LINC00936/microRNA-221-3p Regulates Tumor Progression in Ovarian Cancer by Interacting with LAMA3.

BACKGROUND: Ovarian cancer remains a leading cause of mortality in women. It is known that long non-coding RNA (lncRNA) controls various biological processes and pathogenesis of many diseases, including cancers. This study aimed to determine whether LINC00936 and microRNA-221-3p (miR-221-3p) influence the laminin alpha 3 chain gene (LAMA3) in the development of ovarian cancer. METHODS: The expressions of LINC00936, miR-221-3p, and LAMA3 in ovarian cancer and adjacent tissues were assessed. Furthermore, ovarian cancer cells were transfected with vectors with overexpressed LINC00936, miR-221-3p mimic, miR-221-3p inhibitor, and si-LAMA3 to elucidate their functions in ovarian cancer cell proliferation, migration, invasion, angiogenesis, and tumorigenesis. The binding relationship between LINC00936 and miR-221-3p and the relationship between miR-221-3p and LAMA3 were verified to explore the mechanism of action of LINC00936 in ovarian cancer. LINC00936 binds to miR-221-3p as a ceRNA and regulates the expression of LAMA3. RESULTS: LINC00936 and LAMA3 were poorly expressed, while miR-221-3p was highly expressed in ovarian cancer tissues. Over-expression of LINC00936 contributed to decreasing miR- 221-3p expression and increasing LAMA3 expression. LINC00936 overexpression or miR-221- 3p silencing downregulated the levels of PCNA, MMP-2, MMP-9, and VEGF and decreased cell proliferation, migration, invasion, angiogenesis, and ovarian cancer tumorigenesis. CONCLUSION: Collectively, overexpression of LINC00936 suppressed the development of ovarian cancer by competitively binding to miR-221-3p and controlling LAMA3 expression. These results could serve as a novel theoretical base for the treatment of ovarian cancer.

Cervical Cancer Cells-Derived Extracellular Vesicles Containing microRNA-146a-5p Affect Actin Dynamics to Promote Cervical Cancer Metastasis by Activating the Hippo-YAP Signaling Pathway via WWC2.

Application of extracellular vesicles (EVs) for cancer treatment has been well-documented. We probed into the potential role of cervical cancer cells-secreted EVs by transferring miR-146a-5p in cervical cancer. After characterization of miR-146a-5p expression in clinical cervical cancer tissue samples, gain- and loss-of-function experiments were implemented to test the effect of miR-146a-5p on the invasion, epithelial-mesenchymal transition (EMT), and anoikis in cervical cancer cells. EVs were isolated from high-metastatic cervical cancer cells, after which their effects on the malignant behaviors of low-metastatic cervical cancer cells were assessed in a co-culture system. Luciferase assay was implemented to validate the putative binding relationship between miR-146a-5p and WWC2, followed by further investigation of downstream pathway (Hippo-YAP). Finally, nude mouse lung metastasis model was developed for in vivo validation. miR-146a-5p was elevated in cervical cancer tissues and high miR-146a-5p expression promoted the metastatic potential of cervical cancer cells through enhancing their invasiveness and anoikis resistance, and inducing EMT. Furthermore, miR-146a-5p carried by EVs secreted by highly metastatic cervical cancer cells could promote the metastasis of low-metastatic cervical cancer cells. Mechanistically, miR-146a-5p targeted WWC2 to activate YAP, by which it inhibited the phosphorylation of cofilin, and promoted the process of cofilin-mediated depolymerization of F-actin to G-actin. In vivo data demonstrated that EVs-carried miR-146a-5p promoted tumor metastasis through the WWC2/YAP axis. Cancer-derived EVs delivered pro-metastatic miR-146a-5p to regulate the actin dynamics in cervical cancer, thereby leading to cancer metastasis. This experiment highlighted an appealing therapeutic modality for cervical cancer.

Histone Methyltransferase SETDB1 Promotes Immune Evasion in Colorectal Cancer via FOSB-Mediated Downregulation of MicroRNA-22 through BATF3/PD-L1 Pathway.

Tumors may develop a variety of immune evasion mechanisms during the progression of colorectal cancer (CRC). Here, we intended to explore the mechanism of histone methyltransferase SETDB1 in immune evasion in CRC. The expression of SETDB1, microRNA-22 (miR-22), BATF3, PD-L1, and FOSB in CRC tissues and cells was determined with their interactions analyzed also. Gain-of-function and loss-of-function approaches were employed to evaluate the effects of the SETDB1/FOSB/miR-22/BATF3/PD-L1 axis on T cell function, immune cell infiltration, and tumorigenesis. Aberrant high SETDB1 expression in CRC was positively associated with PD-L1 expression. SETDB1 negatively regulated miR-22 expression by downregulating FOSB expression, while miR-22 downregulated PD-L1 expression via targeting BATF3. Furthermore, SETDB1 silencing promoted the T cell-mediated cytotoxicity to tumor cells via the FOSB/miR-22/BATF3/PD-L1 axis and hindered CRC tumor growth in mice while leading to decreased immune cell infiltration. Taken together, SETDB1 could activate the BATF3/PD-L1 axis by inhibiting FOSB-mediated miR-22 and promote immune evasion in CRC, which provides a better understanding of the mechanisms underlying immune evasion in CRC.

Combination therapy with Nab-paclitaxel and the interleukin-15 fused with anti-human serum albumin nanobody as a synergistic treatment for colorectal cancer.

This study determines the effect of Nab-paclitaxel in combination with IL-15 fusion protein, containing IL-15 and an anti-HSA nanobody domain, on colorectal cancer bearing mice. In vitro binding test of IL15 fusion protein to HSA and Nab-paclitaxel, as well as CTLL-2 cell stimulation assay were performed. The tumor inhibitory effects of Nab-paclitaxel in combination with IL-15 fusion protein was evaluated in the HCT116 bearing murine model. Moreover, the population and function of cytotoxic T cells and M1 macrophages, as well as MDSCs and Treg cells, were also further examined. As a result, combination therapy of Nab-paclitaxel and IL-15 fusion protein effectively inhibits the tumor growth and produced a 78% reduction in tumor size for HCT116, as compared to vehicle group. In the TDLN for the combination group, there were 18% of CD8+ IFN-γ + T-cells and 0.47% CD4+CD25+FOXP3+ regulatory T-cells, as opposed to 5.0% and 5.1%, respectively, for the model control group. Combination therapy further exhibited enhanced suppressive effects on the accumulation of CD11b+GR-1+ MDSC in spleen and bone marrow. Furthermore, Nab-paclitaxel and IL-15 fusion protein showed a significant suppression of NF-κB-mediated immune suppressive markers and increased expression of CD8, Granzyme B, CD62L, CD49b, and CD86 without obvious organ toxicity. In conclusion, combination therapy of Nab-paclitaxel and IL-15 fusion protein can effectively stimulate the antitumor activity of immune effector cells, thereby inhibiting immunosuppressive cells within the TME of colorectal cancer, and the overall therapeutic effect has a significant advantage over monotherapy.AbbreviationsInterleukin 15, IL-15; Human serum albumin, HSA; Myeloid-derived suppressor cells, MDSC; Albumin binding domain, ABD; Tumor drainage lymph node, TDLN; Natural killer (NK); Tumor-draining lymph node (TDLN); Tumor infiltrating lymphocyte, TIL; Immunogenic cell death, ICD; Enhanced permeability retention, EPR; Liposomal doxorubicin, Doxil; 5-fluorouracil, 5-FU.

Intravenous Delivery of RNA Encoding Anti-PD-1 Human Monoclonal Antibody for Treating Intestinal Cancer.

Recently, antibody-based therapeutic agents are becoming most leading biologics for treating many diseases, especially for cancer. However, large-scale application of antibody drugs is still hampered by high cost and complex technical process. Endogenous expression of proteins or antibodies can be achieved by applying in vitro transcription (IVT) technique to produce mRNA and then deliver into body, which supplies opportunity to avoid many disadvantages in antibody production as well as clinical applications. Here, we designed the IVT-mRNA encoding the Pembrolizumab, as a commercial anti-PD-1 monoclonal antibody (mAb). The in vitro functional properties and in vivo antitumor activities of the Pembrolizumab expressed from mRNA were both assessed. Maximized expression level of the Pembrolizumab from IVT-mRNA was achieved via optimizing the usage of signal peptide and molar ratio of heavy/light chain. Then the mRNA was further formulated by lipid nanoparticle (LNP), which enable efficient in vivo delivery and protect mRNA from degradation. Intravenously delivered the single dose of mRNA-LNPs in mice resulted in duration of serum Pembrolizumab level over 25 µg/mL more than 35 days. Pharmacokinetic study exhibited significantly enhanced drug exposure of mRNA-encoded mAbs compared with direct injection of Pembrolizumab at same dose. Chronic treatment of the tumor-bearing mice with LNP-encapsulated Pembrolizumab mRNA effectively downregulated the growth of intestinal tumors and improved the animal survival. In brief, our present research demonstrated that the application of LNP-encapsulated IVT-mRNA can change the human body into a protein drug manufacturing site to express full-size mAbs for treating cancer and hold potential to be a novel alternative to protein-based therapies.

Engineered mRNA-expressed bispecific antibody prevent intestinal cancer via lipid nanoparticle delivery.

The potential of antibodies, especially for the bispecific antibodies, are limited by high cost and complex technical process of development and manufacturing. A cost-effective and rapid platform for the endogenous antibodies expression via using the in vitro transcription (IVT) technique to produce nucleoside-modified mRNA and then encapsulated into lipid nanoparticle (LNP) may turn the body to a manufactory. Coinhibitory pathway of programmed death ligand 1 (PD-L1) and programmed cell death protein 1 receptor (PD-1) could suppress the T-cell mediated immunity. We hypothesized that the coblocking of PD-L1 and PD-1 via bispecific antibodies may achieve more potential antitumor efficacies compare with the monospecific ones. Here, we described the application of mRNA to encode a bispecific antibody with ablated Fc immune effector functions that targets both human PD-L1 and PD-1, termed XA-1, which was further assessed the in vitro functional activities and in vivo antitumor efficacies. The in vitro mRNA-encoded XA-1 held comparable abilities to fully block the PD-1/PD-L1 pathway as well as to enhance functional T cell activation compared to XA-1 protein from CHO cell source. Pharmacokinetic tests showed enhanced area under curve (AUC) of mRNA-encoded XA-1 compared with XA-1 at same dose. Chronic treatment of LNP-encapsulated XA-1 mRNA in the mouse tumor models which were reconstituted with human immune cells effectively induced promising antitumor efficacies compared to XA-1 protein. Current results collectively demonstrated that LNP-encapsulated mRNA represents the viable delivery platform for treating cancer and hold potential to be applied in the treatment of many diseases.Abbreviations: IVT: in vitro transcription; LNP: lipid nanoparticle; hPD-1: human PD-1; hPD-L1: human PD-L1; ITS-G: Insulin-Transferrin-Selenium; Pen/Strep: penicillin-streptomycin; FBS: fetal bovine serum; TGI: tumor growth inhibition; IE1: cytomegalovirus immediate early 1; SP: signal peptide; hIgLC: human immunoglobulin kappa light chain; hIgHC: human IgG1 heavy chain; AUC: area under the curve; Cl: serum clearance; Vss: steady-state distributed volume; MLR: mixed lymphocyte reaction.

Lipopolysaccharides increase the risk of colorectal cancer recurrence and metastasis due to the induction of neutrophil extracellular traps after curative resection.

BACKGROUND: Intra-abdominal infection after curative surgery for colorectal cancer is a serious complication associated with an increased risk of recurrence. Lipopolysaccharides (LPS)-an essential component of the cell wall of Gram-negative bacteria-were found to exert a protumorigenic effect by stimulating the inflammatory pathology and formation of neutrophil extracellular traps (NETs). This study was conducted to test whether LPS-induced formation of NETs promotes the development of cancer and metastasis. METHODS: The clinical characteristics, incidence of relapse, and serum myeloperoxidase-DNA complexes of 40 patients with infection and 40 patients without infection after curative surgery were analyzed. The effects of LPS on the induction of NETs were evaluated in a mouse model of colorectal cancer and liver metastasis. The toll-like receptor 9 (TLR9)-a DNA receptor-was knocked down to assess its effect on the mitogen-activated protein kinase pathway and activities implicated in the formation of NETs. RESULTS: Analysis of the clinical data obtained from these patients showed the significant relation of the formation of NETs and incidence of metastasis and survival rates. Subsequent in vitro experiments revealed an increased level of citrullinated-histone H3 and myeloperoxidase-DNA in LPS-injected mice with colorectal cancer. In the mimic metastatic model, injection of LPS enhanced the metastatic capacity, which was then attenuated by DNase I. This suggested that the formation of NETs was activated by LPS. Injection of TLR9-knockdown HCT116 cells in mice, followed by induction through LPS, mitigated the level of citrullinated-histone H3 and myeloperoxidase-DNA. This finding implied that the formation of NETs was suppressed. CONCLUSION: These findings shed light on the mechanism underlying the relationship between the elevated rate of colorectal cancer recurrence in patients who underwent surgery and the incidence of infection. This mechanism involves the protumorigenic activities of LPS-induced formation of NETs. The NETs which could be mediated by the TLR9 and the mitogen-activated protein kinase signaling pathway.