In Silico Analyses of Vertebrate G-Protein-Coupled Receptor Fusions United With or Without an Additional Transmembrane Sequence Indicate Classification into Three Groups of Linkers.

Natural G-protein-coupled receptors (GPCRs) rarely have an additional transmembrane (TM) helix, such as an artificial TM-linker that can unite two class A GPCRs in tandem as a single-polypeptide chain (sc). Here, we report that three groups of TM-linkers exist in the intervening regions of natural GPCR fusions from vertebrates: (1) the original consensus (i.e., consensus 1) and consensus 2~4 (related to GPCR itself or its receptor-interacting proteins); (2) the consensus but GPCR-unrelated ones, 1~7; and (3) the inability to apply 1/2 that show no similarity to any other proteins. In silico analyses indicated that all natural GPCR fusions from Amphibia lack a TM-linker, and reptiles have no GPCR fusions; moreover, in either the GPCR-GPCR fusion or fusion protein of (GPCR monomer) and non-GPCR proteins from vertebrates, excluding tetrapods, i.e., so-called fishes, TM-linkers differ from previously reported mammalian and are avian sequences and are classified as Groups 2 and 3. Thus, previously reported TM-linkers were arranged: Consensus 1 is [T(I/A/P)(A/S)-(L/N)(I/W/L)(I/A/V)GL(L/G)(A/T)(S/L/G)(I/L)] first identified in invertebrate sea anemone Exaiptasia diaphana (LOC110241027) and (330-SPSFLCI-L-SLL-340) identified in a tropical bird Opisthocomus hoazin protein LOC104327099 (XP_009930279.1); GPCR-related consensus 2~4 are, respectively, (371-prlilyavfc fgtatg-386) in the desert woodrat Neotoma lepida A6R68_19462 (OBS78147.1), (363-lsipfcll yiaallgnfi llfvi-385) in Gavia stellate (red-throated loon) LOC104264164 (XP_009819412.1), and (479-ti vvvymivcvi glvgnflvmy viir-504) in a snailfish GPCR (TNN80062.1); In Mammals Neotoma lepida, Aves Erythrura gouldiae, and fishes protein (respectively, OBS83645.1, RLW13346.1 and KPP79779.1), the TM-linkers are Group 2. Here, we categorized, for the first time, natural TM-linkers as rare evolutionary events among all vertebrates.

HER4 and EGFR Activate Cell Signaling in NRG1 Fusion-Driven Cancers: Implications for HER2-HER3-specific Versus Pan-HER Targeting Strategies.

INTRODUCTION: NRG1 gene fusions are clinically actionable alterations identified in NSCLC and other tumors. Previous studies have reported that NRG1 fusions signal through HER2 and HER3 but, thus far, strategies targeting HER3 specifically or HER2-HER3 signaling have exhibited modest activity in patients with NSCLC bearing NRG1 fusions. Although NRG1 fusion proteins can bind HER4 in addition to HER3, the contribution of HER4 and other HER family members in NRG1 fusion-positive cancers is not fully understood. METHODS: We investigated the role of HER4 and EGFR-HER3 signaling in NRG1 fusion-positive cancers using Ba/F3 models engineered to express various HER family members in combination with NRG1 fusions and in vitro and in vivo models of NRG1 fusion-positive cancer. RESULTS: We determined that NRG1 fusions can stimulate downstream signaling and tumor cell growth through HER4, independent of other HER family members. Moreover, EGFR-HER3 signaling is also activated in cells expressing NRG1 fusions, and inhibition of these receptors is also necessary to effectively inhibit tumor cell growth. We observed that cetuximab, an anti-EGFR antibody, in combination with anti-HER2 antibodies, trastuzumab and pertuzumab, yielded a synergistic effect. Furthermore, pan-HER tyrosine kinase inhibitors were more effective than tyrosine kinase inhibitors with greater specificity for EGFR, EGFR-HER2, or HER2-HER4, although the relative degree of dependence on EGFR or HER4 signaling varied between different NRG1 fusion-positive cancers. CONCLUSIONS: Our findings indicate that pan-HER inhibition including HER4 and EGFR blockade is more effective than selectively targeting HER3 or HER2-HER3 in NRG1 fusion-positive cancers.

Dual-targeting therapy against HER3/MET in human colorectal cancers.

BACKGROUND: Colorectal cancer (CRC) is the most common malignancy in the world, and novel molecular targeted therapies for CRC have been vigorously pursued. We searched for novel combination therapies based on the expression patterns of membrane proteins in CRC cell lines. RESULTS: A positive correlation was observed between the expression of human pidermal growth factor receptor (HER) 3 and mesenchymal-to-epithelial transition factor (MET) on the cell surface of CRC cell lines. The brief stimulation of HER3/MET-high SW1116 CRC cells with both neuregulin-1 (NRG1) and hepatocyte growth factor enhanced ERK phosphorylation and cell proliferation more than each stimulation alone. In addition, a prolonged NRG1 stimulation resulted in the tyrosine phosphorylation of MET. In this context, the Forkhead Box protein M1 (FOXM1)-regulated tyrosine phosphorylation of MET by NRG1 was demonstrated, suggesting the existence of a signaling pathway mediated by FOXM1 upon the NRG1 stimulation. Since the co-expression of HER3 and MET was also demonstrated in in vivo CRC tissues by immunohistochemistry, we investigated whether the co-inhibition of HER3 and MET could be an effective therapy for CRC. We established HER3-and/or MET-KO SW1116 cell lines, and HER3/MET-double KO resulted in the inhibition of in vitro cell proliferation and in vivo tumor growth in nude mice by SW1116 cells. Furthermore, the combination of patritumab, an anti-HER3 fully human mAb, and PHA665752, a MET inhibitor, markedly inhibited in vitro cell proliferation, 3D-colony formation, and in vivo tumor growth in nude mice by SW1116 cells CONCLUSION: The dual targeting of HER3/MET has potential as CRC therapy.

CD98 regulates the phosphorylation of HER2 and a bispecific anti-HER2/CD98 antibody inhibits the growth signal of human breast cancer cells.

Human epidermal growth factor receptor (HER) family proteins are currently major targets of therapeutic monoclonal antibodies against various epithelial cancers. However, the resistance of cancer cells to HER family-targeted therapies, which may be caused by cancer heterogeneity and persistent HER phosphorylation, often reduces overall therapeutic effects. We herein showed that a newly discovered molecular complex between CD98 and HER2 affected HER function and cancer cell growth. The immunoprecipitation of the HER2 or HER3 protein from lysates of SKBR3 breast cancer (BrCa) cells revealed the HER2-CD98 or HER3-CD98 complex. The knockdown of CD98 by small interfering RNAs inhibited the phosphorylation of HER2 in SKBR3 cells. A bispecific antibody (BsAb) that recognized the HER2 and CD98 proteins was constructed from a humanized anti-HER2 (SER4) IgG and an anti-CD98 (HBJ127) single chain variable fragment, and this BsAb significantly inhibited the cell growth of SKBR3 cells. Prior to the inhibition of AKT phosphorylation, BsAb inhibited the phosphorylation of HER2, however, significant inhibition of HER2 phosphorylation was not observed in anti-HER2 pertuzumab, trastuzumab, SER4 or anti-CD98 HBJ127 in SKBR3 cells. The dual targeting of HER2 and CD98 has potential as a new therapeutic strategy for BrCa.

In Vitro Tumor Cell-Binding Assay to Select High-Binding Antibody and Predict Therapy Response for Personalized 64Cu-Intraperitoneal Radioimmunotherapy against Peritoneal Dissemination of Pancreatic Cancer: A Feasibility Study.

Peritoneal dissemination of pancreatic cancer has a poor prognosis. We have reported that intraperitoneal radioimmunotherapy using a 64Cu-labeled antibody (64Cu-ipRIT) is a promising adjuvant therapy option to prevent this complication. To achieve personalized 64Cu-ipRIT, we developed a new in vitro tumor cell-binding assay (64Cu-TuBA) system with a panel containing nine candidate 64Cu-labeled antibodies targeting seven antigens (EGFR, HER2, HER3, TfR, EpCAM, LAT1, and CD98), which are reportedly overexpressed in patients with pancreatic cancer. We investigated the feasibility of 64Cu-TuBA to select the highest-binding antibody for individual cancer cell lines and predict the treatment response in vivo for 64Cu-ipRIT. 64Cu-TuBA was performed using six human pancreatic cancer cell lines. For three cell lines, an in vivo treatment study was performed with 64Cu-ipRIT using high-, middle-, or low-binding antibodies in each peritoneal dissemination mouse model. The high-binding antibodies significantly prolonged survival in each mouse model, while low-and middle-binding antibodies were ineffective. There was a correlation between in vitro cell binding and in vivo therapeutic efficacy. Our findings suggest that 64Cu-TuBA can be used for patient selection to enable personalized 64Cu-ipRIT. Tumor cells isolated from surgically resected tumor tissues would be suitable for analysis with the 64Cu-TuBA system in future clinical studies.

Selective targeting of multiple myeloma cells with a monoclonal antibody recognizing the ubiquitous protein CD98 heavy chain.

Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.

Altered binding avidities and improved growth inhibitory effects of novel anti-HER3 mAb against human cancers in the presence of HER1-or HER2-targeted drugs.

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (KA) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The KA of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of KA, and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high KA by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI-H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high KA by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI-H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI-H1838, the reactivity and KA of Ab4 increased compared with in parent NCI-H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of KA with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116.

Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target.

L-type amino acid transporter 1 (LAT1)/SLC7A5 is the first identified CD98 light chain disulfide linked to the CD98 heavy chain (CD98hc/SLC3A2). LAT1 transports large neutral amino acids, including leucine, which activates mTOR, and is highly expressed in human cancers. We investigated the oncogenicity of human LAT1 introduced to NIH/3T3 cells by retrovirus infection. NIH/3T3 cell lines stably expressing human native (164C) or mutant (164S) LAT1 (naLAT1/3T3 or muLAT1/3T3, respectively) were established. We confirmed that endogenous mouse CD98hc forms a disulfide bond with exogenous human LAT1 in naLAT1/3T3, but not in muLAT1/3T3. Endogenous mouse CD98hc mRNA increased in both naNIH/3T3 and muLAT1/3T3, and a similar amount of exogenous human LAT1 protein was detected in both cell lines. Furthermore, naLAT1/3T3 and muLAT1/3T3 cell lines were evaluated for cell growth-related phenotypes (phosphorylation of ERK, cell-cycle progression) and cell malignancy-related phenotypes (anchorage-independent cell growth, tumor formation in nude mice). naLAT1/3T3 had stronger growth- and malignancy- related phenotypes than NIH/3T3 and muLAT1/3T3, suggesting the oncogenicity of native LAT1 through its interaction with CD98hc. Anti-LAT1 monoclonal antibodies significantly inhibited in vitro cell proliferation and in vivo tumor growth of naLAT1/3T3 cells in nude mice, demonstrating LAT1 to be a promising anti-cancer target.

[Analysis of Target Molecules towards Anti-cancer Therapeutic Antibodies].

Target molecules of existing anti-cancer therapeutic monoclonal antibodies (mAbs) are divided into 1) receptor-type tyrosine kinases, such as human epidermal growth factor receptor (HER) family, 2) differentiation antigens, such as CD20 (Rituxan target), 3) angiogenesis-related molecules, and 4) immune checkpoint molecules (PD-1, etc.). We have recently reported a novel therapy targeting lymphangiogenesis, but not angiogenesis, using an anti-LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) mAb. At present, many transporters are not considered to be target molecules for the cancer therapy; however, our study strongly suggested that the inhibition of cancer metabolism by mAbs against amino acid transporters will play a significant role in future cancer therapies. Most anti-cancer therapeutic mAbs bind cell-surface molecules on viable cancer cells: therefore, it is necessary to produce mAbs recognizing epitopes on the extracellular domains of native and non-denatured proteins. We concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti-cancer therapeutic mAbs. We introduce our efforts to develop seeds for therapeutic mAbs using whole cancer cells and transfectants as the immunogen. As many target candidates in the future are multi-pass membrane proteins, such as 12-pass amino acid transporter proteins belonging to the solute carrier (SLC) family, and their possible immunogenic extracellular regions are small, the production of specific mAbs is highly difficult. In this review, we summarize the successful preparation and characterization of mAbs recognizing the extracellular domain of oncoproteins, including transporters.

Down-regulation of dual-specificity phosphatase 6, a negative regulator of oncogenic ERK signaling, by ACA-28 induces apoptosis in NIH/3T3 cells overexpressing HER2/ErbB2.

Dual-specificity phosphatase 6 (DUSP6) is a key negative feedback regulator of the member of the RAS-ERK MAPK signaling pathway that is associated with cellular proliferation and differentiation. Deterioration of DUSP6 expression could therefore result in deregulated growth activity. We have previously discovered ACA-28, a novel anticancer compound with a unique property to stimulate ERK phosphorylation and induce apoptosis in ERK-active melanoma cells. However, the mechanism of cancer cell-specific-apoptosis by ACA-28 remains obscure. Here, we investigated the involvement of DUSP6 in the mechanisms of the ACA-28-mediated apoptosis by using the NIH/3T3 cells overexpressing HER2/ErbB2 (A4-15 cells), as A4-15 exhibited higher ERK phosphorylation and are more susceptible to ACA-28 than NIH/3T3. We showed that A4-15 exhibited high DUSP6 protein levels, which require ERK activation. Notably, the silencing of the DUDSP6 gene by siRNA inhibited proliferation and induced apoptosis in A4-15, but not in NIH/3T3, indicating that A4-15 requires high DUSP6 expression for growth. Importantly, ACA-28 preferentially down-regulated the DUSP6 protein and proliferation in A4-15 via the proteasome, while it stimulated ERK phosphorylation. Collectively, the up-regulation of DUSP6 may exert a growth-promoting role in cancer cells overexpressing HER2. DUSP6 down-regulation in ERK-active cancer cells might have the potential as a novel cancer measure.

Antitumor effects of novel mAbs against cationic amino acid transporter 1 (CAT1) on human CRC with amplified CAT1 gene.

Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer-related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference-mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1-GFP in a GFP expression-dependent manner were selected from established hybridoma clones. Novel anti-CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno-histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti-CAT1 mAbs exhibited internalization activity, antibody-dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW-C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs.

Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers.

Resistance of progressive cancers against chemotherapy is a serious clinical problem. In this context, human epidermal growth factor receptor 3 (HER3) can play important roles in drug resistance to HER1- and HER2- targeted therapies. Since clinical testing of anti-HER3 monoclonal antibodies (mAbs) such as patritumab could not show remarkable effect compared with existing drugs, we generated novel mAbs against anti-HER3. Novel rat mAbs reacted with HEK293 cells expressing HER3, but not with cells expressing HER1, HER2 or HER4. Specificity of mAbs was substantiated by the loss of mAb binding with knockdown by siRNA and knockout of CRISPR/Cas9-based genome-editing. Analyses of CDR sequence and germline segment have revealed that seven mAbs are classified to four groups, and the binding of patritumab was inhibited by one of seven mAbs. Seven mAbs have shown reactivity with various human epithelial cancer cells, strong internalization activity of cell-surface HER3, and inhibition of NRG1 binding, NRG1-dependent HER3 phosphorylation and cell growth. Anti-HER3 mAbs were also reactive with in vivo tumor tissues and cancer tissue-originated spheroid. Ab4 inhibited in vivo tumor growth of human colon cancer cells in nude mice. Present mAbs may be superior to existing anti-HER3 mAbs and support existing anti-cancer therapeutic mAbs.

Anti-tumor effects of an antagonistic mAb against the ASCT2 amino acid transporter on KRAS-mutated human colorectal cancer cells.

KRAS mutations are detected in numerous human cancers, but there are few effective drugs for KRAS-mutated cancers. Transporters for amino acids and glucose are highly expressed on cancer cells, possibly to maintain rapid cell growth and metabolism. Alanine-serine-cysteine transporter 2 (ASCT2) is a primary transporter for glutamine in cancer cells. In this study, we developed a novel monoclonal antibody (mAb) recognizing the extracellular domain of human ASCT2, and investigated whether ASCT2 can be a therapeutic target for KRAS-mutated cancers. Rats were immunized with RH7777 rat hepatoma cells expressing human ASCT2 fused to green fluorescent protein (GFP). Splenocytes from the immunized rats were fused with P3X63Ag8.653 mouse myeloma cells, and selected and cloned hybridoma cells secreting Ab3-8 mAb were established. This mAb reacted with RH7777 transfectants expressing ASCT2-GFP proteins in a GFP intensity-dependent manner. Ab3-8 reacted with various human cancer cells, but not with non-cancer breast epithelial cells or ASCT2-knocked out HEK293 and SW1116 cells. In SW1116 and HCT116 human colon cancer cells with KRAS mutations, treatment with Ab3-8 reduced intracellular glutamine transport, phosphorylation of AKT and ERK, and inhibited in vivo tumor growth of these cells in athymic mice. Inhibition of in vivo tumor growth by Ab3-8 was not observed in HT29 colon and HeLa uterus cancer cells with wild-type KRAS. These results suggest that ASCT2 is an excellent therapeutic target for KRAS-mutated cancers.

Epigenomic Profiling Discovers Trans-lineage SOX2 Partnerships Driving Tumor Heterogeneity in Lung Squamous Cell Carcinoma.

Molecular characterization of lung squamous cell carcinoma (LUSC), one of the major subtypes of lung cancer, has not sufficiently improved its nonstratified treatment strategies over decades. Accumulating evidence suggests that lineage-specific transcriptional regulators control differentiation states during cancer evolution and underlie their distinct biological behaviors. In this study, by investigating the super-enhancer landscape of LUSC, we identified a previously undescribed "neural" subtype defined by Sox2 and a neural lineage factor Brn2, as well as the classical LUSC subtype defined by Sox2 and its classical squamous partner p63. Robust protein-protein interaction and genomic cooccupancy of Sox2 and Brn2, in place for p63 in the classical LUSC, indicated their transcriptional cooperation imparting this unique lineage state in the "neural" LUSC. Forced expression of p63 downregulated Brn2 in the "neural" LUSC cells and invoked the classical LUSC lineage with more squamous/epithelial features, which were accompanied by increased activities of ErbB/Akt and MAPK-ERK pathways, suggesting differential dependency. Collectively, our data demonstrate heterogeneous cell lineage states of LUSC featured by Sox2 cooperation with Brn2 or p63, for which distinct therapeutic approaches may be warranted. SIGNIFICANCE: Epigenomic profiling reveals a novel subtype of lung squamous cell carcinoma with neural differentiation.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/24/6084/F1.large.jpg.

Glutaminolysis-related genes determine sensitivity to xCT-targeted therapy in head and neck squamous cell carcinoma.

Targeting the function of membrane transporters in cancer stemlike cells is a potential new therapeutic approach. Cystine-glutamate antiporter xCT expressed in CD44 variant (CD44v)-expressing cancer cells contributes to the resistance to oxidative stress as well as cancer therapy through promoting glutathione (GSH)-mediated antioxidant defense. Amino acid transport by xCT might, thus, be a promising target for cancer treatment, whereas the determination factors for cancer cell sensitivity to xCT-targeted therapy remain unclear. Here, we demonstrate that high expression of xCT and glutamine transporter ASCT2 is correlated with undifferentiated status and diminished along with cell differentiation in head and neck squamous cell carcinoma (HNSCC). The cytotoxicity of the xCT inhibitor sulfasalazine relies on ASCT2-dependent glutamine uptake and glutamate dehydrogenase (GLUD)-mediated α-ketoglutarate (α-KG) production. Metabolome analysis revealed that sulfasalazine treatment triggers the increase of glutamate-derived tricarboxylic acid cycle intermediate α-KG, in addition to the decrease of cysteine and GSH content. Furthermore, ablation of GLUD markedly reduced the sulfasalazine cytotoxicity in CD44v-expressing stemlike HNSCC cells. Thus, xCT inhibition by sulfasalazine leads to the impairment of GSH synthesis and enhancement of mitochondrial metabolism, leading to reactive oxygen species (ROS) generation and, thereby, triggers oxidative damage. Our findings establish a rationale for the use of glutamine metabolism (glutaminolysis)-related genes, including ASCT2 and GLUD, as biomarkers to predict the efficacy of xCT-targeted therapy for heterogeneous HNSCC tumors.

Endothelial-Mesenchymal Transition Drives Expression of CD44 Variant and xCT in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) pathogenesis shares similarities with carcinogenesis. One CD44 variant (CD44v) isoform, CD44v8-10, binds to and stabilizes the cystine transporter subunit (xCT), producing reduced glutathione and thereby enhancing the antioxidant defense of cancer stem cells. Pharmacological inhibition of xCT by sulfasalazine suppresses tumor growth, survival, and resistance to chemotherapy. We investigated whether the CD44v-xCT axis contributes to PAH pathogenesis. CD44v was predominantly expressed on endothelial-to-mesenchymal transition (EndMT)-like cells in the neointimal layer of PAH affected pulmonary arterioles. In vitro, CD44 standard form and CD44v were induced as a result of EndMT. Among human pulmonary artery endothelial cells that have undergone EndMT, CD44v+ cells showed high levels of xCT expression on their cell surfaces and high concentrations of glutathione for survival. This made CD44v+ cells the most vulnerable target for sulfasalazine. CD44v+xCThi cells showed the highest expression levels of proinflammatory cytokines, antioxidant enzymes, antiapoptotic molecules, and cyclin-dependent kinase inhibitors. In the Sugen5416/hypoxia mouse model, CD44v+ cells were present in the thickened pulmonary vascular wall. The administration of sulfasalazine started either at the same time as "Sugen5416" administration (a prevention model) or after the development of pulmonary hypertension (a reversal model) attenuated the muscularization of the pulmonary vessels, decreased the expression of markers of inflammation, and reduced the right ventricular systolic pressure, while reducing CD44v+ cells. In conclusion, CD44v+xCThi cells appear during EndMT and in pulmonary hypertension tissues. Sulfasalazine is expected to be a novel therapeutic agent for PAH, most likely targeting EndMT-derived CD44v+xCThi cells.

Anti-tumor effects of mAb against L-type amino acid transporter 1 (LAT1) bound to human and monkey LAT1 with dual avidity modes.

L-Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti-LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody-dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side-effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney-derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti-CD98hc mAb, suggesting anti-LAT1 mAbs detect an epitope on LAT1-CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE-1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE-1, and investigated the roles of LYVE-1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)-fused mouse LYVE-1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP-fused mouse LYVE-1 proteins in a GFP expression-dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE-1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA-MB-231 xenograft models. This shows that LYVE-1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

Promotion of malignant phenotype after disruption of the three-dimensional structure of cultured spheroids from colorectal cancer.

Individual and small clusters of cancer cells may detach from the edges of a main tumor and invade vessels, which can act as the origin of metastasis; however, the mechanism for this phenomenon is not well understood. Using cancer tissue-originated spheroids, we studied whether disturbing the 3D architecture of cancer spheroids can provoke the reformation process and progression of malignancy. We developed a mechanical disruption method to achieve homogenous disruption of the spheroids while maintaining cell-cell contact. After the disruption, 9 spheroid lines from 9 patient samples reformed within a few hours, and 3 of the 9 lines exhibited accelerated spheroid growth. Marker expression, spheroid forming capacity, and tumorigenesis indicated that stemness increased after spheroid disruption. In addition, the spheroid forming capacity increased in 6 of 11 spheroid lines. The disruption signature determined by gene expression profiling supported the incidence of remodeling and predicted the prognosis of patients with colorectal cancer. Furthermore, WNT and HER3 signaling were increased in the reformed spheroids, and suppression of these signaling pathways attenuated the increased proliferation and stemness after the disruption. Overall, the disruption and subsequent reformation of cancer spheroids promoted malignancy-related phenotypes through the activation of the WNT and ERBB pathways.

Negative regulation of amino acid signaling by MAPK-regulated 4F2hc/Girdin complex.

Amino acid signaling mediated by the activation of mechanistic target of rapamycin complex 1 (mTORC1) is fundamental to cell growth and metabolism. However, how cells negatively regulate amino acid signaling remains largely unknown. Here, we show that interaction between 4F2 heavy chain (4F2hc), a subunit of multiple amino acid transporters, and the multifunctional hub protein girders of actin filaments (Girdin) down-regulates mTORC1 activity. 4F2hc interacts with Girdin in mitogen-activated protein kinase (MAPK)- and amino acid signaling-dependent manners to translocate to the lysosome. The resultant decrease in cell surface 4F2hc leads to lowered cytoplasmic glutamine (Gln) and leucine (Leu) content, which down-regulates amino acid signaling. Consistently, Girdin depletion augments amino acid-induced mTORC1 activation and inhibits amino acid deprivation-induced autophagy. These findings uncovered the mechanism underlying negative regulation of amino acid signaling, which may play a role in tightly regulated cell growth and metabolism.