 106  cells) also inhibited splenomegaly. Moreover, an increased percentage of CD4+ CD25+ FoxP3+ regulatory T (Treg) cells and decreased percentage of Th1 and Th17 cells were found in mice treated with MDSCs. Taken together, these results imply that MDSCs have immunomodulatory and immunosuppressive effects on disease progression in a murine model of psoriasis and that MDSCs could be used in preventive or therapeutic strategies for the management of autoimmune inflammatory skin disorders, such as psoriasis.

Selective killing of circulating tumor cells prevents metastasis and extends survival.

Distant metastasis is initiated by circulating tumor cells (CTCs), which are considered to be a determining factor for the degree of metastasis and the survival of cancer patients. Although CTC-based diagnostic approaches are being rapidly developed, limited studies have proven the benefits of CTC elimination, with most studies providing only hypothetical inference because of the technical difficulty in examining the effects of CTC elimination in vivo. We modified photodynamic therapy to specifically eliminate green fluorescent protein (GFP)-expressing CTCs and evaluated the therapeutic efficacy of CTC elimination. When circulating blood is illuminated with a blue laser (λ = 473 nm), the combination of GFP and photosensitizers induces a selective elimination of GFP-expressing CTCs, with limited effect on normal cells. In GFP-expressing cancer cell-infused or transplanted mice models, the treatment suppressed distant metastasis and extended the survival of the tumor-bearing mice. Taken together, CTCs are a core seed to be metastasized into secondary organs and elimination of CTCs may improve the survival of cancer patients.

The novel hsa-miR-12528 regulates tumourigenesis and metastasis through hypo-phosphorylation of AKT cascade by targeting IGF-1R in human lung cancer.

Lung cancer cases are increasing yearly; however, few novel therapeutic strategies for treating this disease have been developed. Here the dysregulation between microRNAs and oncogenes or tumour-suppressor genes forms a close connection-loop to the development or progression in human lung carcinogenesis. That is, the relationship between microRNAs and carcinogenic mechanism may find the critical clue to improve the treatment efficacy. Accordingly, we identified and characterised a novel microRNA, hsa-miR-12528, in A549 cells. The miR-12528 expression was aberrantly downregulated in cancer cell lines and in the patient tissues derived from human non-small cell lung cancer. In addition, we found that miR-12528 post-transcriptionally controls the translation of the insulin-like growth factor 1 receptor (IGF-1R) gene by directly targeting the 3'-untranslated region of IGF-1R mRNA. Notably, the IGF-1R gene is elevated in the majority of cancers and may be an attractive therapeutic target for anticancer therapy because elevated IGF-1R mediates the signalling amplification of a major oncogenic pathway in neoplasia. In A549 cells, miR-12528 overexpression epigenetically altered the downstream phosphorylation of the primary IGF-1R networks, negatively regulated proliferation, apoptosis and migratory activity, and consequently inhibited tumourigenesis and metastasis in vivo. Therefore, our discovery of hsa-miR-12528 may be able to be applied to the development of molecular-target therapeutic strategies and diagnosis-specific biomarkers for human lung cancer.

Protective effects of agonistic anti-4-1BB antibody on the development of imiquimod-induced psoriasis-like dermatitis in mice.

Agonistic anti-4-1BB antibodies (Abs) play a central role in immunomodulatory conditions that control the pathogenesis of immune-mediated autoimmune and allergic diseases. However, the effects of agonistic anti-4-1BB Abs have not been examined in an experimental mouse model of psoriasis. Therefore, we investigated the protective effects of agonistic anti-4-1BB Abs, using imiquimod (IMQ)-induced psoriasis-like dermatitis in mice, a condition histologically and clinically similar to human psoriasis. We found that administration of agonistic anti-4-1BB Abs (10mg/kg) significantly alleviated the severity of IMQ-induced psoriasis-like skin inflammation in mice, with reduced histologic symptoms, including inflammatory infiltration, parakeratosis, and hyperkeratosis. Subsequent analyses revealed that the production of Th17 cytokines (IL-17A and IL-23) in the serum and skin of IMQ-induced mice was significantly inhibited by agonistic anti-4-1BB Abs (10mg/kg), although Th1 cytokines (TNF-α and IFN-γ) were not. Moreover, administration of agonistic anti-4-1BB Abs (10mg/kg) induced a relative increase of CD4(+)FoxP3(+) regulatory T (Treg) cells in the spleen and draining lymph node (DLN). Taken together, our data provide evidence that agonistic anti-4-1BB Abs possesses immunosuppressive properties in IMQ-induced psoriasis-like skin inflammation, providing insight into the immunomodulatory effect of agonistic anti-4-1BB Abs for psoriasis immunotherapy.

3'-UTR Polymorphisms in the MiRNA Machinery Genes DROSHA, DICER1, RAN, and XPO5 Are Associated with Colorectal Cancer Risk in a Korean Population.

MicroRNAs play an important role in cancer initiation and development. The aim of this study was to investigate whether polymorphisms in miRNA machinery genes are associated with the development of colorectal cancer (CRC). RAN rs14035 CT heterozygotes and T allele carriers (CT + TT) genotypes had lower risk of CRC, while the DICER1 rs3742330, DROSHA rs10719, and XPO5 rs11077 polymorphisms were not associated with CRC in the full study sample. Specifically, male RAN rs14035 CT heterozygotes and XPO5 rs11077 AA genotype (CT/AA) carriers experienced reduced CRC susceptibility (both colon and rectal). Subgroup analysis demonstrated that the combined RAN rs14035 CT + TT genotype was associated with rectal cancer, but not colon cancer. In addition, the DICER1 rs3742330 AG genotype was associated with a significantly increased risk of colon cancer. Stratified analysis revealed the RAN rs14035 combined CT+TT genotype was associated with decreased CRC risk in male patients without diabetes mellitus (DM) and in patients with rectal cancer. In addition, we found the RAN rs14035 CC genotype was related to a decreased risk of CRC with respect to tumor size and metabolism of homocysteine and folate. Furthermore, patients diagnosed with hypertension or DM who carried the DROSHA rs10719 CC genotype showed increased CRC risk, while the XPO5 rs11077 AC+CC genotype led to increased CRC risk in patients with hypertension only. Our results indicate variations in RAN rs14035, DICER1 rs3742330, XPO5 rs11077, and DROSHA rs10719 of Korean patients are significantly associated with their risk of CRC.

Discovery and characterization of miRNA during cellular senescence in bone marrow-derived human mesenchymal stem cells.

Cellular senescence is an irreversible cell cycle arrest in which specific mRNAs and miRNAs are involved in senescence progression. miRNAs interact with specific mRNAs to regulate various cellular mechanisms, including metabolism, proliferation, apoptosis, senescence and differentiation. In this study, we identify and characterize miRNAs during cellular senescence in mesenchymal stem cells (MSCs). Using previously reported miRNAs, expression profiling of 23 miRNAs was performed using real-time PCR analysis. Among these miRNAs, 19 miRNAs showed upregulated expression patterns in senescent MSCs compared with young MSCs, and 5 miRNAs were downregulated. These miRNAs have not been previously identified as being related to cellular senescence but seem to be related. miR-103-2*, miR-140-5p and miR-330-5p are highly upregulated, while miR-29b and miR-199b-5p are significantly downregulated in senescent MSCs. We identify unique functions of 5 miRNAs and predict putative target genes of 5 miRNAs using our previous report. Among them, miR-199b-5p directly suppressed LAMC1 expression, as shown in a luciferase assay. miR-199b-5p significantly regulates translational activity but does not control post-transcriptional activity. Likewise, miR-199b-5p modulates LAMC networks, which demonstrates the resulting phenomenon during cellular senescence, namely, that miR-199b-5p indirectly regulates cellular senescence in MSCs.

Expression profiles of subtracted mRNAs during cellular senescence in human mesenchymal stem cells derived from bone marrow.

Cellular senescence is an irreversible cell cycle arrest that limits the replicative lifespan of cells. Senescence suppresses development of tumors by regulating aging factors, such as cyclin dependent kinase inhibitor (CKI) and telomerase. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between young human mesenchymal stem cells (Y-hMSCs) and senescent human mesenchymal stem cells (S-hMSCs). We selected positive clones that were functionally characterized by referring to public databases using NCBI BLAST tool. This search revealed that 19 genes were downregulated, and 43 genes were upregulated in S-hMSCs relative to Y-hMSCs. Among subtracted clones in Y-hMSCs, most of genes markedly were related to metabolic functions. These genes, PDIA3, WDR1, FSTL1, COPG1, LMAN1, and PDIA6, significantly downregulated. Conversely, genes for subtracted clones in S-hMSCs were mostly associated with cell adhesion. In particular, the expression levels of 9 genes, HSP90B1, EID1, ATP2B4, DDAH1, PRNP, RAB1A, PGS5, TM4SF1 and SSR3, gradually increased during senescence. These genes have not previously been identified as being related to cellular senescence, but they seemed to be potentially affected during cellular senescence.

miR-7641 modulates the expression of CXCL1 during endothelial differentiation derived from human embryonic stem cells.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that negatively regulate gene expression through binding to 3' untranslated region. We identified and characterized the novel miRNA, miR-7641, in human mesenchymal stem cells. The expression of miR-7641 was downregulated during differentiation from human embryonic stem cells to endothelial cells. The CXCL1, a member of the CXC chemokine family, is known as promoting neovascularization by binding G-protein coupled receptors and is related to endothelial cells biogenesis such as angiogenesis, and it was predicted as target gene of miR-7641 by computerized analysis and the luciferase reporter assay. The miR-7641 significantly suppressed CXCL1 of transcriptional and post-translational levels. These data suggest that miR-7641 might be related with differentiation of human endothelial cells.

The novel miR-7515 decreases the proliferation and migration of human lung cancer cells by targeting c-Met.

MicroRNAs (miRNA) are small noncoding RNAs that regulate gene expression in human diseases, including lung cancer. miRNAs have oncogenic and nononcogenic functions in lung cancer. In this study, we report the identification of a novel miRNA, miR-7515, from lung cancer cells. The novel miR-7515 was characterized using various predictive programs and experimental methods. miR-7515 was able to forming a stem-loop structure and its sequence was conserved in mammals. The expression level of miR-7515 in lung cancer cells and tissues was profiled using TaqMan miRNA assays. miR-7515 was downregulated in lung cancer compared with normal human lung cells and tissues. The target of miR-7515 was determined using a dual luciferase reporter assay. Expression of the target gene was determined by quantitative RT-PCR and Western blot analysis after transfection with miR-7515. miR-7515 directly suppressed human mesenchymal-epithelial transition factor (c-Met) by binding to the 3' untranslated region (UTR). Overexpression of miR-7515 significantly decreased cell-cycle-related proteins downstream of c-Met through c-Met inhibition. Cell proliferation and migration were examined using the XTT proliferation assay and the Transwell migration assay. miR-7515 led to decreased cell proliferation, migration and invasion in a lung cancer cell line. These results suggest that miR-7515 plays an important role in the proliferation and migration of lung cancer cells through c-Met regulation.

Discovery and characterization of novel microRNAs during endothelial differentiation of human embryonic stem cells.

MicroRNAs (miRNAs) are small RNAs that participate in the regulation of genes associated with the differentiation and proliferation. In this study, 5 novel miRNAs were identified from human mesenchymal stem cells and characterized using various analyses. To investigate the potential functions associated with the regulation of cell differentiation, the differences in miRNA expression were examined in undifferentiated and differentiated human embryonic stem (ES) cells using reverse transcription (RT)-PCR analysis. Specifically, 3 miRNAs exhibited decreased expression levels in human umbilical vein endothelial cells (HUVECs) and endothelial cells derived from human ES cells. Putative target genes related to differentiation or maturation of endothelial cells were predicted by seed sequences of 2 novel miRNAs and analyzed for their expression via miRNA-mediated regulation using a luciferase assay. In HUVECs, CDH5 gene expression was directly repressed by hsa-miR-6086. Similarly, hsa-miR-6087 significantly downregulated endoglin expression. Therefore, the roles of these 2 miRNAs may be to directly suppress their target genes, popularly known as endothelial cell markers. Taken together, our results demonstrate that several novel miRNAs perform critical roles in human endothelial cell development.

The hsa-miR-5787 represses cellular growth by targeting eukaryotic translation initiation factor 5 (eIF5) in fibroblasts.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate diverse biological processes. We cloned novel small RNA from human mesenchymal stem cells (hMSCs) and termed microRNA-5787 (hsa-miR-5787) that met the criteria for a miRNA. The level of miR-5787 was elevated in senescent fibroblasts. Based on the target prediction algorithm and results that were obtained, we find that eukaryotic translation initiation factor 5 (eIF5) is a target of miR-5787. Similar to the over-expression of miR-5787, we showed that repression of eIF5 in fibroblasts negatively affected cell growth. Therefore, we propose that the miR-5787 represses cell growth, in part, by targeting eIF5.