Molecular Target For Anticancer Therapy Research Articles

Depolymerization of tubulin as the main molecular mechanism of the cytotoxic and antitumor activity of pyrrole-containing heterocyclic compounds

Introduction. Microtubules are highly dynamic polymers of α, β-tubulin dimers involves in a broad spectrum of the processes, such as intracellular transport and cell proliferation. This makes them an attractive molecular target for anti-cancer therapies. Substances that affect the dynamic state of tubulin microtubules are known as the mitotic poisons that are effectiveand widely used in the chemotherapy of various tumors. Mitotic poisons are able to interfere with polymerization (stabilization) or depolymerization of tubulin, which in turn leads to the arrest of cells in the M-phase (named as a mitotic catastrophe) and their subsequent death via activation of apoptotic mechanisms. However, the effectiveness of MP-based therapies is gradually decreasing over the time due to development of multiple drug resistance mechanisms in cancer cells. Thus, development of novel compounds selectively targeting tubulin and effectively overcoming multiple drugresistance phenotype in cancer is an urgent need in current oncology. Aim. To examine the cytotoxic and antitumor activities of several pyrrole-containing heterocyclic compounds (EPC-91, EPC-92 and PCA-93) against cancer cell lines with epithelial and mesenchymal origin, including those with multiple drug resistance phenotype. Materials and methods. Studies were performed on parental human cancer cell lines – triple-negative breast cancer HCC1806, gastrointestinal stromal tumor GIST T-1, osteosarcoma SaOS-2, – sensitive to chemotherapy (paclitaxel, doxorubicin) and their resistant sublines (HCC1806 Tx-R, GIST T-1 Tx-R, SaOS-2 Dox-R), as well as on murine colorectal adenocarcinoma cell line Colon-26, exhibiting primary resistance to the aforementioned chemotherapeutic agents. Results. The cytotoxic activities of EPC-91 and PCA-93 were due to their abilities to depolymerize tubulin. The results of immunofluorescence microscopy and Western blotting indicated that the compounds disrupt assembly of tubulin microtubules and prevent polymerization of α-tubulin in cancer cells. Inhibition of tubulin polymerizations led to significant increasein number of round-shaped and phospho-histone 3 (e. g. mitotic) cells, followed by their death through apoptosis. PCA-93 also exhibited potent anti-tumor effect against Colon-26 cells due to its anti-proliferative and proapoptotic activities. Conclusion. The data shown here illustrates potent cytotoxic activities of EPC-91 and PCA-93 against multiple cancer cell lines in vitro including those with multiple drug resistance phenotype. Similarly, PCA-93 was found to be highly effective against Colon-26 cell in vivo, thereby illustrating the attractive platform for the development of novel pyrrole-based agents exhibiting potent anti-tumor activities.

Mechanism of cinobufacin anti-metastatic colorectal cancer based on integrated mining of proteomics and bioinformatics

In this study, the effector proteins of anti-metastatic colorectal cancer (CRC) were mined through proteomics based on the preliminary screening of cinobufacin anti-cancer genes using bioinformatics and the role of cinobufacin anti-metastatic CRC was explored. Proteomics was used to analyze the differential expression of proteins in biopsy tissues of patients with metastatic CRC and bioinformatics was adopted to screen for cinobufacin tumor suppressor genes. The effects of proteomics-mined effector protein CyclinD1 on cell proliferation and colony formation were evaluated by MTT assay and colony formation assay. The cinobufacin sensitivity of metastatic CRC cells were also assessed. After proteomic analysis, the common targets of “Cinobufacin-metastatic CRC” were screened out, among which the tumor-associated CyclinD1 was listed as its level was increased in biopsy tissues of patients with metastatic CRC. The preliminary screening of cinobufacin anti-cancer genes based on bioinformatics found that tumor suppressor gene P53 was closely related to metastatic CRC. CyclinD1 had a relation with P53 mRNA. Further studies showed that P53 was the direct target of CyclinD1 and CyclinD1 overexpression enhanced the sensitivity of metastatic CRC cells to cinobufacin. Using bioinformatics-based gene database analysis technology and proteomics mining technology, the differential expression of proteomics in the biopsy tissues of patients with metastatic CRC and the tumor suppressor genes regulated by cinobufacin were identified. CyclinD1 and P53 found by bioinformatics might be biomarkers for metastatic CRC prognosis, providing a new possible molecular target for anticancer therapy.

Synergistic Effect of Co-Culturing Breast Cancer Cells and Fibroblasts in the Formation of Tumoroid Clusters and Design of In Vitro 3D Models for the Testing of Anticancer Agents.

Breast cancer is still the leading cause of women's death due to relapse and metastasis. In vitro tumor models are considered reliable tools for drug screening and understanding cancer-driving mechanisms due to the possibility of mimicking tumor heterogeneity. Herein, a 3D breast cancer model (3D-BCM) is developed based on enzymatically-crosslinked silk fibroin (eSF) hydrogels. Human MCF7 breast cancer cells are encapsulated into eSF hydrogels, with and without human mammary fibroblasts. The spontaneously occurring conformational change from random coil to β-sheet is correlated with increased eSF hydrogels' stiffness over time. Moreover, mechanical properties analysis confirms that the cells can modify the stiffness of the hydrogels, mimicking the microenvironment stiffening occurring in vivo. Fibroblasts support cancer cells growth and assembly in the eSF hydrogels up to 14 days of culture. Co-cultured 3D-BCM exhibits an upregulated expression of genes related to extracellular matrix remodeling and fibroblast activation. The 3D-BCM is subjected to doxorubicin and paclitaxel treatments, showing differential drug response. Overall, these results suggest that the co-culture of breast cancer cells and fibroblasts in eSF hydrogels allow the development of a mimetic in vitro platform to study cancer progression. This opens up new research avenues to investigate novel molecular targets for anti-cancer therapy.

Oncogenic Role of ADAM32 in Hepatoblastoma: A Potential Molecular Target for Therapy.

Simple SummaryHepatoblastoma (HBL) is a rare hepatic malignancy occurring mainly in early childhood. Although recently developed treatment regimens have improved prognosis, many refractory cases still exist, and the anticancer drug cis-diamminedichloroplatinum—cisplatin—primarily used in HBL treatment often induces severe side effects. Therefore, more effective and safer therapies are needed. In this study, we demonstrate that the expression level of ADAM32 is particularly high in HBL tissue samples and is associated with poor prognosis in various cancer types through the regulation of cancer cell proliferation, stemness, migration, invasion, and acquired resistance to chemotherapy. Our results thus indicate that ADAM32 could be a promising candidate therapeutic target molecule, and provide new insights into the molecular mechanisms of HBL carcinogenesis.Outcomes of pediatric hepatoblastoma (HBL) have improved, but refractory cases still occur. More effective and safer drugs are needed that are based on molecular mechanisms. A disintegrin and metalloproteases (ADAMs) are expressed with high frequency in various human carcinomas and play an important role in cancer progression. In this study, we analyzed expression of ADAMs in HBL with a cDNA microarray dataset and found that the expression level of ADAM32 is particularly high. To investigate the role of ADAM32 in cancer, forced expression or knockdown experiments were conducted with HepG2 and HBL primary cells. Colony formation, cell migration and invasion, and cell viability were increased in HepG2 expressing ADAM32, whereas knockdown of ADAM32 induced a decrease in these cellular functions. Quantitative RT-PCR demonstrated an association between ADAM32 expression and the expression of genes related to cancer stem cells and epithelial–mesenchymal transition (EMT), suggesting a role of ADAM32 in cancer stemness and EMT. Furthermore, knockdown of ADAM32 increased cisplatin-induced apoptosis, and this effect was attenuated by a caspase-8 inhibitor, suggesting that ADAM32 plays a role in extrinsic apoptosis signaling. We conclude that ADAM32 plays a crucial role in progression of HBL, so it might be a promising molecular target in anticancer therapy.

Proteasome Inhibitors and Their Potential Applicability in Osteosarcoma Treatment.

Simple SummaryBone cancer has seen minimal benefits in therapeutic options in the past 30 years. Proteasome inhibitors present a new avenue of research for the treatment of bone cancer. Proteasome inhibitors impair the function of the proteasome, a structure within the cell that removes unwanted and misfolded proteins. Bone cancer cells heavily rely on the proteasome to properly function and survive. Impairing the proteasome function can have detrimental consequences and lead to cell death. This review provides a thorough summary of the in vitro, in vivo, and clinical research that has explored proteasome inhibitors for the treatment of bone cancer.Osteosarcoma (OS) is the most common type of bone cancer, with ~30% of patients developing secondary/metastatic tumors. The molecular complexity of tumor metastasis and the lack of effective therapies for OS has cultivated interest in exploiting the proteasome as a molecular target for anti-cancer therapy. As our understanding towards the behavior of malignant cells expands, it is evident that cancerous cells display a greater reliance on the proteasome to maintain homeostasis and sustain efficient biological activities. This led to the development and approval of first- and second-generation proteasome inhibitors (PIs), which have improved outcomes for patients with multiple myeloma and mantle cell lymphoma. Researchers have since postulated the therapeutic potential of PIs for the treatment of OS. As such, this review aims to summarize the biological effects and latest findings from clinical trials investigating PI-based treatments for OS. Integrating PIs into current treatment regimens may better outcomes for patients diagnosed with OS.

Role of the Transcription Factor FOSL1 in Organ Development and Tumorigenesis.

The transcription factor FOSL1 plays an important role in cell differentiation and tumorigenesis. Primarily, FOSL1 is crucial for the differentiation of several cell lineages, namely adipocytes, chondrocytes, and osteoblasts. In solid tumors, FOSL1 controls the progression of tumor cells through the epithelial–mesenchymal transformation. In this review, we summarize the available data on FOSL1 expression, stabilization, and degradation in the cell. We discuss how FOSL1 is integrated into the intracellular signaling mechanisms and provide a comprehensive analysis of FOSL1 influence on gene expression. We also analyze the pathological changes caused by altered Fosl1 expression in genetically modified mice. In addition, we dedicated a separate section of the review to the role of FOSL1 in human cancer. Primarily, we focus on the FOSL1 expression pattern in solid tumors, FOSL1 importance as a prognostic factor, and FOSL1 perspectives as a molecular target for anticancer therapy.

Composites of Nucleic Acids and Boron Clusters (C2B10H12) as Functional Nanoparticles for Downregulation of EGFR Oncogene in Cancer Cells.

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO’s properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.

The Urokinase Receptor: A Multifunctional Receptor in Cancer Cell Biology. Therapeutic Implications.

Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.

USP32 confers cancer cell resistance to YM155 via promoting ER-associated degradation of solute carrier protein SLC35F2.

Background: The most commonly preferred chemotherapeutic agents to treat cancers are small-molecule drugs. However, the differential sensitivity of various cancer cells to small molecules and untargeted delivery narrow the range of potential therapeutic applications. The mechanisms responsible for drug resistance in a variety of cancer cells are also largely unknown. Several deubiquitinating enzymes (DUBs) are the main determinants of drug resistance in cancer cells.Methods: We used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs) and systematically screened for DUBs resistant to the clinically evaluated anticancer compound YM155. A series of in vitro and in vivo experiments were conducted to reveal the relationship between USP32 and SLC35F2 on YM155-mediated DNA damage in cancer cells.Results: CRISPR-based dual-screening method identified USP32 as a novel DUB that governs resistance for uptake of YM155 by destabilizing protein levels of SLC35F2, a solute-carrier protein essential for the uptake of YM155. The expression of USP32 and SLC35F2 was negatively correlated across a panel of tested cancer cell lines. YM155-resistant cancer cells in particular exhibited elevated expression of USP32 and low expression of SLC35F2.Conclusion: Collectively, our DUB-screening strategy revealed a resistance mechanism governed by USP32 associated with YM155 resistance in breast cancers, one that presents an attractive molecular target for anti-cancer therapies. Targeted genome knockout verified that USP32 is the main determinant of SLC35F2 protein stability in vitro and in vivo, suggesting a novel way to treat tumors resistant to small-molecule drugs.

28 Poster Discussion - DS-7300a, a novel B7-H3-targeting antibody-drug conjugate with a novel DNA topoisomerase I inhibitor DXd, exhibits potent anti-tumor effects in nonclinical models

Background: B7-H3 (CD276) is a type I transmembrane protein belonging to the B7 family which includes immune checkpoint molecules such as CTLA-4 ligands and PD-L1. B7-H3 is highly expressed in various types of solid tumors with low expression in normal tissues. Several studies have reported that B7-H3 overexpression is associated with poor prognosis in several types of cancers. These findings support B7-H3 as an attractive molecular target for anti-cancer therapies. We generated DS-7300a, a B7-H3-targeting antibody-drug conjugate (ADC), which is composed of a humanized anti-B7-H3 monoclonal antibody, an enzymatically cleavable peptide-based linker, and a novel exatecan derivative (DXd) that is a potent DNA topoisomerase I inhibitor. In this study, we evaluated the pharmacological activities of DS-7300a in nonclinical models.

Expression of CXCR7 in colorectal adenoma and adenocarcinoma: Correlation with clinicopathological parameters

Colorectal carcinoma (CRC) is one of the most lethal malignancies, it ranks third in cancer-related morbidity and mortality. Although great progress has been made in early diagnosis and combined treatment of CRC, the prognosis of patients remains poor owing to the high rate of recurrence and distant metastasis. CXCR7 belongs to chemokine receptor family and has been identified as a novel receptor for CXCL12. It plays an important role in development and in progression of cancer to metastatic stage. The aim of studyTo evaluate the immunohistochemical expression of CXCR7 in colorectal adenoma and carcinoma and to analyze its correlation with clinicopathological factors. This is retrospective study including 58 colonic adenocarcinoma specimens and 18 cases of colonic adenoma. ResultsCXCR7 showed positive cytoplasmic expression in two out 18 cases of colorectal adenoma (11%) and 42 out of 58 cases of CRC (72.4%) with a significant difference between both (p < 0.001). We found a significant correlation between upregulation of CXCR7 and presence of lymphovascular tumor emboli, presence of lymph node metastasis and advanced TNM stage of the CRC. The association of the CXCR7 with patient age, sex, tumor size, depth of invasion and tumor cell differentiation was found to be non-significant. Regarding colonic adenoma, we found no significant association between CXCR7 expression on one hand and patient age, sex, tumor size, histologic type and degree of dysplasia on the other hand. ConclusionCXCR7 in CRC may act as a novel predictive indicator for prognosis and even be a potential molecular target for anticancer therapies.

Long noncoding RNA NR2F1-AS1 promotes the malignancy of non-small cell lung cancer via sponging microRNA-493-5p and thereby increasing ITGB1 expression.

Several studies have reported that the long noncoding ribonucleic acid (lncRNA) NR2F1 antisense RNA 1 (NR2F1-AS1) affects multiple cellular pathways that are involved in tumorigenesis and tumor progression. The present study aimed to detect NR2F1-AS1 expression in non-small cell lung cancer (NSCLC), investigate the role of NR2F1-AS1 in promoting the tumorigenic behavior of NSCLC cells, and elucidate the mechanism underlying the effect of NR2F1-AS1 on NSCLC progression. Our results showed that NR2F1-AS1 expression was upregulated in NSCLC cells, and notably, its upregulation was correlated with adverse clinical characteristics and shorter overall survival in patients with NSCLC. The absence of NR2F1-AS1 functionally decreased NSCLC cell proliferation, migration, and invasion and promoted tumor cell apoptosis. In addition, the tumor growth of NSCLC cells in vivo was inhibited after NR2F1-AS1 silencing. Mechanistically, NR2F1-AS1 functioned as a competing endogenous RNA for miR-493-5p and consequently increased ITGB1 expression. Rescue assays further validated that an increased output of the miR-493-5p/ITGB1 axis could neutralize the regulatory impact of NR2F1-AS1 knockdown on the malignant phenotype of NSCLC cells. In summary, the NR2F1-AS1/miR-493-5p/ITGB1 pathway initiates pro-oncogenic behavior in NSCLC tumor progression, and the NR2F1-AS1/miR-493-5p/ITGB1 axis may provide new molecular targets for anticancer therapy against NSCLC.

Association between hTERT Polymorphisms and Female Papillary Thyroid Carcinoma.

A previous genome-wide association study showed that hTERT rs10069690 and rs2736100 polymorphisms were associated with thyroid cancer risk. This study further investigated the association between increased risk and clinicopathologic characteristics for Papillary Thyroid Carcinoma (PTC) and hTERT polymorphisms rs10069690 or rs2736100 in a Chinese female population. The hTERT genotypes of 276 PTC patients and 345 healthy subjects were determined with regard to SNPs rs10069690 and rs2736100. The association between these SNPs and the risk of PTC and clinicopathologic characteristics was investigated by logistic regression. We found a significant difference between PTC and rs10069690 (Odds Ratio (OR) = 1.515; P = 0.005), but not between PTC and rs2736100. When the analysis was limited to females, rs10069690 and rs2736100 were both associated with increased risk for PTC in female individuals (OR = 1.647, P = 0.007; OR = 1.339, P = 0.041, respectively). Further haplotype analysis revealed a stimulative effect of haplotypes TC and CA of TERT rs10069690-rs2736100, which increased risk for PTC in female individuals (OR = 1.579, P = 0.014; OR = 0.726, P = 0.025, respectively). Furthermore, the heterozygote A/C of rs2736100 showed significant difference for age (OR = 0.514, P = 0.047). Our finding suggests that hTERT polymorphisms rs10069690 and rs2736100 are associated with increased risk for PTC in Chinese female population and rs2736100 may be related to age. Consistent with US20170360914 and US20170232075, they are expected to be a potential molecular target for anti-cancer therapy.

PD-L1 Expression on Cell Block Preparations of Pancreatic Adenocarcinoma

Abstract Objectives Programmed death-ligand 1 (PD-L1) is an emerging molecular target in anticancer therapy, most notably non–small cell lung cancers. PD-L1 expression in pancreatic adenocarcinomas (PDAs) on surgical specimens is highly variable, with 10% to 60% of tumors showing expression. PD-L1 expression in PDA on endoscopic ultrasound-guided fine needle aspirations (EUS-FNAs) has been rarely studied. Methods Formalin-fixed, paraffin embedded (FFPE) cell blocks (CBs) from 65 EUS-FNA samples of 55 patients, with a diagnosis of PDA, with at least 20% tumor cellularity were retrieved. The cell blocks were originally fixed in CytoRich fixative. Immunohistochemical staining (IHC) for PD-L1 was performed using the M365329-1 (22C3) clone according to the manufacturer’s approved protocol and optimized for the fixation method using appropriate controls. A combined positive score (CPS) defined per CAP guidelines as the total number of positive tumor cells and inflammatory cells as a percentage of the total number of tumor cells was assessed for each case and was grouped as &lt;1, 1-20, or &gt;20. Results Twenty-five samples (38%) from 21 patients showed immunoreactivity to PD-L1, with 21 (32%) having a CPS of &lt;1 and four (6%) having a CPS of 1-20. Eight of these 25 samples had surgical correlates, of which concordant staining was noted in five (62.5%). Of the discordant three, decreased tumor cell sampling in the core biopsy was noted in one. Overall, 20 of 21 patients (95%) with PD-L1 immunoreactivity had disease progression with 17 (81%) associated with metastatic disease and three (14%) with locally advanced disease. Conclusion Immunohistochemical analysis for PD-L1 is feasible on EUS-FNA CB samples when optimized and validated for the fixation method using appropriate controls. PD-L1 expression is seen in only a minority of PDAs, albeit with a very low CPS. The potential role of PD-L1 as a prognostic marker for disease progression needs further exploration.

Recent advances in the development of legumain-selective chemical probes and peptide prodrugs.

Legumain, which is also known as vacuolar processing enzyme (VPE) or asparaginyl endopeptidase (AEP), is a cysteine protease that was first discovered and characterized in the leguminous seeds of the moth bean in the early 1990s. Later, this enzyme was also detected in higher organisms, including eukaryotes. This pH-dependent protease displays the highest activity in acidic endolysosomal compartments; however, legumain also displays nuclear, cytosolic and extracellular activity when stabilized by other proteins or intramolecular complexes. Based on the results from over 25 years of research, this protease is involved in multiple cellular events, including protein degradation and antigen presentation. Moreover, when dysregulated, this protease contributes to the progression of several diseases, with cancer being the well-studied example. Research on legumain biology was undoubtedly facilitated by the use of small molecule chemical tools. Therefore, in this review, I present the historical perspectives and most current strategies for the development of small molecule substrates, inhibitors and activity-based probes for legumain. These tools are of paramount importance in elucidating the roles of legumain in multiple biological processes. Finally, as this enzyme appears to be a promising molecular target for anticancer therapies, the development of legumain-activated prodrugs is also described.

Mucin-1 (MUC1) as a promising molecular target in anticancer therapy

Mucin 1 (MUC1) has been recognized by the National Cancer Institute as one of the most promising molecular targets in cancer therapy. Its overexpression has been demonstrated in many epithelial tumors,especially in breast cancer, whichis associated with poor prognosis. Mucin 1 is an important barrier to the penetration of drugs and takes part in the inhibition of apoptosis in tumor cells. MUC1 triggers the activation of several pathways of intracellular signaling. MUC1 interactions with ICAM-1, E-selectin, galectin-3, EGFR, ERα estrogen receptor, p53 protein, heat shock proteins HSP70 and HSP90 have been demonstrated. The MUC1 membrane subunit contributes to the activation of the ERK1 and ERK2 kinases by the induction of the Ras-Raf-Mek-Erk pathway. In addition, the role of MUC1 in the activation of the WNT/β-catenin/TCF7L2 pathway and the induction of transcription of the cyclin D1 gene was confirmed. Numerous studies have shown that blockade of MUC1 by monoclonal antibodies or small molecule inhibitors may promote therapeutic effects and contribute to increased susceptibility of tumor cells to chemotherapeutic agents. The combined effect of the anti-MUC1 antibody with novel anticancer agents may have a better therapeutic effect than monotherapy. This article reviews the current knowledge about the role of MUC1 in the development and progression of cancer as well as potential novel strategies based on mucin 1 in antineoplastic therapy.

Phosphorylation of cortactin by cyclin-dependent kinase5 modulates actin bundling by the dynamin 1-cortactin ring-like complex and formation of filopodia and lamellipodia in NG108-15 glioma-derived cells.

Dynamin copolymerizes with cortactin to form a ring-like complex that bundles and stabilizes actin filaments. Actin bundle formation is crucial for generation of filopodia and lamellipodia, which guide migration, invasion, and metastasis of cancer cells. However, it is unknown how the dynamin-cortactin complex regulates actin bundle formation. The present study investigated phosphorylation of cortactin by cyclin-dependent kinase 5 (CDK5) and its effect on actin bundle formation by the dynamin-cortactin complex. CDK5 directly phosphorylated cortactin at T145/T219 in vitro. Phosphomimetic mutants in which one or both of these threonine residues was substituted by aspartate were used. The three phosphomimetic mutants (T145D, T219D and T145DT219D) had a decreased affinity for F-actin. Furthermore, electron microscopy demonstrated that these phosphomimetic mutants could not form a ring-like complex with dynamin 1. Consistently, the dynamin 1-phosphomimetic cortactin complexes exhibited decreased actin-bundling activity. Expression of the phosphomimetic mutants resulted in not only aberrant lamellipodia and short filopodia but also cell migration in NG108-15 glioma-derived cells. These results indicate that phosphorylation of cortactin by CDK5 regulates formation of lamellipodia and filopodia by modulating dynamin 1/cortactin-dependent actin bundling. Taken together, these findings suggest that CDK5 is a potential molecular target for anticancer therapy.

Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy.

γ-Glutamylcyclotransferase (GGCT), which is one of the major enzymes involved in glutathione metabolism, is upregulated in a wide range of cancers—glioma, breast, lung, esophageal, gastric, colorectal, urinary bladder, prostate, cervical, ovarian cancers and osteosarcoma—and promotes cancer progression; its depletion leads to the suppression of proliferation, invasion, and migration of cancer cells. It has been demonstrated that the suppression or inhibition of GGCT has an antitumor effect in cancer-bearing xenograft mice. Based on these observations, GGCT is now recognized as a promising therapeutic target in various cancers. This review summarizes recent advances on the mechanisms of the antitumor activity of GGCT inhibition.

High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer

Human Tousled-like kinases (TLKs) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their possible involvement in cancer via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein–drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. We have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

VEGF-R1 as a Potential Molecular Target for Anticancer Therapy.

The possibility of using VEGF-R1 receptor for targeted therapy in oncology was investigated. Using the approach to measuring the protein content in intact nuclei of cells, which was developed by us, we showed the presence of this receptor in the nuclei of tumor, but not normal cells. A direct correlation between the level of VEGF-R1 expression in the nucleus and the degree of malignancy of tumor cells, indicating the prognostic value of this parameter, was found. The mechanisms of the functioning of this receptor and the pathways of inhibiting its activity are discussed, and the validity of the selection of VEGF-R1 as a molecular target for anticancer therapy is conformed.