Evolution of cell therapy for renal cell carcinoma.

Treatment for renal cell carcinoma (RCC) has improved dramatically over the last decade, shifting from high-dose cytokine therapy in combination with surgical resection of tumors to targeted therapy, immunotherapy, and combination therapies. However, curative treatment, particularly for advanced-stage disease, remains rare. Cell therapy as a "living drug" has achieved hematological malignancy cures with a high response rate, and significant research efforts have been made to facilitate its translation to solid tumors. Herein, we overview the cellular therapies for RCC focusing on allogeneic hematopoietic stem cell transplantation, T cell receptor gene-modified T cells, chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) cells, lymphokine-activated killer (LAK) cells, γδ T cells, and dendritic cell vaccination. We have also included perspectives for using other recent approaches, such as CAR macrophages, dendritic cell-cytokine induced killer cells and regulatory CAR-T cells to shed light on preclinical development of cell therapy and advancing cell therapy into clinic to achieve cures for RCC.

Isolated Peptide from Spider Venom Modulates Dendritic Cells In Vitro: A Possible Application in Oncoimmunotherapy for Glioblastoma.

Dendritic cells (DCs) vaccine is a potential tool for oncoimmunotherapy. However, it is known that this therapeutic strategy has failed in solid tumors, making the development of immunoadjuvants highly relevant. Recently, we demonstrated that Phoneutria nigriventer spider venom (PnV) components are cytotoxic to glioblastoma (GB) and activate macrophages for an antitumor profile. However, the effects of these molecules on the adaptive immune response have not yet been evaluated. This work aimed to test PnV and its purified fractions in DCs in vitro. For this purpose, bone marrow precursors were collected from male C57BL6 mice, differentiated into DCs and treated with venom or PnV-isolated fractions (F1-molecules < 3 kDa, F2-3 to 10 kDa and F3->10 kDa), with or without costimulation with human GB lysate. The results showed that mainly F1 was able to activate DCs, increasing the activation-dependent surface marker (CD86) and cytokine release (IL-1ß, TNF-α), in addition to inducing a typical morphology of mature DCs. From the F1 purification, a molecule named LW9 was the most effective, and mass spectrometry showed it to be a peptide. The present findings suggest that this molecule could be an immunoadjuvant with possible application in DC vaccines for the treatment of GB.

Functional characterization and development of novel human kinase insert domain receptor chimeric antigen receptor T-cells for immunotherapy of non-small cell lung cancer.

CAR-T cell therapy, in which T cells are transfected or transduced with a chimeric antigen receptor (CAR), is a transformative type of cancer immunotherapy. Despite outstanding success in hematological malignancies, their efficacy against solid tumors has been limited. Here, we aimed to explore whether T cells modified by a CAR targeting the vascular endothelial growth factor 2 receptor/ kinase insert domain receptor (KDR) could destroy tumors and their vasculature. A second-generation KDR-CAR was constructed and transfected into T cells using lentivirus. The 3D structure of the CAR construct and target antigen was predicted. Moreover, in silico analysis, including molecular docking and molecular dynamics (MD) simulation, were used to evaluate the minimum energy of interaction and stability of the complex. The anti-cancer effect of KDR-specific CAR-T cells was tested with KDR-expressing and KDR overexpressing A549 cell line. The in-silico study suggested that this CAR construct could be effective for lung cancer therapy. We evaluated this using both in vitro and in vivo experiments. The KDR-CAR-T cells targeted and killed KDR-A549 with high efficiency by expressing IFN-γ and releasing granzyme B. The in vivo study showed that KDR-CAR-T cells dramatically inhibited the growth of lung cancer KDR-A549 xenografts in BALB/c-nu mice at day 10. The characterization of T cells modified by KDR-CAR by computational biology and wet-lab experiments suggested its applicability as a new treatment strategy for lung cancer and, potentially, for other vascularized solid tumors.

Carbonic Anhydrase IX: A Renewed Target for Cancer Immunotherapy.

The carbonic anhydrase isoform IX (CAIX) enzyme is constitutively overexpressed in the vast majority of clear cell renal cell carcinoma (ccRCC) and can also be induced in hypoxic microenvironments, a major hallmark of most solid tumors. CAIX expression is restricted to a few sites in healthy tissues, positioning this molecule as a strategic target for cancer immunotherapy. In this review, we summarized preclinical and clinical data of immunotherapeutic strategies based on monoclonal antibodies (mAbs), fusion proteins, chimeric antigen receptor (CAR) T, and NK cells targeting CAIX against different types of solid malignant tumors, alone or in combination with radionuclides, cytokines, cytotoxic agents, tyrosine kinase inhibitors, or immune checkpoint blockade. Most clinical studies targeting CAIX for immunotherapy were performed using G250 mAb-based antibodies or CAR T cells, developed primarily for bioimaging purposes, with a limited clinical response for ccRCC. Other anti-CAIX mAbs, CAR T, and NK cells developed with therapeutic intent presented herein offered outstanding preclinical results, justifying further exploration in the clinical setting.

Comprehensive analysis of ceRNA networks to determine genes related to prognosis, overall survival, and immune infiltration in clear cell renal carcinoma.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the common subtypes of kidney cancer. Circular RNAs (circRNAs) act as competing endogenous RNAs (ceRNAs) to affect the expression of microRNAs (miRNAs), and hence the expression of genes involved in the development and progression of ccRCC. However, these interactions have not been sufficiently explored. METHODS: The differential expression of circRNAs (DEC) was extracted from the GEO database, and the expression of circRNAs was analyzed by the Limma R package. The interaction of miRNAs with circRNAs was predicted using (cancer-specific circRNA database) CSCD and circinteractome database. The genes affected by the miRNAs were predicted by miRwalk version 3, and the differential expression was retrieved using TCGA. Functional enrichment was assessed and a PPI network was created using DAVID and Cytoscape, respectively. The genes with significant interactions (hub-genes) were screened, and the total survival rate of ccRCC patients was extracted from the Gene Expression Profiling Interactive Analysis (GEPIA) database. To confirm the expression of OS genes we used the Immunohistochemistry (IHC) data and TCGA database. The correlation between gene expression and immune cell infiltration was investigated using TIMER2.0. Finally, potential drug candidates were predicted by the cMAP database. RESULTS: Four DECs (hsa_circ_0003340, hsa_circ_0007836, hsa_circ_0020303, and hsa_circ_0001873) were identified, along with 11 interacting miRNAs (miR-1224-3p, miR-1294, miR-1205, miR-1231, miR-615-5p, miR-940, miR-1283, and miR-1305). These miRNAs were predicted to affect 1282 target genes, and function enrichment was used to identify the genes involved in cancer biology. 18 hub-genes (CCR1, VCAM1, NCF2, LAPTM5, NCKAP1L, CTSS, BTK, LILRB2, CD53, MPEG1, C3AR1, GPR183, C1QA, C1QC, P2RY8, LY86, CYBB, and IKZF1) were identified from a PPI network. VCAM1, NCF2, CTSS, LILRB2, MPEG1, C3AR1, P2RY8, and CYBB could affect the survival of ccRCC patients. The hub-gene expression was correlated with tumor immune cell infiltration and patient prognosis. Two potantial drug candidates, naphazoline and lithocholic acid could play a role in ccRCC therapy, as well other cancers. CONCLUSION: This bioinformatics analysis brings a new insight into the role of circRNA/miRNA/mRNA interactions in ccRCC pathogenesis, prognosis, and possible drug treatment or immunotherapy.

Integrative and in-vitro analysis reveal hsa_circ_001787 can act as a diagnostic biomarker for colorectal cancer.

BACKGROUND: Circular RNAs (circRNAs) are a new kind of non-coding RNA(ncRNA). Throughout research, we see an increase in the number of studies demonstrating that circRNAs occupy a pivotal role in the growth and advancement of human tumors. Nevertheless, hsa_circ_001787's role in the evolution of colorectal cancer (CRC) remains unclear. This current study ascertained the expression level of circRNA001787 in CRC specimens and neighboring healthy tissues, and investigated the miRNAs associate with hsa_circ_001787, as well as the relationship between hsa_circ_001787 and pathological factors. METHOD: First, the expression level of hsa_circ_001787 was measured in 43 matched Tissues from CRC and normal tissues through using real-time quantitative reverse transcription PCR (qRT-PCR). Second, based on circular RNA-microRNA and microRNA-mRNA pairs, a circRNA-miRNA-mRNA network was created. The survival rate of mRNAs was investigated through the GEPIA in the network. Regarding the elucidated function analysis of hsa_circ_001787, The biological, molecular, cellular function (GO) and pathway (KEGG) enrichment was obtained. RESULT: We detected that hsa_circ_001787 expression level was significantly down expressed in CRC tissue versus paired CRC histological normal tissue. The area under the curve (AUC) was 0.83. The expression level of hsa_circ_001787 was significantly associated with pathological factors such as tumor grade and the primary site of the tumor. Based on the hsa_circ_001787, a novel circRNA/miRNA/mRNA network has been built up, four miRNAs, and 24 mRNA. The pathway of mRNAs analyzed in the pathogenesis of CRC. Four genes distinguished via the GEPIA database were positively linked to the overall survival of CRC patients. CONCLUSION: Our study suggested that hsa_circ_001787 was significantly down-regulated in CRC. We might be able to use this as a new biomarker in the screening of CRC. Furthermore, our finding achieves a broader understanding of the regulatory mechanisms by which hsa_circ_001787 acts as ceRNA in colorectal cancer.

CAR-T cells: Early successes in blood cancer and challenges in solid tumors.

New approaches to cancer immunotherapy have been developed, showing the ability to harness the immune system to treat and eliminate cancer. For many solid tumors, therapy with checkpoint inhibitors has shown promise. For hematologic malignancies, adoptive and engineered cell therapies are being widely developed, using cells such as T lymphocytes, as well as natural killer (NK) cells, dendritic cells, and potentially others. Among these adoptive cell therapies, the most active and advanced therapy involves chimeric antigen receptor (CAR)-T cells, which are T cells in which a chimeric antigen receptor is used to redirect specificity and allow T cell recognition, activation and killing of cancers, such as leukemia and lymphoma. Two autologous CAR-T products have been approved by several health authorities, starting with the U.S. Food and Drug Administration (FDA) in 2017. These products have shown powerful, inducing, long-lasting effects against B cell cancers in many cases. In distinction to the results seen in hematologic malignancies, the field of using CAR-T products against solid tumors is in its infancy. Targeting solid tumors and trafficking CAR-T cells into an immunosuppressive microenvironment are both significant challenges. The goal of this review is to summarize some of the most recent aspects of CAR-T cell design and manufacturing that have led to successes in hematological malignancies, allowing the reader to appreciate the barriers that must be overcome to extend CAR-T therapies to solid tumors successfully.

The Nuclear Factor Kappa B (NF-kB) signaling in cancer development and immune diseases.

The nuclear factor kappa B (NF-kB) family of transcription factors plays an essential role as stressors in the cellular environment, and controls the expression of important regulatory genes such as immunity, inflammation, death, and cell proliferation. NF-kB protein is located in the cytoplasm, and can be activated by various cellular stimuli. There are two pathways for NF-kB activation, as the canonical and non-canonical pathways, which require complex molecular interactions with adapter proteins and phosphorylation and ubiquitinase enzymes. Accordingly, this increases NF-kB translocation in the nucleus and regulates gene expression. In this study, the concepts that emerge in different cellular systems allow the design of NF-kB function in humans. This would not only allow the development for rare diseases associated with NF-kB, but would also be used as a source of useful information to eliminate widespread consequences such as cancer or inflammatory/immune diseases.

Components from spider venom activate macrophages against glioblastoma cells: new potential adjuvants for anticancer immunotherapy.

Immunomodulation has been considered an important approach in the treatment of malignant tumours. However, the modulation of innate immune cells remains an underexplored tool. Studies from our group demonstrated that the Phoneutria nigriventer spider venom (PnV) administration increased the infiltration of macrophage in glioblastoma, in addition to decreasing the tumour size in a preclinical model. The hypothesis that PnV would be modulating the innate immune system led us to the main objective of the present study: to elucidate the effects of PnV and its purified fractions on cultured macrophages. Results showed that PnV and the three fractions activated macrophages differentiated from bone marrow precursors. Further purification generated 23 subfractions named low weight (LW-1 to LW-12) and high weight (HW-1 to HW-11). LW-9 presented the best immunomodulatory effect. Treated cells were more phagocytic, migrated more, showed an activated morphological profile and induced an increased cytotoxic effect of macrophages on tumour cells. However, while M1-controls (LPS) increased IL-10, TNF-alpha and IL-6 release, PnV, fractions and subfractions did not alter any cytokine, with the exception of LW-9 that stimulated IL-10 production. These findings suggest that molecules present in LW-9 have the potential to be used as immunoadjuvants in the treatment of cancer.

The Hippo Tumor Suppressor Pathway (YAP/TAZ/TEAD/MST/LATS) and EGFR-RAS-RAF-MEK in cancer metastasis.

Hippo Tumor Suppressor Pathway is the main pathway for cell growth that regulates tissue enlargement and organ size by limiting cell growth. This pathway is activated in response to cell cycle arrest signals (cell polarity, transduction, and DNA damage) and limited by growth factors or mitogens associated with EGF and LPA. The major pathway consists of the central kinase of Ste20 MAPK (Saccharomyces cerevisiae), Hpo (Drosophila melanogaster) or MST kinases (mammalian) that activates the mammalian AGC kinase dmWts or LATS effector (MST and LATS). YAP in the nucleus work as a cofactor for a wide range of transcription factors involved in proliferation (TEA domain family, TEAD1-4), stem cells (Oct4 mononuclear factor and SMAD-related TGFß effector), differentiation (RUNX1), and Cell cycle/apoptosis control (p53, p63, and p73 family members). This is due to the diverse roles of YAP and may limit tumor progression and establishment. TEAD also coordinates various signal transduction pathways such as Hippo, WNT, TGFß and EGFR, and effects on lack of regulation of TEAD cancerous genes, such as KRAS, BRAF, LKB1, NF2 and MYC, which play essential roles in tumor progression, metastasis, cancer metabolism, immunity, and drug resistance. However, RAS signaling is a pivotal factor in the inactivation of Hippo, which controls EGFR-RAS-RAF-MEK-ERK-mediated interaction of Hippo signaling. Thus, the loss of the Hippo pathway may have significant consequences on the targets of RAS-RAF mutations in cancer.

Encapsulated Checkpoint Blocker Before Chemotherapy: The Optimal Sequence of Anti-CTLA-4 and Doxil Combination Therapy.

INTRODUCTION: Today, a new paradigm has emerged for cancer treatment introducing combination therapies. Doxil, a liposomal doxorubicin serving as a chemotherapeutic agent, is an effective immunogenic killer of cancer cells. Anti-CTLA-4 has been approved for the treatment of some cancers, including melanoma, but side effects have limited its therapeutic potential. METHODS: In this study, two approaches were utilized to increase treatment efficiency and decrease the side effects of anti-CTLA-4, combining it with chemotherapy and encapsulation in a PEGylated liposome. A different sequence of anti-CTLA-4 and Doxil was assessed in combination therapy using non-liposomal and liposomal anti-CTLA-4. RESULTS: Our results showed that liposomal anti-CTLA-4 reduced the size of established tumors and increased survival in comparison with non-liposomal anti-CTLA-4 in a well-established B16 mouse melanoma model. In combination therapy with Doxil, only the administration of anti-CTLA-4 before Doxil showed synergism in both non-liposomal and liposomal form and increased the CD8+/regulatory T cell ratio. DISCUSSION: In summary, our results demonstrate the potential of utilizing a nanocarrier system for the delivery of checkpoint blockers, such as anti-CTLA-4 which further showed potential in a combination therapy, especially when administered before chemotherapy.

A New World of Biomarkers and Therapeutics for Female Reproductive System and Breast Cancers: Circular RNAs.

As one of the most recently (re)discovered types of non-coding RNAs (ncRNA), circular RNAs (circRNAs) differentiate from other ncRNAs by a specific biogenesis, high stability, and distinct functions. The biogenesis of circRNAs can be categorized into three mechanisms that permit the back-splicing reaction: exon-skipping, pairing of neighboring introns, and dimerization of RNA-binding proteins. Regarding their stability, circRNAs have no free ends, specific to linear RNA molecules, prompting a longer half-life and resistance to exonuclease-mediated activity by RNase R, bypassing the common RNA turnover process. Regarding their functions, circular transcripts can be categorized into four broad roles: miRNA sponging, protein binding, regulation of transcription, and coding for proteins and peptides. Female reproductive system (including mainly ovarian, corpus, and cervix uteri cancers) and breast cancers are the primary causes of death in women worldwide, accounting for over 1,212,772 deaths in 2018. We consider that a better understanding of the molecular pathophysiology through the study of coding and non-coding RNA regulators could improve the diagnosis and therapeutics of these cancers. Developments in the field of circRNA in regard to breast or gynecological cancers are recent, with most circRNA-related discoveries having been made in the last 2 years. Therefore, in this review we summarize the newly detected roles of circRNAs in female reproductive system (cervical cancer, ovarian cancer, and endometrial cancer) and breast cancers. We argue that circRNAs can become essential elements of the diagnostic and therapeutic tools for female reproductive system cancers in the future.

The Role and Function of Ras-association domain family in Cancer: A Review.

Ras gene mutation has been observed in more than 30% of cancers, and 90% of pancreatic, lung and colon cancers. Ras proteins (K-Ras, H-Ras, N-Ras) act as molecular switches which are activated by binding to GTP. They play a role in the cascade of cell process control (proliferation and cell division). In the inactive state, transforming GTP to GDP leads to the activation of GTpase in Ras gene. However, the mutation in Ras leads to the loss of internal GTPase activity and permanent activation of the protein. The activated Ras can promote the cell death or stop cell growth, which are facilitated by Ras-association domain family. Various studies have been conducted to determine the importance of losing RASSF proteins in Ras-induced tumors. This paper examines the role of Ras and RASSF proteins. In general, RASSF proteins can be used as a suitable means for targeting a large group of Ras-induced tumors.

Biological function and molecular mechanism of piRNA in cancer.

Cancer is the second leading cause of death globally. piRNAs, which are a novel type of identified small noncoding RNA (ncRNA), play a crucial role in cancer genomics. In recent years, a relatively large number of studies have demonstrated that several piRNA are aberrantly expressed in various kinds of cancers including gastric cancer, bladder cancer, breast cancer, colorectal cancer and Lung cancer and may probably serve as a novel therapeutic target and biomarker for cancer treatment. The present review summarized current advances in our knowledge of the roles of piRNAs in cancer.

Molecular Mechanisms and Biological Functions of siRNA.

One of the most important advances in biology has been the discovery that siRNA (small interfering RNA) is able to regulate the expression of genes, by a phenomenon known as RNAi (RNA interference). The discovery of RNAi, first in plants and Caenorhabditis elegans and later in mammalian cells, led to the emergence of a transformative view in biomedical research. siRNA has gained attention as a potential therapeutic reagent due to its ability to inhibit specific genes in many genetic diseases. siRNAs can be used as tools to study single gene function both in vivo and in-vitro and are an attractive new class of therapeutics, especially against undruggable targets for the treatment of cancer and other diseases. The siRNA delivery systems are categorized as non-viral and viral delivery systems. The non-viral delivery system includes polymers; Lipids; peptides etc. are the widely studied delivery systems for siRNA. Effective pharmacological use of siRNA requires 'carriers' that can deliver the siRNA to its intended site of action. The carriers assemble the siRNA into supramolecular complexes that display functional properties during the delivery process.