Switching the Chemoselectivity in the Preparation of [18F]FNA-N-CooP, a Free Thiol-Containing Peptide for Targeted Positron Emission Tomography Imaging of Fatty Acid Binding Protein 3.

Fatty acid binding protein 3 (FABP3) is expressed both in tumor cells and in the tumor vasculature, making it a potential target for medical imaging and therapy. In this study, we aimed to radiolabel a CooP peptide with a free amino and thiol group, and evaluate the radiolabeled product [18F]FNA-N-CooP for imaging FABP3 expression in breast cancer brain metastases by positron emission tomography. [18F]FNA-N-CooP was prepared by highly chemoselective N-acylation and characterized using different chemical approaches. We validated its binding to the target using in vitro tissue section autoradiography and performed stability tests in vitro and in vivo. [18F]FNA-N-CooP was successfully synthesized in 16.8% decay-corrected radiochemical yield with high radiochemical purity (98.5%). It exhibited heterogeneous binding on brain metastasis tissue sections from a patient with breast cancer, with foci of radioactivity binding corresponding to FABP3 positivity. Furthermore, the tracer binding was reduced by 55% in the presence of nonradioactive FNA-N-CooP a blocker, indicating specific tracer binding and that FABP3 is a viable target for [18F]FNA-N-CooP. Favorably, the tracer did not bind to necrotic tumor tissue. However, [18F]FNA-N-CooP displayed limited stability both in vitro in mouse plasma or human serum and in vivo in mouse, therefore further studies are needed to improve the stability [18F]FNA-N-CooP to be used for in vivo applications.

IGSF3 tissue expression in squamous cell carcinoma of the oropharynx: a novel tool for prognosis assessment in HPV-related and HPV-unrelated disease.

Biomarkers are not broadly used in the management of head and neck cancers (HNCs). Biomarkers have been beneficial in the management of other cancers, however, not in HNCs. Therefore, we observed the immunopositivity of a novel biomarker called immunoglobulin superfamily member 3 (IGSF3) in tumor tissues in HPV-related and HPV-unrelated OPSCC. Two patient cohorts (C1 and C2) from separate time periods were available for this study (total N = 282). Both consisted of OPSCC patients treated at the Helsinki University Hospital (HUS, Helsinki, Finland) during 2000-2016. For HPV determination, HPV mRNA in situ hybridization was used. Immunohistochemistry was used to assess IGSF3 immunopositivity in cancer tissues. Overall survival (OS) was used as endpoint in the statistical analysis. In C1, stronger immunopositivity of IGSF3 in tumor-infiltrating lymphocytes (TILs) correlated with favorable OS (p = 0.005). Stronger IGSF3 immunopositivity in tumor cells (TCs) was associated with HPV negativity (p = 0.017). Stronger IGSF3 immunopositivity in TILs correlated with HPV positivity (p < 0.001). Elevated IGSF3 immunopositivity in TILs associates with HPV-related tumors and may signify favorable prognosis. The immunopositivity of IGSF3 differs between HPV-related and HPV-unrelated OPSCC.

GNAS mutation inhibits growth and induces phosphodiesterase 4D expression in colorectal cancer cell lines.

Approximately 5% of colorectal cancers (CRCs) have a gain-of-function mutation in the GNAS gene, which leads to the activation of cAMP-dependent signaling pathways and associates with poor prognosis. We investigated the effect of an activating GNAS mutation in CRC cell lines on gene expression and cell proliferation in vitro, and tumor growth in vivo. GNAS-mutated (GNASmt) HCT116 cells showed stimulated synthesis of cAMP as compared to parental (Par) cells. The most upregulated gene in the GNASmt cells was cAMP-hydrolyzing phosphodiesterase 4D (PDE4D) as detected by RNA sequencing. To further validate our finding, we analyzed PDE4D expression in a set of human CRC tumors (n = 35) and demonstrated overexpression in GNAS mutant CRC tumors as compared to GNAS wild-type tumors. The GNASmt HCT116 cells proliferated more slowly than the Par cells. PDE4 inhibitor Ro 20-1724 and PDE4D subtype selective inhibitor GEBR-7b further suppressed the proliferation of GNASmt cells without an effect on Par cells. The growth inhibitory effect of these inhibitors was also seen in the intrinsically GNAS-mutated SK-CO-1 CRC cell line having high levels of cAMP synthesis and PDE4D expression. In vivo, GNASmt HCT116 cells formed smaller tumors than the Par cells in nude mice. In conclusion, our findings demonstrate that GNAS mutation results in the growth suppression of CRC cells. Moreover, the GNAS mutation-induced overexpression of PDE4D provides a potential avenue to impede the proliferation of CRC cells through the use of PDE4 inhibitors.

Comparison of trastuzumab emtansine, trastuzumab deruxtecan, and disitamab vedotin in a multiresistant HER2-positive breast cancer lung metastasis model.

Human epidermal growth factor 2 (HER2)-positive breast cancer with lung metastases resistant to targeted agents is a common therapeutic challenge. Absence of preclinical lung metastasis models that are resistant to multiple anti-HER2 targeted drugs hampers the development of novel therapies. We established a novel HER2-positive breast cancer cell line (L-JIMT-1) with a high propensity to form lung metastases from the parenteral JIMT-1 cell line by injecting JIMT-1 cells into immunodeficient SCID mice. Lung metastases developed in all mice injected with L-JIMT-1 cells, and more rapidly and in greater numbers compared with the parental JIMT-1 cells. L-JIMT-1 cells expressed more epidermal growth factor receptor and HER2 than JIMT-1 cells. L-JIMT-1 cells were resistant to all five tyrosine kinase inhibitors tested in vitro (afatinib, erlotinib, lapatinib, sapitinib, and tucatinib). When we compared JIMT-1 and L-JIMT-1 sensitivity to three HER2-targeting antibody-drug conjugates (ADCs) trastuzumab emtansine (T-DM1), trastuzumab deruxtecan (T-DXd), and disitamab vedotin (DV) in vitro, JIMT-1 cells were resistant T-DXd, partially sensitive to T-DM1, and sensitive to DV, while L-JIMT-1 cells were resistant to both T-DM1 and T-DXd, but moderately sensitive to DV. In a mouse model, all three ADCs inhibited the growth of L-JIMT-1 lung metastases compared to a vehicle, but DV and T-DXd more strongly than T-DM1, and DV treatment led to the smallest tumor burden. The L-JIMT breast cancer lung metastasis model developed may be useful in the evaluation of anti-cancer agents for multiresistant HER2-positive advanced breast cancer.

Radiosynthesis, structural identification and in vitro tissue binding study of [18F]FNA-S-ACooP, a novel radiopeptide for targeted PET imaging of fatty acid binding protein 3.

BACKGROUND: Fatty acid binding protein 3 (FABP3) is a target with clinical relevance and the peptide ligand ACooP has been identified for FABP3 targeting. ACooP is a linear decapeptide containing a free amino and thiol group, which provides opportunities for conjugation. This work is to develop methods for radiolabeling of ACooP with fluorine-18 (18F) for positron emission tomography (PET) applications, and evaluate the binding of the radiolabeled ACooP in human tumor tissue sections with high FABP3 expression. RESULTS: The prosthetic compound 6-[18F]fluoronicotinic acid 4-nitrophenyl ester was conveniently prepared with an on-resin 18F-fluorination in 29.9% radiochemical yield and 96.6% radiochemical purity. Interestingly, 6-[18F]fluoronicotinic acid 4-nitrophenyl ester conjugated to ACooP exclusively by S-acylation instead of the expected N-acylation, and the chemical identity of the product [18F]FNA-S-ACooP was confirmed. In the in vitro binding experiments, [18F]FNA-S-ACooP exhibited heterogeneous and high focal binding in malignant tissue sections, where we also observed abundant FABP3 positivity by immunofluorescence staining. Blocking study further confirmed the [18F]FNA-S-ACooP binding specificity. CONCLUSIONS: FABP3 targeted ACooP peptide was successfully radiolabeled by S-acylation using 6-[18F]fluoronicotinic acid 4-nitrophenyl ester as the prosthetic compound. The tissue binding and blocking studies together with anti-FABP3 immunostaining confirmed [18F]FNA-S-ACooP binding specificity. Further preclinical studies of [18F]FNA-S-ACooP are warranted.

Immunoglobulin superfamily member 3 is required for the vagal neural crest cell migration and enteric neuronal network organization.

The immunoglobulin (Ig) superfamily members are involved in cell adhesion and migration, complex multistep processes that play critical roles in embryogenesis, wound healing, tissue formation, and many other processes, but their specific functions during embryonic development remain unclear. Here, we have studied the function of the immunoglobulin superfamily member 3 (IGSF3) by generating an Igsf3 knockout (KO) mouse model with CRISPR/Cas9-mediated genome engineering. By combining RNA and protein detection methodology, we show that during development, IGSF3 localizes to the neural crest and a subset of its derivatives, suggesting a role in normal embryonic and early postnatal development. Indeed, inactivation of Igsf3 impairs the ability of the vagal neural crest cells to migrate and normally innervate the intestine. The small intestine of Igsf3 KO mice shows reduced thickness of the muscularis externa and diminished number of enteric neurons. Also, misalignment of neurons and smooth muscle cells in the developing intestinal villi is detected. Taken together, our results suggest that IGSF3 functions contribute to the formation of the enteric nervous system. Given the essential role of the enteric nervous system in maintaining normal gastrointestinal function, our study adds to the pool of information required for further understanding the mechanisms of gut innervation and etiology behind bowel motility disorders.

Efficacy and Safety of Glycosphingolipid SSEA-4 Targeting CAR-T Cells in an Ovarian Carcinoma Model.

Chimeric antigen receptor (CAR) T-cell immunotherapies for solid tumors face critical challenges such as heterogeneous antigen expression. We characterized stage-specific embryonic antigen-4 (SSEA-4) cell-surface glycolipid as a target for CAR T-cell therapy. SSEA-4 is mainly expressed during embryogenesis but is also found in several cancer types making it an attractive tumor-associated antigen. Anti-SSEA-4 CAR-T cells were generated and assessed preclinically in vitro and in vivo for antitumor response and safety. SSEA-4 CAR-T cells effectively eliminated SSEA-4-positive cells in all the tested cancer cell lines, whereas SSEA-4-negative cells lines were not targeted. In vivo efficacy and safety studies using NSG mice and the high-grade serous ovarian cancer cell line OVCAR4 demonstrated a remarkable and specific antitumor response at all the CAR T-cell doses used. At high T-cell doses, CAR T cell-treated mice showed signs of health deterioration after a follow-up period. However, the severity of toxicity was reduced with a delayed onset when lower CAR T-cell doses were used. Our data demonstrate the efficacy of anti-SSEA-4 CAR T-cell therapy; however, safety strategies, such as dose-limiting and/or equipping CAR-T cells with combinatorial antigen recognition should be implemented for its potential clinical translation.

KSHV infection of endothelial precursor cells with lymphatic characteristics as a novel model for translational Kaposi's sarcoma studies.

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.

Bivalent EGFR-Targeting DARPin-MMAE Conjugates.

Epidermal growth factor receptor (EGFR) is a validated tumor marker overexpressed in various cancers such as squamous cell carcinoma (SSC) of the head and neck and gliomas. We constructed protein-drug conjugates based on the anti-EGFR Designed Ankyrin Repeat Protein (DARPin) E01, and compared the bivalent DARPin dimer (DD1) and a DARPin-Fc (DFc) to the monomeric DARPin (DM) and the antibody derived scFv425-Fc (scFvFc) in cell culture and a mouse model. The modular conjugation system, which was successfully applied for the preparation of protein-drug and -dye conjugates, uses bio-orthogonal protein-aldehyde generation by the formylglycine-generating enzyme (FGE). The generated carbonyl moiety is addressed by a bifunctional linker with a pyrazolone for a tandem Knoevenagel reaction and an azide for strain-promoted azide-alkyne cycloaddition (SPAAC). The latter reaction with a PEGylated linker containing a dibenzocyclooctyne (DBCO) for SPAAC and monomethyl auristatin E (MMAE) as the toxin provided the stable conjugates DD1-MMAE (drug-antibody ratio, DAR = 2.0) and DFc-MMAE (DAR = 4.0) with sub-nanomolar cytotoxicity against the human squamous carcinoma derived A431 cells. In vivo imaging of Alexa Fluor 647-dye conjugates in A431-xenografted mice bearing subcutaneous tumors as the SCC model revealed unspecific binding of bivalent DARPins to the ubiquitously expressed EGFR. Tumor-targeting was verified 6 h post-injection solely for DD1 and scFvFc. The total of four administrations of 6.5 mg/kg DD1-MMAE or DFc-MMAE twice weekly did not cause any sequela in mice. MMAE conjugates showed no significant anti-tumor efficacy in vivo, but a trend towards increased necrotic areas (p = 0.2213) was observed for the DD1-MMAE (n = 5).

Hepsin regulates TGFß signaling via fibronectin proteolysis.

Transforming growth factor-beta (TGFß) is a multifunctional cytokine with a well-established role in mammary gland development and both oncogenic and tumor-suppressive functions. The extracellular matrix (ECM) indirectly regulates TGFß activity by acting as a storage compartment of latent-TGFß, but how TGFß is released from the ECM via proteolytic mechanisms remains largely unknown. In this study, we demonstrate that hepsin, a type II transmembrane protease overexpressed in 70% of breast tumors, promotes canonical TGFß signaling through the release of latent-TGFß from the ECM storage compartment. Mammary glands in hepsin CRISPR knockout mice showed reduced TGFß signaling and increased epithelial branching, accompanied by increased levels of fibronectin and latent-TGFß1, while overexpression of hepsin in mammary tumors increased TGFß signaling. Cell-free and cell-based experiments showed that hepsin is capable of direct proteolytic cleavage of fibronectin but not latent-TGFß and, importantly, that the ability of hepsin to activate TGFß signaling is dependent on fibronectin. Altogether, this study demonstrates a role for hepsin as a regulator of the TGFß pathway in the mammary gland via a novel mechanism involving proteolytic downmodulation of fibronectin.

Circumventing Drug Treatment? Intrinsic Lethal Effects of Polyethyleneimine (PEI)-Functionalized Nanoparticles on Glioblastoma Cells Cultured in Stem Cell Conditions.

Glioblastoma (GB) is the most frequent malignant tumor originating from the central nervous system. Despite breakthroughs in treatment modalities for other cancer types, GB remains largely irremediable due to the high degree of intratumoral heterogeneity, infiltrative growth, and intrinsic resistance towards multiple treatments. A sub-population of GB cells, glioblastoma stem cells (GSCs), act as a reservoir of cancer-initiating cells and consequently, constitute a significant challenge for successful therapy. In this study, we discovered that PEI surface-functionalized mesoporous silica nanoparticles (PEI-MSNs), without any anti-cancer drug, very potently kill multiple GSC lines cultured in stem cell conditions. Very importantly, PEI-MSNs did not affect the survival of established GB cells, nor other types of cancer cells cultured in serum-containing medium, even at 25 times higher doses. PEI-MSNs did not induce any signs of apoptosis or autophagy. Instead, as a potential explanation for their lethality under stem cell culture conditions, we demonstrate that the internalized PEI-MSNs accumulated inside lysosomes, subsequently causing a rupture of the lysosomal membranes. We also demonstrate blood-brain-barrier (BBB) permeability of the PEI-MSNs in vitro and in vivo. Taking together the recent indications for the vulnerability of GSCs for lysosomal targeting and the lethality of the PEI-MSNs on GSCs cultured under stem cell culture conditions, the results enforce in vivo testing of the therapeutic impact of PEI-functionalized nanoparticles in faithful preclinical GB models.

Redox responsive Pluronic micelle mediated delivery of functional siRNA: a modular nano-assembly for targeted delivery.

There is an unmet need to develop strategies that allow site-specific delivery of short interfering RNA (siRNA) without any associated toxicity. To address this challenge, we have developed a novel siRNA delivery platform using chemically modified pluronic F108 as an amphiphilic polymer with a releasable bioactive disulfide functionality. The micelles exhibited thermoresponsive properties and showed a hydrodynamic size of ∼291 nm in DLS and ∼200-250 nm in SEM at 37 °C. The grafting of free disulfide pyridyl groups enhanced the transfection efficiency and was successfully demonstrated in human colon carcinoma (HCT116; 88%) and glioma cell lines (U87; 90%), non-cancerous human dermal fibroblast (HDF; 90%) cells as well as in mouse embryonic stem (mES; 54%) cells. To demonstrate the versatility of our modular nanocarrier design, we conjugated the MDGI receptor targeting COOP peptide on the particle surface that allowed the targeted delivery of the cargo molecules to human patent-derived primary BT-13 gliospheres. Transfection experiments with this design resulted in ∼65% silencing of STAT3 mRNA in BT-13 gliospheres, while only ∼20% of gene silencing was observed in the absence of the peptide. We believe that our delivery method solves current problems related to the targeted delivery of RNAi drugs for potential in vivo applications.

CD109-GP130 interaction drives glioblastoma stem cell plasticity and chemoresistance through STAT3 activity.

Glioma stem cells (GSCs) drive propagation and therapeutic resistance of glioblastomas, the most aggressive diffuse brain tumors. However, the molecular mechanisms that maintain the stemness and promote therapy resistance remain poorly understood. Here we report CD109/STAT3 axis as crucial for the maintenance of stemness and tumorigenicity of GSCs and as a mediator of chemoresistance. Mechanistically, CD109 physically interacts with glycoprotein 130 to promote activation of the IL-6/STAT3 pathway in GSCs. Genetic depletion of CD109 abolished the stemness and self-renewal of GSCs and impaired tumorigenicity. Loss of stemness was accompanied with a phenotypic shift of GSCs to more differentiated astrocytic-like cells. Importantly, genetic or pharmacologic targeting of CD109/STAT3 axis sensitized the GSCs to chemotherapy, suggesting that targeting CD109/STAT3 axis has potential to overcome therapy resistance in glioblastoma.

Peptidotriazolamers Inhibit Aß(1-42) Oligomerization and Cross a Blood-Brain-Barrier Model.

In peptidotriazolamers every second peptide bond is replaced by a 1H-1,2,3-triazole. Such foldamers are expected to bridge the gap in molecular weight between small-molecule drugs and protein-based drugs. Amyloid ß (Aß) aggregates play an important role in Alzheimer's disease. We studied the impact of amide bond replacements by 1,4-disubstituted 1H-1,2,3-triazoles on the inhibitory activity of the aggregation "hot spots" K16 LVFF20 and G39 VVIA42 in Aß(1-42). We found that peptidotriazolamers act as modulators of the Aß(1-42) oligomerization. Some peptidotriazolamers are able to interfere with the formation of toxic early Aß oligomers, depending on the position of the triazoles, which is also supported by computational studies. Preliminary in vitro results demonstrate that a highly active peptidotriazolamer is also able to cross the blood-brain-barrier.

Peptide-Based Strategies for Targeted Tumor Treatment and Imaging.

Cancer is one of the leading causes of death worldwide. The development of cancer-specific diagnostic agents and anticancer toxins would improve patient survival. The current and standard types of medical care for cancer patients, including surgery, radiotherapy, and chemotherapy, are not able to treat all cancers. A new treatment strategy utilizing tumor targeting peptides to selectively deliver drugs or applicable active agents to solid tumors is becoming a promising approach. In this review, we discuss the different tumor-homing peptides discovered through combinatorial library screening, as well as native active peptides. The different structure-function relationship data that have been used to improve the peptide's activity and conjugation strategies are highlighted.

Monotherapy efficacy of blood-brain barrier permeable small molecule reactivators of protein phosphatase 2A in glioblastoma.

Glioblastoma is a fatal disease in which most targeted therapies have clinically failed. However, pharmacological reactivation of tumour suppressors has not been thoroughly studied as yet as a glioblastoma therapeutic strategy. Tumour suppressor protein phosphatase 2A is inhibited by non-genetic mechanisms in glioblastoma, and thus, it would be potentially amendable for therapeutic reactivation. Here, we demonstrate that small molecule activators of protein phosphatase 2A, NZ-8-061 and DBK-1154, effectively cross the in vitro model of blood-brain barrier, and in vivo partition to mouse brain tissue after oral dosing. In vitro, small molecule activators of protein phosphatase 2A exhibit robust cell-killing activity against five established glioblastoma cell lines, and nine patient-derived primary glioma cell lines. Collectively, these cell lines have heterogeneous genetic background, kinase inhibitor resistance profile and stemness properties; and they represent different clinical glioblastoma subtypes. Moreover, small molecule activators of protein phosphatase 2A were found to be superior to a range of kinase inhibitors in their capacity to kill patient-derived primary glioma cells. Oral dosing of either of the small molecule activators of protein phosphatase 2A significantly reduced growth of infiltrative intracranial glioblastoma tumours. DBK-1154, with both higher degree of brain/blood distribution, and more potent in vitro activity against all tested glioblastoma cell lines, also significantly increased survival of mice bearing orthotopic glioblastoma xenografts. In summary, this report presents a proof-of-principle data for blood-brain barrier-permeable tumour suppressor reactivation therapy for glioblastoma cells of heterogenous molecular background. These results also provide the first indications that protein phosphatase 2A reactivation might be able to challenge the current paradigm in glioblastoma therapies which has been strongly focused on targeting specific genetically altered cancer drivers with highly specific inhibitors. Based on demonstrated role for protein phosphatase 2A inhibition in glioblastoma cell drug resistance, small molecule activators of protein phosphatase 2A may prove to be beneficial in future glioblastoma combination therapies.

Tumor-Targeting Peptides: The Functional Screen of Glioblastoma Homing Peptides to the Target Protein FABP3 (MDGI).

We recently identified the glioblastoma homing peptide CooP (CGLSGLGVA) using in vivo phage display screen. The mammary-derived growth inhibitor (MDGI/FABP3) was identified as its interacting partner. Here, we present an alanine scan of A-CooP to investigate the contribution of each amino acid residue to the binding to FABP3 by microscale thermophoresis (MST) and surface plasmon resonance (SPR). We also tested the binding affinity of the A-CooP-K, KA-CooP, and retro-inverso A-CooP analogues to the recombinant FABP3. According to the MST analysis, A-CooP showed micromolar (KD = 2.18 µM) affinity to FABP3. Alanine replacement of most of the amino acids did not affect peptide affinity to FABP3. The A-CooP-K variant showed superior binding affinity, while A-[Ala5]CooP and A-[Ala7]CooP, both replacing a glycine residue with alanine, showed negligible binding to FABP3. These results were corroborated in vitro and in vivo using glioblastoma models. Both A-CooP-K and A-CooP showed excellent binding in vitro and homing in vivo, while A-[Ala5]CooP and control peptides failed to bind the cells or home to the intracranial glioblastoma xenografts. These results provide insight into the FABP3-A-CooP interaction that may be important for future applications of drug conjugate design and development.

Prostate-specific membrane antigen expression in the vasculature of primary lung carcinomas associates with faster metastatic dissemination to the brain.

Glioblastomas and brain metastases (BM) of solid tumours are the most common central nervous system neoplasms associated with very unfavourable prognosis. In this study, we report the association of prostate-specific membrane antigen (PSMA) with various clinical parameters in a large cohort of primary and secondary brain tumours. A tissue microarray containing 371 cases of ascending grades of gliomas pertaining to astrocytic origin and samples of 52 cases of primary lung carcinomas with matching BM with follow-up time accounting to 10.4 years was evaluated for PSMA expression using immunohistochemistry. In addition, PSMA expression was studied in BM arising from melanomas and breast carcinomas. Neovascular expression of PSMA was evident alongside with high expression in the proliferating microvasculature of glioblastomas when compared to the tumour cell expression. This result correlated with the results obtained from the in silico (cancer genome databases) analyses. In gliomas, only the vascular expression of PSMA associated with poor overall survival but not the tumour cell expression. In the matched primary lung cancers and their BM (n = 52), vascular PSMA expression in primary tumours associated with significantly accelerated metastatic dissemination to the brain with a tendency towards poor overall survival. Taken together, we report that the vascular expression of PSMA in the primary and secondary brain tumours globally associates with the malignant progression and poor outcome of the patients.

Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is a biomarker of poor prognosis in primary melanomas, and its depletion inhibits melanoma cell invasion and disrupts tumor blood vessel walls.

Melanoma is an unpredictable, highly metastatic malignancy, and treatment of advanced melanoma remains challenging. Novel molecular markers based on the alterations in gene expression and the molecular pathways activated or deactivated during melanoma progression are needed for predicting the course of the disease already in primary tumors and for providing new targets for therapy. Here, we sought to identify genes whose expression in primary melanomas correlate with patient disease-specific survival using global gene expression profiling. Many of the identified potential markers of poor prognosis were associated with the epithelial-mesenchymal transition, extracellular matrix formation, and angiogenesis. We studied further the significance of one of the genes, prolyl 4-hydroxylase subunit alpha 1 (P4HA1), in melanoma progression. P4HA1 depletion in melanoma cells reduced cell adhesion, invasion, and viability in vitro. In melanoma xenograft assays, we found that P4HA1 knockdown reduced melanoma tumor invasion as well as the deposition of collagens, particularly type IV collagen, in the interstitial extracellular matrix and in the basement membranes of tumor blood vessels, leading to vessel wall rupture and hemorrhages. Further, P4HA1 knockdown reduced the secretion of collagen triple helix repeat containing 1 (CTHRC1), an important mediator of melanoma cell migration and invasion, in vitro and its deposition around tumor blood vessels in vivo. Taken together, P4HA1 is an interesting potential prognostic marker and therapeutic target in primary melanomas, influencing many aspects of melanoma tumor progression.

ARX788, a novel anti-HER2 antibody-drug conjugate, shows anti-tumor effects in preclinical models of trastuzumab emtansine-resistant HER2-positive breast cancer and gastric cancer.

The majority of HER2-positive breast or gastric cancers treated with T-DM1 eventually show resistance to this agent. We compared the effects of T-DM1 and ARX788, a novel anti-HER2 antibody-drug conjugate, on cell growth and apoptosis in HER2-positive breast cancer and gastric cancer cell lines sensitive to T-DM1, gastric cancer cell lines resistant to T-DM1, HER2-negative breast cancer cell lines, and T-DM1-resistant xenograft models. ARX788 was effective in T-DM1-resistant in vitro and in vivo models of HER2-positive breast cancer and gastric cancer. ARX788 showed a pronounced growth inhibitory effect on all five HER2-positive cell lines tested, of which two gastric cancer cell lines had acquired resistance to T-DM1. ARX788 evoked more apoptotic events compared to T-DM1. While JIMT-1 and RN-87 xenograft tumors progressed on T-DM1 treatment, all such tumors responded to ARX788, and four out of the six JIMT-1 tumors and nine out of the twelve RN-87 tumors disappeared during the ARX788 treatment. Mice treated with ARX788 survived longer than those treated with T-DM1. The data support evaluation of ARX788 in patients with HER2-positive breast cancer or gastric cancer including cancers that progress during T-DM1 therapy.