Cancer-Specific Sequences in the Diagnosis and Treatment of NUT Carcinoma.

PURPOSE: NUT carcinoma (NC) is a solid tumor caused by the rearrangement of NUTM1 that usually develops in midline structures, such as the thorax. No standard treatment has been established despite high lethality. Thus, we investigated whether targeting the junction region of NUTM1 fusion breakpoints could serve as a potential treatment option for NC. Materials and Methods: We designed and evaluated a series of small interfering RNAs (siRNAs) targeting the junction region of BRD4-NUTM1 fusion (B4N), the most common form of NUTM1 fusion. Droplet digital polymerase chain reaction using the blood of patients was also tested to evaluate the treatment responses by the junction sequence of the B4N fusion transcripts. RESULTS: As expected, the majority of NC fusion types were B4N (12 of 18, 67%). B4N fusion-specific siRNA treatment on NC cells showed specific inhibitory effects on the B4N fusion transcript and fusion protein without affecting the endogenous expression of the parent genes, resulting in decreased relative cell growth and attenuation of tumor size. In addition, the fusion transcript levels in platelet-rich-plasma samples of the NC patients with systemic metastasis showed a negative correlation with therapeutic effect, suggesting its potential as a measure of treatment responsiveness. CONCLUSION: This study suggests that tumor-specific sequences could be used to treat patients with fusion genes as part of precision medicine for a rare but deadly disease.

Homologous Recombination Deficiency in Ovarian, Breast, Colorectal, Pancreatic, Non-Small Cell Lung and Prostate Cancers, and the Mechanisms of Resistance to PARP Inhibitors.

Homologous recombination (HR) is a highly conserved DNA repair mechanism that protects cells from exogenous and endogenous DNA damage. Breast cancer 1 (BRCA1) and breast cancer 2 (BRCA2) play an important role in the HR repair pathway by interacting with other DNA repair proteins such as Fanconi anemia (FA) proteins, ATM, RAD51, PALB2, MRE11A, RAD50, and NBN. These pathways are frequently aberrant in cancer, leading to the accumulation of DNA damage and genomic instability known as homologous recombination deficiency (HRD). HRD can be caused by chromosomal and subchromosomal aberrations, as well as by epigenetic inactivation of tumor suppressor gene promoters. Deficiency in one or more HR genes increases the risk of many malignancies. Another conserved mechanism involved in the repair of DNA single-strand breaks (SSBs) is base excision repair, in which poly (ADP-ribose) polymerase (PARP) enzymes play an important role. PARP inhibitors (PARPIs) convert SSBs to more cytotoxic double-strand breaks, which are repaired in HR-proficient cells, but remain unrepaired in HRD. The blockade of both HR and base excision repair pathways is the basis of PARPI therapy. The use of PARPIs can be expanded to sporadic cancers displaying the "BRCAness" phenotype. Although PARPIs are effective in many cancers, their efficacy is limited by the development of resistance. In this review, we summarize the prevalence of HRD due to mutation, loss of heterozygosity, and promoter hypermethylation of 35 DNA repair genes in ovarian, breast, colorectal, pancreatic, non-small cell lung cancer, and prostate cancer. The underlying mechanisms and strategies to overcome PARPI resistance are also discussed.

Visualization of a novel human monoclonal antibody against Claudin-3 for targeting ovarian cancer.

INTRODUCTION: Claudin-3 (CLDN3), a tight junction protein, regulates cell-to-cell interactions in epithelial or endothelial cell sheets. During tumorigenesis, epithelial cells are transformed, and tumor cells proliferate through out-of-plane division, resulting in external exposure of CLDN3. Since alterations of CLDN3 expression are associated with cancer progression and higher CLDN3 expression is observed in most ovarian cancers, we tested the feasibility of using a CLDN3-specific antibody as a novel imaging tracer. MATERIALS AND METHODS: After reducing the CLDN3-specific antibodies to expose the -SH groups, click chemistry was used to conjugate the radioactive isotope 111In or the fluorescent protein FNR648. Human ovarian cancer OVCAR-3 and glioblastoma U87MG cells were used as CLDN3-positive and -negative cells. Flow cytometry was used to determine the CLDN3 IgG1 monoclonal antibody binding to both cell lines. OVCAR-3 cells were injected subcutaneously into mice to establish a xenograft model. 111In-labeled CLDN3 antibodies (370 kBq/50 µL) were administered intravenously into mice. After 24 h, organs, including tumors, were excised and measured with a γ-counter. Images were acquired with the IVIS optical imaging system and SPECT/CT. RESULTS: The labeling efficiency of NOTA-111In and antibody-NOTA-111In was 98.52% and 100%, respectively. FNR648-labeled CLDN3 antibody bound to the cell surface of OVCAR-3 and U87MG with 83.4% and 5.7% specificity, respectively. In OVCAR-3 tumor xenografted mice, CLDN3 IgG1 antibody showed a 2.5-fold higher tumor uptake (20.4 ± 7.4% ID/g) than human IgG1 (8.8 ± 2.6% ID/g) at 24 h post injection. The CLDN3 antibody fluorescence signal in the tumor peaked at 24 h post injection. CONCLUSION: We have successfully conjugated a radioisotope and a fluorescent protein with CLDN3-specific antibodies and verified the specific binding of labeled antibodies to OVCAR-3 tumors in a mouse model. Our data suggested that CLDN3-specific human monoclonal antibodies could be used as a useful theranostic tracer.

Relevance of Circulating Tumor Cells as Predictive Markers for Cancer Incidence and Relapse.

Shedding of cancer cells from the primary site or undetectable bone marrow region into the circulatory system, resulting in clinically overt metastasis or dissemination, is the hallmark of unfavorable invasive cancers. The shed cells remain in circulation until they extravasate to form a secondary metastatic lesion or undergo anoikis. The circulating tumor cells (CTCs) found as single cells or clusters carry a plethora of information, are acknowledged as potential biomarkers for predicting cancer prognosis and cancer progression, and are supposed to play key roles in determining tailored therapies for advanced diseases. With the advent of novel technologies that allow the precise isolation of CTCs, more and more clinical trials are focusing on the prognostic and predictive potential of CTCs. In this review, we summarize the role of CTCs as a predictive marker for cancer incidence, relapse, and response to therapy.

Matrix metalloproteinase 11 (MMP11) in macrophages promotes the migration of HER2-positive breast cancer cells and monocyte recruitment through CCL2-CCR2 signaling.

Matrix metalloproteinase 11 (MMP11), a member of the MMP family involved in the degradation of the extracellular matrix, has been implicated in cancer progression. Despite the stromal expression of MMP11 in breast cancer, the prognostic significance and role of MMP11 in immune or stromal cells of breast cancer remain unclear. Based on the immunohistochemical analysis of breast cancer tissues from 497 patients, we demonstrated that MMP11 expression in mononuclear inflammatory cells (predominantly macrophages) is an independent negative prognostic factor in breast cancer, whereas MMP11 expression in tumor cells and fibroblasts is not associated with patient survival. Enforced MMP11 expression in breast cancer cells did not promote cell proliferation and migration. However, MMP11-overexpressing macrophages enhanced the migration of HER2-positive (HER2+) breast cancer cells, recruitment of monocytes, and tube formation of endothelial cells. Furthermore, we found that the chemokine CCL2 secreted from MMP11-overexpressing macrophages activated the MAPK pathway via its receptor CCR2 in breast cancer cells, thereby promoting the migration of HER2+ breast cancer cells through MMP9 upregulation. We also found that MMP11 expression in macrophages was stimulated by MMP11-overepressing HER2+ breast cancer cells. Collectively, our findings provide evidence that MMP11 in macrophages may play a pro-tumoral role in HER2+ breast cancer through interaction with cancer cells, monocytes, and endothelial cells.

Selection Strategies and Practical Application of BRAF V600E-Mutated Non-Small Cell Lung Carcinoma.

PURPOSE: The incidence of BRAF V600E mutation in non-small cell lung carcinoma (NSCLC) is lower than 2%, which poses difficulties in finding legitimate patients for targeted therapy. We investigated the predictive factors pertaining to BRAF V600E and the effectiveness of the VE1 antibody as a screening method for patient selection. MATERIALS AND METHODS: The study was designed into two steps. In a first group, BRAF-mutated NSCLCs were identified from sequencing data to determine the features of BRAF V600E mutation. The results of the first group helped the collection of adenocarcinomas with a papillary or micropapillary pattern but without EGFR or ALK alterations as a second group so that the frequency of BRAF V600E mutation could be calculated. The sensitivity and specificity of the VE1 were compared with BRAF V600E status. RESULTS: Among 39 BRAF-mutated NSCLCs in the first group, 20 (51%) were V600E. BRAF V600E mutation was more common in female patients and showed no significant correlation with smoking status. Nineteen cases were adenocarcinomas without EGFR and ALK alterations. The most common patterns of invasion were papillary and micropapillary along with central fibrosis. The sensitivity and specificity of the VE1 were 90.0% and 92.3%, respectively. In the second group, 6.7% of cases were VE1-positive, indicating that the prevalence was significantly higher than that reported in previous studies (0.3-1.8%). CONCLUSION: BRAF V600E-mutated NSCLCs could be enriched with the application of clinicopathologic parameters, which are not perfect. Therefore, additional VE1 immunohistochemistry may be useful as a screening method.

Macrosphelide A Exhibits a Specific Anti-Cancer Effect by Simultaneously Inactivating ENO1, ALDOA, and FH.

Aerobic glycolysis in cancer cells, also known as the Warburg effect, is an indispensable hallmark of cancer. This metabolic adaptation of cancer cells makes them remarkably different from normal cells; thus, inhibiting aerobic glycolysis is an attractive strategy to specifically target tumor cells while sparing normal cells. Macrosphelide A (MSPA), an organic small molecule, is a potential lead compound for the design of anti-cancer drugs. However, its role in modulating cancer metabolism remains poorly understood. MSPA target proteins were screened using mass spectrometry proteomics combined with affinity chromatography. Direct and specific interactions of MSPA with its candidate target proteins were confirmed by in vitro binding assays, competition assays, and simulation modeling. The siRNA-based knockdown of MSPA target proteins indirectly confirmed the cytotoxic effect of MSPA in HepG2 and MCF-7 cancer cells. In addition, we showed that MSPA treatment in the HEPG2 cell line significantly reduced glucose consumption and lactate release. MSPA also inhibited cancer cell proliferation and induced apoptosis by inhibiting critical enzymes involved in the Warburg effect: aldolase A (ALDOA), enolase 1 (ENO1), and fumarate hydratase (FH). Among these enzymes, the purified ENO1 inhibitory potency of MSPA was further confirmed to demonstrate the direct inhibition of enzyme activity to exclude indirect/secondary factors. In summary, MSPA exhibits anti-cancer effects by simultaneously targeting ENO1, ALDOA, and FH.

Molecular engineering of antibodies for site-specific conjugation to lipid polydopamine hybrid nanoparticles.

Conjugation of antibodies to nanoparticles allows specific cancer targeting, but conventional conjugation methods generate heterogeneous conjugations that cannot guarantee the optimal orientation and functionality of the conjugated antibody. Here, a molecular engineering technique was used for site-specific conjugation of antibodies to nanoparticles. We designed an anti-claudin 3 (CLDN3) antibody containing a single cysteine residue, h4G3cys, then linked it to the maleimide group of lipid polydopamine hybrid nanoparticles (LPNs). Because of their negatively charged lipid coating, LPNs showed high colloidal stability and provided a functional surface for site-specific conjugation of h4G3cys. The activity of h4G3cys was tested by measuring the binding of h4G3cys-conjugated LPNs (C-LPNs) to CLDN3-positive tumor cells and assessing its subsequent photothermal effects. C-LPNsspecifically recognized CLDN3-overexpressing T47D breast cancer cells but not CLDN3-negative Hs578T breast cancer cells. High binding of C-LPNs to CLDN3-overexpressing T47D cells resulted in significantly higher temperature generation upon NIR irradiation and potent anticancer photothermal efficacy. Consistent with this, intravenous injection of C-LPNsin a T47D xenograft mouse model followed by NIR irradiation caused remarkable tumor ablation compared with other treatments through high temperature increases. Our results establish an accurate antibody-linking method and demonstrate the possibility of developing therapeutics using antibody-guided nanoparticles.

Development of Human Monoclonal Antibody for Claudin-3 Overexpressing Carcinoma Targeting.

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell-cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of FcγRIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.