Biology subjects

Semba, K.

Publications and source records attributed to Semba, K..

10 recordsLinked to original sources

bioRxiv2025

Preclinical Prediction of Resistance and Optimization of Sequential Therapy for ALK-positive Lung Cancer Using Next-generation ALK Inhibitors

BackgroundAnaplastic lymphoma kinase (ALK) gene rearrangements occur in approximately 5% of non-small cell lung cancers (NSCLCs). Although ALK tyrosine kinase inhibitors provide substantial clinical benefits, acquired resistance-conferring mutations frequently emerge, leading to disease progression. Preclinical prediction of these mutations might help guide the development of more effective sequential treatment strategies prior to clinical application. ObjectiveTo predict the emergence of resistance mutations to the investigational ALK inhibitors zotizalkib (TPX-0131), gilteritinib (ASP2215), and neladalkib (NVL-655) following resistance to first-line alectinib and assess the potential of these drugs as second-line therapies. MethodsA polymerase chain reaction (PCR)-based mutagenesis system was used to introduce random mutations into ALK cDNA harboring representative alectinib-resistant mutations. Mutant libraries were expressed in Ba/F3 cells, which were exposed to each inhibitor. Drug-resistant clones were isolated, sequenced, and evaluated for drug sensitivity using viability assays and immunoblotting. ResultsSeveral resistance mutations against zotizalkib, gilteritinib, and neladalkib were identified. Sequential use of these agents effectively suppressed all predicted resistance patterns with G1202R or I1171N. ConclusionsThis PCR-based platform provides a valuable approach for anticipating resistance mutations and guiding the design of optimized sequential therapies. Zotizalkib, gilteritinib, and neladalkib might represent promising alternatives to lorlatinib as second-line treatments for ALK-positive NSCLC. Key PointsO_LIPCR-based mutation prediction system was successfully applied to fourth-generation ALK inhibitors. C_LIO_LIshowed efficacy against G1202R-positive relapses with minimal evidence of secondary resistance mutations. C_LIO_LIcombinations of gilteritinib with either neladalkib or ensartinib may sustain efficacy and delay resistance in I1171N-positive relapses. C_LI

cancer biology↗

bioRxiv2023

Metastatic potentials classified with hypoxia-inducible factor 1 downstream gene in pan-cancer cell lines

Hypoxia-inducible factor 1 (HIF1) gene codes a transcription factor that is stabilized under hypoxia conditions via post-translational modifications. HIF1 regulates tumor malignancy and metastasis by gene transcriptions, such as Warburg effect- and angiogenesis-related genes, in cancer cells. However, the HIF1 downstream genes show varied expressional patterns in different cancer types. Herein, we performed the hierarchical clustering based on the HIF1 downstream gene expression patterns using 1,406 cancer cell lines crossing 30 types of cancer to understand the relationship between HIF1 downstream genes and the metastatic potential of cancer cell lines. Four types of cancer were classified by HIF1 downstream genes with significantly altered metastatic potentials. Furthermore, different HIF1 downstream gene subsets were extracted to discriminate each subtype for the four cancer types. HIF1 downstream subtyping classification will help understand the novel insight into tumor malignancy and metastasis in each cancer type. FundingThis work was supported by Project for JSPS KAKENHI (Grant-in-Aid for Scientific Research (C): JP23K06665 to JN, Grant-in-Aid for Early-Carrier Scientists: JP21K15562 to JN), and in part by translational research program from Fukushima Prefecture (KS). Competing Interests statementThe authors have declared that no conflict of interest exists.

cancer biology↗

bioRxiv2022

Targeting c-Jun is a potential therapy of luminal breast cancer bone metastasis

Luminal breast cancer has the highest bone metastasis frequency among all breast cancer subtypes, but its metastatic mechanism has not been elucidated because of the lack of appropriate metastatic cell lines. The study aim was to characterize high-osteolytic bone metastatic MCF7-BM cell lines and extract c-Jun, a novel bone metastasis marker. We found that c-Jun was upregulated in MCF7-BM cells, and its deficiency was associated with suppression of the cell migration, transformation, and stemness of BM cells. In vivo, c-Jun-deficient MCF7-TAM67 cells exhibited weaker bone metastatic ability. Additionally, c-Jun overexpression in MCF7-BM cells led to a tumor-migration promotion cycle in the bone microenvironment possibly by enhancing calcium-induced migration and releasing the osteoclast activator BMP5. Inhibition of c-Jun by JNK-IN-8, a JNK inhibitor, effectively reduced tumorigenesis activities and bone metastatic tumors. Our results indicate the potential benefits of a therapy that targets c-Jun to prevent or minimize luminal breast cancer bone metastasis.

cancer biology↗

bioRxiv2022

Prediction of Resistant Mutations against Upcoming ALK-TKIs, Repotrectinib (TPX-0005) and Ensartinib (X-396)

ALK gene rearrangement is observed in approximately 4% of patients with non-small cell lung cancer. These individuals benefit clinically from a range of approved ALK-TKIs; however, using many ALK-TKIs in a row will always result in resistant compound mutations in the kinase domain. Therefore, next-generation ALK-TKIs which are potent to these resistant mutations are still under development. In this context, preclinical prediction of resistant mutations generated by developing ALK-TKIs will provide useful information about the effective sequential treatment of ALK-TKIs. In this study, we developed a simple error-prone PCR-based mutation prediction system, and as a model study, we predicted resistant mutations against upcoming ALK-TKI, repotrectinib, and ensartinib. According to the predictive mutation patterns, repotrectinib and ensartinib may be used as second-line therapeutic options following the first-line alectinib treatment.

cancer biology↗

bioRxiv2021

Metalloproteinase-dependent and TMPRSS2-independnt cell surface entry pathway of SARS-CoV-2 requires the furin-cleavage site and the S2 domain of spike protein

The ongoing global vaccination program to prevent SARS-CoV-2 infection, the causative agent of COVID-19, has had significant success. However, recently virus variants have emerged that can evade the immunity in a host achieved through vaccination. Consequently, new therapeutic agents that can efficiently prevent infection from these new variants, and hence COVID-19 spread are urgently required. To achieve this, extensive characterization of virus-host cell interactions to identify effective therapeutic targets is warranted. Here, we report a cell surface entry pathway of SARS-CoV-2 that exists in a cell type-dependent manner is TMPRSS2-independent but sensitive to various broad-spectrum metalloproteinase inhibitors such as marimastat and prinomastat. Experiments with selective metalloproteinase inhibitors and gene-specific siRNAs revealed that a disintegrin and metalloproteinase 10 (ADAM10) is partially involved in the metalloproteinase pathway. Consistent with our finding that the pathway is unique to SARS-CoV-2 among highly pathogenic human coronaviruses, both the furin cleavage motif in the S1/S2 boundary and the S2 domain of SARS-CoV-2 spike protein are essential for metalloproteinase-dependent entry. In contrast, the two elements of SARS-CoV-2 independently contributed to TMPRSS2-dependent S2 priming. The metalloproteinase pathway is involved in SARS-CoV-2-induced syncytia formation and cytopathicity, leading us to theorize that it is also involved in the rapid spread of SARS-CoV-2 and the pathogenesis of COVID-19. Thus, targeting the metalloproteinase pathway in addition to the TMPRSS2 and endosome pathways could be an effective strategy by which to cure COVID-19 in the future. Author SummaryTo develop effective therapeutics against COVID-19, it is necessary to elucidate in detail the infection mechanism of the causative agent, SARS-CoV-2, including recently emerging variants. SARS-CoV-2 binds to the cell surface receptor ACE2 via the Spike protein, and then the Spike protein is cleaved by host proteases to enable entry. Selection of target cells by expression of these tissue-specific proteases contributes to pathogenesis. Here, we found that the metalloproteinase-mediated pathway is important for SARS-CoV-2 infection, variants included. This pathway requires both the prior cleavage of Spike into two domains and a specific sequence in the second domain S2, conditions met by SARS-CoV-2 but lacking in the related human coronavirus SARS-CoV. The contribution of several proteases, including metalloproteinases, to SARS-CoV-2 infection was cell type dependent, especially in cells derived from kidney, ovary, and endometrium, in which SARS-CoV-2 infection was metalloproteinase-dependent. In these cells, inhibition of metalloproteinases by treatment with marimastat or prinomastat, whose safety was previously confirmed in clinical trials, was important in preventing cell death. Our study provides new insights into the complex pathogenesis unique to COVID-19 and relevant to the development of effective therapies.

microbiology↗

bioRxiv2021

Spatial transcriptomics reveals the two cancer stem cell-like populations in triple-negative breast cancer

Gene expression analysis at the single-cell level by next generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell-cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-seq (scRNA-seq) datasets confirmed that the two CSC-like populations existed both in TNBC xenograft models and TNBC patients. In addition, the gene signature of the HMGA1-high CSC-like cluster has the potential to serve as a novel biomarker for diagnosis. The diversity of these multiple CSC-like populations may cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

cancer biology↗

bioRxiv2021

Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer (TNBC) cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections (CAI) using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain proliferation ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study. Statements and DeclarationsO_ST_ABSFundingC_ST_ABSThis study was supported by JSPS KAKENHI (grant no. 18K16269: Grant-in-Aid for Early-Career Scientist to J.N.; grant no. 20J01794, Grant-in-Aid for JSPS fellows to J.N.; grant no. 20J23297, Grant-in-Aid for JSPS fellows to Y.H.) and partially supported by the grants for translational research programs from Fukushima Prefecture (S.W. and K.S.). AuthorshipYH and KA performed the in vivo experiments and bioinformatical analyses. SW, and KS interpreted the data. YH, KA, and JN wrote the manuscript. JN conceived and designed the study. All the authors reviewed and edited the manuscript. Competing InterestsThe authors declare that they have no competing interests. Ethical approvalThe animal experiments were conducted under the approval of the ethics committee of Waseda University (2020-A067, 2021-A074).

cancer biology↗

bioRxiv2021

Aberrant accumulation of NIK promotes tumorigenicity by dysregulating post-translational modifications in breast cancer

Post-translational modifications and mRNA translation are frequently altered in human cancers. However, investigations to understand their roles in the cancer progression mechanism remain insufficient. In this research, we explored protein levels altered by translational or post-translational regulation by analyzing transcriptome and western blotting data of the highly malignant breast cancer cell lines. From these analyses, NIK was found to be upregulated at the protein level to predominantly activate the non-canonical NF-{kappa}B pathway in a breast cancer cell line. Furthermore, the increase in NIK protein production was attributed to the dysregulation of ubiquitin-proteasome system caused by a decrease in the translation of cIAP1. NIK upregulation contributed to tumorigenicity by regulating the expression of inflammatory response-related genes. Collectively, our study suggests that NIK is post-translationally modified and has the potential to be a therapeutic target and diagnostic marker for breast cancer.

cancer biology↗

bioRxiv2021

HOXB7 induces oncogenic transformation in NMuMG cells via JAK2-STAT3 signaling

The homeobox family genes are often dysregulated in a various cancer type. Particularly HOXB7 amplification and overexpression correlate with poor prognosis in various cancer such as gastric, pancreatic, and lung cancers. Moreover, HOXB7 is known to contribute to cancer progression by promoting epithelial to mesenchymal transition, anti-cancer drug resistance, and angiogenesis. In this study, we show that HOXB7 is coamplified with ERBB2 in a subset of breast cancer patients and HOXB7 expression correlates with poor prognosis in HER2-positive breast cancer patients. This clinical observation is supported by the following results: HOXB7 overexpression in an immortalized murine mammary gland epithelial cell line NMuMG induces cellular transformation in vitro, tumorigenesis and lung metastasis through the activation of JAK-STAT signaling.

cancer biology↗

bioRxiv2020

The anticoagulant nafamostat potently inhibits SARS-CoV-2 infection in vitro: an existing drug with multiple possible therapeutic effects

Although infection by SARS-CoV-2, the causative agent of COVID-19, is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked MERS-CoV S protein-initiated cell fusion by targeting TMPRSS2, and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on SARS-CoV-2 S protein, ACE2 and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an EC50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. These findings, together with accumulated clinical data regarding its safety, make nafamostat a likely candidate drug to treat COVID-19.

microbiology↗