The Role of Endoglin in Hepatocellular Carcinoma

Endoglin (CD105) is a type-1 integral transmembrane glycoprotein and coreceptor for transforming growth factor-β (TGF-β) ligands. The endoglin/TGF-β signaling pathway regulates hemostasis, cell proliferation/migration, extracellular matrix (ECM) synthesis and angiogenesis. Angiogenesis contributes to early progression, invasion, postoperative recurrence, and metastasis in hepatocellular carcinoma (HCC), one of the most widespread malignancies globally. Endoglin is overexpressed in newly formed HCC microvessels. It increases microvessel density in cirrhotic and regenerative HCC nodules. In addition, circulating endoglin is present in HCC patients, suggesting potential for use as a diagnostic or prognostic factor. HCC angiogenesis is dynamic and endoglin expression varies by stage. TRC105 (carotuximab) is an antibody against endoglin, and three of its clinical trials were related to liver diseases. A partial response was achieved when combining TRC105 with sorafenib. Although antiangiogenic therapy still carries some risks, combination therapy with endoglin inhibitors or other targeted therapies holds promise.     

Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein

Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin.     

Endoglin/CD105-Based Imaging of Cancer and Cardiovascular Diseases: A Systematic Review

Molecular imaging of pathologic lesions can improve efficient detection of cancer and cardiovascular diseases. A shared pathophysiological feature is angiogenesis, the formation of new blood vessels. Endoglin (CD105) is a coreceptor for ligands of the Transforming Growth Factor-β (TGF-β) family and is highly expressed on angiogenic endothelial cells. Therefore, endoglin-based imaging has been explored to visualize lesions of the aforementioned diseases. This systematic review highlights the progress in endoglin-based imaging of cancer, atherosclerosis, myocardial infarction, and aortic aneurysm, focusing on positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), near-infrared fluorescence (NIRF) imaging, and ultrasound imaging. PubMed was searched combining the following subjects and their respective synonyms or relevant subterms: “Endoglin”, “Imaging/Image-guided surgery”. In total, 59 papers were found eligible to be included: 58 reporting about preclinical animal or in vitro models and one ex vivo study in human organs. In addition to exact data extraction of imaging modality type, tumor or cardiovascular disease model, and tracer (class), outcomes were described via a narrative synthesis. Collectively, the data identify endoglin as a suitable target for intraoperative and diagnostic imaging of the neovasculature in tumors, whereas for cardiovascular diseases, the evidence remains scarce but promising.     

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling

Background: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib^®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. Methods: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. Results: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. Conclusions: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.     

Endoglin in the Spotlight to Treat Cancer

A spotlight has been shone on endoglin in recent years due to that fact of its potential to serve as both a reliable disease biomarker and a therapeutic target. Indeed, endoglin has now been assigned many roles in both physiological and pathological processes. From a molecular point of view, endoglin mainly acts as a co-receptor in the canonical TGFβ pathway, but also it may be shed and released from the membrane, giving rise to the soluble form, which also plays important roles in cell signaling. In cancer, in particular, endoglin may contribute to either an oncogenic or a non-oncogenic phenotype depending on the cell context. The fact that endoglin is expressed by neoplastic and non-neoplastic cells within the tumor microenvironment suggests new possibilities for targeted therapies. Here, we aimed to review and discuss the many roles played by endoglin in different tumor types, as well as the strong evidence provided by pre-clinical and clinical studies that supports the therapeutic targeting of endoglin as a novel clinical strategy.     

Insights into the Mechanisms of Action of MDA-7/IL-24: A Ubiquitous Cancer-Suppressing Protein

Melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24), a secreted protein of the IL-10 family, was first identified more than two decades ago as a novel gene differentially expressed in terminally differentiating human metastatic melanoma cells. MDA-7/IL-24 functions as a potent tumor suppressor exerting a diverse array of functions including the inhibition of tumor growth, invasion, angiogenesis, and metastasis, and induction of potent “bystander” antitumor activity and synergy with conventional cancer therapeutics. MDA-7/IL-24 induces cancer-specific cell death through apoptosis or toxic autophagy, which was initially established in vitro and in preclinical animal models in vivo and later in a Phase I clinical trial in patients with advanced cancers. This review summarizes the history and our current understanding of the molecular/biological mechanisms of MDA-7/IL-24 action rendering it a potent cancer suppressor.     

[68Ga]Ga-DFO-c(RGDyK): Synthesis and Evaluation of Its Potential for Tumor Imaging in Mice

Angiogenesis has a pivotal role in tumor growth and the metastatic process. Molecular imaging was shown to be useful for imaging of tumor-induced angiogenesis. A great variety of radiolabeled peptides have been developed to target αvβ3 integrin, a target structure involved in the tumor-induced angiogenic process. The presented study aimed to synthesize deferoxamine (DFO)-based c(RGD) peptide conjugate for radiolabeling with gallium-68 and perform its basic preclinical characterization including testing of its tumor-imaging potential. DFO-c(RGDyK) was labeled with gallium-68 with high radiochemical purity. In vitro characterization including stability, partition coefficient, protein binding determination, tumor cell uptake assays, and ex vivo biodistribution as well as PET/CT imaging was performed. [⁶⁸Ga]Ga-DFO-c(RGDyK) showed hydrophilic properties, high stability in PBS and human serum, and specific uptake in U-87 MG and M21 tumor cell lines in vitro and in vivo. We have shown here that [⁶⁸Ga]Ga-DFO-c(RGDyK) can be used for αvβ3 integrin targeting, allowing imaging of tumor-induced angiogenesis by positron emission tomography.     

The Efficacy of Anti-PD-L1 Treatment in Melanoma Is Associated with the Expression of the ECM Molecule EMILIN2

The use of immune checkpoint inhibitors has revolutionized the treatment of melanoma patients, leading to remarkable improvements in the cure. However, to ensure a safe and effective treatment, there is the need to develop markers to identify the patients that would most likely respond to the therapies. The microenvironment is gaining attention in this context, since it can regulate both the immunotherapy efficacyand angiogenesis, which is known to be affected by treatment. Here, we investigated the putative role of the ECM molecule EMILIN-2, a tumor suppressive and pro-angiogenic molecule. We verified that the EMILIN2 expression is variable among melanoma patients and is associated with the response to PD-L1 inhibitors. Consistently, in preclinical settings, the absence of EMILIN-2 is associated with higher PD-L1 expression and increased immunotherapy efficacy. We verified that EMILIN-2 modulates PD-L1 expression in melanoma cells through indirect immune-dependent mechanisms. Notably, upon PD-L1 blockage, Emilin2^−/− mice displayed improved intra-tumoral vessel normalization and decreased tumor hypoxia. Finally, we provide evidence indicating that the inclusion of EMILIN2 in a number of gene expression signatures improves their predictive potential, a further indication that the analysis of this molecule may be key for the development of new markers to predict immunotherapy efficacy.     

CD146+ Pericytes Subset Isolated from Human Micro-Fragmented Fat Tissue Display a Strong Interaction with Endothelial Cells: A Potential Cell Target for Therapeutic Angiogenesis

Pericytes (PCs) are mesenchymal stromal cells (MSCs) that function as support cells and play a role in tissue regeneration and, in particular, vascular homeostasis. PCs promote endothelial cells (ECs) survival which is critical for vessel stabilization, maturation, and remodeling. In this study, PCs were isolated from human micro-fragmented adipose tissue (MFAT) obtained from fat lipoaspirate and were characterized as NG2⁺/PDGFRβ⁺/CD105⁺ cells. Here, we tested the fat-derived PCs for the dispensability of the CD146 marker with the aim of better understanding the role of these PC subpopulations on angiogenesis. Cells from both CD146-positive (CD146⁺) and negative (CD146⁻) populations were observed to interact with human umbilical vein ECs (HUVECs). In addition, fat-derived PCs were able to induce angiogenesis of ECs in spheroids assay; and conditioned medium (CM) from both PCs and fat tissue itself led to the proliferation of ECs, thereby marking their role in angiogenesis stimulation. However, we found that CD146⁺ cells were more responsive to PDGF-BB-stimulated migration, adhesion, and angiogenic interaction with ECs, possibly owing to their higher expression of NCAM/CD56 than the corresponding CD146⁻ subpopulation. We conclude that in fat tissue, CD146-expressing cells may represent a more mature pericyte subpopulation that may have higher efficacy in controlling and stimulating vascular regeneration and stabilization than their CD146-negative counterpart.     

CD39 Regulation and Functions in T Cells

CD39 is an enzyme which is responsible, together with CD73, for a cascade converting adenosine triphosphate into adenosine diphosphate and cyclic adenosine monophosphate, ultimately leading to the release of an immunosuppressive form of adenosine in the tumor microenvironment. Here, we first review the environmental and genetic factors shaping CD39 expression. Second, we report CD39 functions in the T cell compartment, highlighting its role in regulatory T cells, conventional CD4⁺ T cells and CD8⁺ T cells. Finally, we compile a list of studies, from preclinical models to clinical trials, which have made essential contributions to the discovery of novel combinatorial approaches in the treatment of cancer.     

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12–CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12–CXCR4/CXCR7 axis as a treatment strategy for CRC.     

The Role of Chemokine Receptor CXCR3 and Its Ligands in Renal Cell Carcinoma

The major invasive subtype of kidney cancer is renal cell carcinoma (RCC). The essential components of cancer development are chronic inflammation and neoangiogenesis. It has been suggested that the chemokine ligand 9, -10, –11 (CXCL9–11) and chemokine receptor 3 (CXCR3) chemokines receptor expressed on monocytes, T and NK cells may be involved in the inhibition of angiogenesis. However, to date, little is known about the potential clinical significance of these chemokines and their receptor in renal cell carcinoma. Therefore, in this review, we described the role of CXCR3 and its ligands in pathogenesis of RCC. We performed an extensive search of the current literature in our investigation, using the MEDLINE/PubMed database. The changes of chemokines and their specific receptor in renal cell carcinoma were observed. Published studies revealed an increased expression of CXCR3 and elevated concentration of its ligands in RCC. The association between treatment of RCC and CXCL9–11/CXCR3 concentration and expression was also observed. Moreover, CXCR3 and its ligands levels were related to patient’s prognosis, risk of metastasis and tumor growth. This review describes the potential role of CXCR3 and its ligands in pathogenesis of RCC, as well as their potential immune-therapeutic significance. However, future studies should aim to confirm the clinical and prognostic role of CXCL9–11/CXCR3 in renal cell carcinoma.     

Translating Research for the Radiotheranostics of Nanotargeted 188Re-Liposome

Nanoliposomes are one of the leading potential nano drug delivery systems capable of targeting chemotherapeutics to tumor sites because of their passive nano-targeting capability through the enhanced permeability and retention (EPR) effect for cancer patients. Recent advances in nano-delivery systems have inspired the development of a wide range of nanotargeted materials and strategies for applications in preclinical and clinical usage in the cancer field. Nanotargeted ¹⁸⁸Re-liposome is a unique internal passive radiotheranostic agent for nuclear imaging and radiotherapeutic applications in various types of cancer. This article reviews and summarizes our multi-institute, multidiscipline, and multi-functional studied results and achievements in the research and development of nanotargeted ¹⁸⁸Re-liposome from preclinical cells and animal models to translational clinical investigations, including radionuclide nanoliposome formulation, targeted nuclear imaging, biodistribution, pharmacokinetics, radiation dosimetry, radiation tumor killing effects in animal models, nanotargeted radionuclide and radio/chemo-combination therapeutic effects, and acute toxicity in various tumor animal models. The systemic preclinical and clinical studied results suggest ¹⁸⁸Re-liposome is feasible and promising for in vivo passive nanotargeted radionuclide theranostics in future cancer care applications.     

Role of Cyclooxygenase-2 in Head and Neck Tumorigenesis

The cyclooxygenase-2 (COX-2) is a potent enzyme that converts arachidonic acid to prostaglandins (PG), including PGE2, a key mediator of inflammation and angiogenesis. Importantly, COX-2 is activated in response to inflammatory stimuli, where it is also believed to promote the development and progression of head and neck cancers (HNC). COX-2 can mediate its protumorigenic effect through various mechanisms, such as inducing cell proliferation, inhibition of apoptosis, and suppressing the host’s immune response. Furthermore, COX-2 can induce the production of vascular endothelial growth factors, hence, promoting angiogenesis. Indeed, the ability of COX-2 inhibitors to selectively restrict the proliferation of tumor cells and mediating apoptosis provides promising therapeutic targets for cancer patients. Thus, in this comprehensive review, we summarized the reported differential expression patterns of COX-2 in different stages of head and neck carcinogenesis—from potentially premalignant lesions to invasive carcinomas. Furthermore, we examined the available meta-analysis evidence for COX-2 role in the carcinogenesis of HNC. Finally, further understanding of the biological processes of COX-2 and its role in orchestrating cell proliferation, apoptosis, and angiogenesis may give therapeutically beneficial insight to develop the management plan of HNC patients and improve their clinical outcomes.     

High Levels of KAP1 Expression Are Associated with Aggressive Clinical Features in Ovarian Cancer

KAP1 is an universal corepressor for Kruppel-associated box zinc finger proteins in both normal and tumor cells. In this study, the biological function and clinical significance of KAP1 expression in ovarian cancer were investigated. Immunohistological staining of KAP1 was evaluated in 111 patients with ovarian epithelial cancer, 15 with ovarian borderline tumor, and 20 normal ovarian tissue. The correlations of KAP1 expression with clinicopathological features were studied. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess overall survival to analyze the effect of KAP1 expression on the prognosis of ovarian cancer. The positive rates of KAP1 were significantly higher in ovarian epithelial cancer (55.7%) and borderline tumor (20.0%) than in normal ovarian tissue (5.0%) (all p < 0.01). KAP1 expression correlated significantly with clinical stage (χ² = 14.57, p < 0.0001), pathological grade (χ² = 6.06, p = 0.048) and metastases (χ² =10.38, p = 0.001). Patients with high KAP 1 levels showed poor survival (p < 0.0001). Multivariate analysis showed that KAP1 high expression was an independent predictor for ovarian cancer patients (hazard ratio = 0.463; 95% confidence interval = 0.230–0.9318, p = 0.031). Functionally, depletion of KAP1 by siRNA inhibited ovarian cancer cell proliferation, cell migration. KAP1 expression correlated with aggressive clinical features in ovarian cancer. High KAP1 expression was a prognostic factor of ovarian cancer.     

Peroxisome Proliferator-Activated Receptor γ Expression Is Inversely Associated with Macroscopic Vascular Invasion in Human Hepatocellular Carcinoma

Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated nuclear receptor that regulates cellular lipid and glucose metabolism and also plays an inhibitory role in various cancers. However, the role of PPARγ in hepatocellular carcinoma (HCC) remains controversial. This study aimed to investigate the prognostic value of PPARγ in HCC and its role in inhibiting tumor progression, namely, HCC cell growth, migration, and angiogenesis. Immunohistochemical PPARγ staining was examined in 83 HCC specimens to investigate the clinicopathological correlations between PPARγ expression and various parameters. The functional role of PPARγ was determined via PPARγ overexpression and knockdown in HCC cells. Patients with low HCC tissue PPARγ expression were significantly younger (p = 0.006), and exhibited more tumor numbers (p = 0.038), more macroscopic vascular invasion (MVI) (p = 0.008), and more advanced TNM (size of primary tumor, number of regional lymph nodes, and distant metastasis) stages at diagnosis (p = 0.013) than patients with high HCC tissue PPARγ expression. PPARγ knockdown increased HCC cell growth, migration, and angiogenesis, while PPARγ overexpression reduced HCC cell growth, migration, and angiogenesis. These results suggest that low PPARγ expression is an independent predictor of more MVI in HCC patients. PPARγ contributes to the suppression of HCC cell growth, migration, and angiogenesis. Therefore, PPARγ may be a therapeutic target in HCC patients.     

Relationship between VEGF Family Members,Their Receptors and Cell Death in the Neoplastic Transformation of Colorectal Cancer

Colorectal cancer (CRC) is the second most common cause of cancer death in the world. Both modifiable and nonmodifiable risk factors play a significant role in the pathogenesis of this tumor. The diagnosis is usually made late due to limitations of screening tests; therefore, the scientists are looking for new diagnostic tools such as gene or miRNA expression or different proteins’ concentrations, e.g., vascular endothelial growth factor (VEGF) family members. The VEGF family (VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF) plays a key role in the processes of blood vessel formation in embryonic development as well as in pathological angiogenesis and lymphangiogenesis, which allow the tumor to grow exponentially. Blockage of VEGF-related pathways seems to be a valid therapeutic target. It was suggested in recent studies, that besides already used drugs, e.g., bevacizumab, there are other agents with potential usefulness in anticancer activity such as miRNAs, TMEA, granzyme K, baicalein and arginine. Moreover, VEGF proteins were assessed to induce the expression of anti-apoptotic proteins such as BCL-2 and BAX. Therefore, investigations concerning the usefulness of VEGF family members, not only in the development but also in the therapy of CRC, in order to fully elucidate their role in carcinogenesis, are extremely important.     

The Role of Gut Microbiota in Overcoming Resistance to Checkpoint Inhibitors in Cancer Patients: Mechanisms and Challenges

The introduction of immune checkpoint inhibitors has constituted a major revolution in the treatment of patients with cancer. In contrast with the traditional cytotoxic therapies that directly kill tumor cells, this treatment modality enhances the ability of the host’s immune system to recognize and target cancerous cells. While immune checkpoint inhibitors have been effective across multiple cancer types, overcoming resistance remains a key area of ongoing research. The gut microbiota and its role in cancer immunosurveillance have recently become a major field of study. Gut microbiota has been shown to have direct and systemic effects on cancer pathogenesis and hosts anti-tumor immune response. Many studies have also shown that the host microbiota profile plays an essential role in the response to immunotherapy, especially immune checkpoint inhibitors. As such, modulating this microbial environment has offered a potential path to overcome the resistance to immune checkpoint inhibitors. In this review, we will talk about the role of microbiota in cancer pathogenesis and immune-system activity. We will also discuss preclinical and clinical studies that have increased our understanding about the roles and the mechanisms through which microbiota influences the response to treatment with immune checkpoint inhibitors.