Spatial and single-cell colocalisation analysis reveals MDK-mediated immunosuppressive environment with regulatory T cells in colorectal carcinogenesis.

BACKGROUND: Cell-cell interaction factors that facilitate the progression of adenoma to sporadic colorectal cancer (CRC) remain unclear, thereby hindering patient survival. METHODS: We performed spatial transcriptomics on five early CRC cases, which included adenoma and carcinoma, and one advanced CRC. To elucidate cell-cell interactions within the tumour microenvironment (TME), we investigated the colocalisation network at single-cell resolution using a deep generative model for colocalisation analysis, combined with a single-cell transcriptome, and assessed the clinical significance in CRC patients. FINDINGS: CRC cells colocalised with regulatory T cells (Tregs) at the adenoma-carcinoma interface. At early-stage carcinogenesis, cell-cell interaction inference between colocalised adenoma and cancer epithelial cells and Tregs based on the spatial distribution of single cells highlighted midkine (MDK) as a prominent signalling molecule sent from tumour epithelial cells to Tregs. Interaction between MDK-high CRC cells and SPP1+ macrophages and stromal cells proved to be the mechanism underlying immunosuppression in the TME. Additionally, we identified syndecan4 (SDC4) as a receptor for MDK associated with Treg colocalisation. Finally, clinical analysis using CRC datasets indicated that increased MDK/SDC4 levels correlated with poor overall survival in CRC patients. INTERPRETATION: MDK is involved in the immune tolerance shown by Tregs to tumour growth. MDK-mediated formation of the TME could be a potential target for early diagnosis and treatment of CRC. FUNDING: Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Science Research; OITA Cancer Research Foundation; AMED under Grant Number; Japan Science and Technology Agency (JST); Takeda Science Foundation; The Princess Takamatsu Cancer Research Fund.

Metastases and treatment-resistant lineages in patient-derived cancer cells of colorectal cancer.

Circulating tumor cells (CTCs) play an important role in metastasis and recurrence. However, which cells comprise the complex tumor lineages in recurrence and are key in metastasis are unknown in colorectal cancer (CRC). CRC with high expression of POU5F1 has a poor prognosis with a high incidence of liver metastatic recurrence. We aim to reveal the key cells promoting metastasis and identify treatment-resistant lineages with established EGFP-expressing organoids in two-dimensional culture (2DOs) under the POU5F1 promotor. POU5F1-expressing cells are highly present in relapsed clinical patients' blood as CTCs. Sorted POU5F1-expressing cells from 2DOs have cancer stem cell abilities and abundantly form liver metastases in vivo. Single-cell RNA sequencing of 2DOs identifies heterogeneous populations derived from POU5F1-expressing cells and the Wnt signaling pathway is enriched in POU5F1-expressing cells. Characteristic high expression of CTLA4 is observed in POU5F1-expressing cells and immunocytochemistry confirms the co-expression of POU5F1 and CTLA4. Demethylation in some CpG islands at the transcriptional start sites of POU5F1 and CTLA4 is observed. The Wnt/ß-catenin pathway inhibitor, XAV939, prevents the adhesion and survival of POU5F1-expressing cells in vitro. Early administration of XAV939 also completely inhibits liver metastasis induced by POU5F1-positive cells.

Wnt activation disturbs cell competition and causes diffuse invasion of transformed cells through NF-κB-MMP21 pathway.

Normal epithelial cells exert their competitive advantage over RasV12-transformed cells and eliminate them into the apical lumen via cell competition. However, the internal or external factors that compromise cell competition and provoke carcinogenesis remain elusive. In this study, we examine the effect of sequential accumulation of gene mutations, mimicking multi-sequential carcinogenesis on RasV12-induced cell competition in intestinal epithelial tissues. Consequently, we find that the directionality of RasV12-cell extrusion in Wnt-activated epithelia is reversed, and transformed cells are delaminated into the basal lamina via non-cell autonomous MMP21 upregulation. Subsequently, diffusively infiltrating, transformed cells develop into highly invasive carcinomas. The elevated production of MMP21 is elicited partly through NF-κB signaling, blockage of which restores apical elimination of RasV12 cells. We further demonstrate that the NF-κB-MMP21 axis is significantly bolstered in early colorectal carcinoma in humans. Collectively, this study shows that cells with high mutational burdens exploit cell competition for their benefit by behaving as unfit cells, endowing them with an invasion advantage.

Topological data analysis of protein structure and inter/intra-molecular interaction changes attributable to amino acid mutations.

The presence of some amino acid mutations in the amino acid sequence that determines a protein's structure can significantly affect that 3D structure and its biological function. However, the effects upon structural and functional changes differ for each displaced amino acid, and it is very difficult to predict these changes in advance. Although computer simulations are very effective at predicting conformational changes, they struggle to determine whether the amino acid mutation of interest induces sufficient conformational changes, unless the researcher is a specialist in molecular structure calculations. Therefore, we created a framework that efficiently utilizes molecular dynamics and persistent homology methods to identify amino acid mutations that induce structural changes. We show that this framework can be used not only to predict conformational changes produced by amino acid mutations but also to extract groups of mutations that significantly alter similar molecular interactions, by capturing the resultant protein-protein interaction changes.

Increased flexibility of the SARS-CoV-2 RNA-binding site causes resistance to remdesivir.

Mutations continue to accumulate within the SARS-CoV-2 genome, and the ongoing epidemic has shown no signs of ending. It is critical to predict problematic mutations that may arise in clinical environments and assess their properties in advance to quickly implement countermeasures against future variant infections. In this study, we identified mutations resistant to remdesivir, which is widely administered to SARS-CoV-2-infected patients, and discuss the cause of resistance. First, we simultaneously constructed eight recombinant viruses carrying the mutations detected in in vitro serial passages of SARS-CoV-2 in the presence of remdesivir. We confirmed that all the mutant viruses didn't gain the virus production efficiency without remdesivir treatment. Time course analyses of cellular virus infections showed significantly higher infectious titers and infection rates in mutant viruses than wild type virus under treatment with remdesivir. Next, we developed a mathematical model in consideration of the changing dynamic of cells infected with mutant viruses with distinct propagation properties and defined that mutations detected in in vitro passages canceled the antiviral activities of remdesivir without raising virus production capacity. Finally, molecular dynamics simulations of the NSP12 protein of SARS-CoV-2 revealed that the molecular vibration around the RNA-binding site was increased by the introduction of mutations on NSP12. Taken together, we identified multiple mutations that affected the flexibility of the RNA binding site and decreased the antiviral activity of remdesivir. Our new insights will contribute to developing further antiviral measures against SARS-CoV-2 infection.

Spatial and single-cell transcriptomics decipher the cellular environment containing HLA-G+ cancer cells and SPP1+ macrophages in colorectal cancer.

The cellular interactions in the tumor microenvironment of colorectal cancer (CRC) are poorly understood, hindering patient treatment. In the current study, we investigate whether events occurring at the invasion front are of particular importance for CRC treatment strategies. To this end, we analyze CRC tissues by combining spatial transcriptomics from patients with a public single-cell transcriptomic atlas to determine cell-cell interactions at the invasion front. We show that CRC cells are localized specifically at the invasion front. These cells induce human leukocyte antigen G (HLA-G) to produce secreted phosphoprotein 1 (SPP1)+ macrophages while conferring CRC cells with anti-tumor immunity, as well as proliferative and invasive properties. Taken together, these findings highlight the signaling between CRC cell populations and stromal cell populations at the cellular level.

Bayesian statistical method for detecting structural and topological diversity in polymorphic proteins.

Polymorphisms in immune-related proteins and viral spike proteins are high and complicate host-virus interactions. Therefore, diversity analysis of such protein structures is essential to understand the mechanism of the immune system. However, experimental methods, including X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy, have several problems: (i) they are conducted under different conditions from the actual cellular environment, (ii) they are laborious, time-consuming, and expensive, and (iii) they do not provide information on the thermodynamic behaviors. In this paper, we propose a computational method to solve these problems by using MD simulations, persistent homology, and a Bayesian statistical model. We apply our method to eight types of HLA-DR complexes to evaluate the structural diversity. The results show that our method can correctly discriminate the intrinsic structural variations caused by amino acid mutations from the random fluctuations caused by thermal vibrations. In the end, we discuss the applicability of our method in combination with existing deep learning-based methods for protein structure analysis.

UVB-Induced Skin Autoinflammation Due to Nlrp1b Mutation and Its Inhibition by Anti-IL-1ß Antibody.

NLRP1 (NACHT and leucine-rich repeat-containing protein family, pyrin domain-containing protein 1) is an innate immune sensor that is involved in the formation of inflammasome complexes. NLRP1 hyperactivity has been reported to cause inherited autoinflammatory diseases including familial keratosis lichenoides chronica and NLRP1-associated autoinflammation with arthritis and dyskeratosis. We generated Nlrp1b (the mouse homologue of human NLRP1) gain-of-function knock-in (Nlrp1b KI) mice with UVB irradiation-induced autoinflammatory skin lesions. We demonstrated that UVB irradiation induces IL-1ß upregulation and IL-1ß-dependent inflammation via caspase-1 activation in these Nlrp1b KI mice. RNA sequencing revealed the upregulation of inflammasome pathway-related genes, keratinocyte stress marker genes, and keratinocyte differentiation marker genes in the Nlrp1b KI mice after UVB irradiation. The skin inflammation and hyperkeratosis from UVB irradiation in the Nlrp1b KI mice were inhibited by both intraperitoneal and subcutaneous administration of anti-IL-1ß antibodies before UVB irradiation. UVB irradiation and the IL-1ß pathway are important in the pathogenesis of NLRP1-associated autoinflammatory skin lesions.

Impact of sarcopenia in patients with advanced or recurrent colorectal cancer treated with regorafenib.

BACKGROUND: Regorafenib is a key agent for patients with advanced or recurrent colorectal cancer. Sarcopenia represented by skeletal muscle depletion is closely related to frailty and predicts oncological prognoses. We hypothesized that sarcopenia negatively affects the time to treatment failure (TTF) or overall survival (OS) of patients treated with regorafenib. METHODS: We retrospectively reviewed the medical records of all patients treated with regorafenib between May 2013 and April 2019 at our institution. The cross-sectional area of the psoas muscle at the level of the third lumbar vertebra on baseline computed tomography (CT) was assessed to calculate the psoas muscle index (PMI). Sarcopenia was defined based on PMI cut-off values for Asian adults (6.36 cm2/m2 for males and 3.92 cm2/m2 for females). RESULTS: Thirty-four patients were analyzed. The prevalence of sarcopenia was 44.1%. Sarcopenia was significantly associated with poorer OS (median 3.2 vs. 5.3 months, p = 0.031). Less 75% 1-Month Relative Dose Intensity patients experienced significantly shorter TTF and OS than the rest, as did patients receiving total regorafenib dose of < 3360 mg (median 3.1 and 9.4 months, p < 0.001). Multivariate analysis showed that sarcopenia was a significant predictor of prognosis. CONCLUSION: Sarcopenia was a predictive marker of negative outcome for patients with advanced or recurrent colorectal cancer treated with regorafenib. Screening for sarcopenia can be used to identify patients more likely to benefit from regorafenib in routine clinical practice.

Theoretical analyses and experimental validation of the effects caused by the fluorinated substituent modification of DNA.

Halogen-modified nucleic acid molecules, such as trifluorothymidine (FTD) and 5-fluorouracil, are widely used in medical science and clinical site. These compounds have a very similar nucleobase structure. It is reported that both of these compounds could be incorporated into DNA. The incorporation of FTD produces highly anti-tumor effect. However, it is not known whether to occur a significant effect by the incorporation of 5-fluorouracil. Nobody knows why such a difference will occur. To understand the reason why there is large differences between trifluorothymidine and 5-fluorouracil, we have performed the molecular dynamics simulations and molecular orbital calculations. Although the active interaction energy between Halogen-modified nucleic acids or and complementary adenine was increased, in only FTD incorporated DNA, more strongly dispersion force interactions with an adjacent base were detected in many thermodynamic DNA conformations. As the results, the conformational changes occur even if it is in internal body temperature. Then the break of hydrogen bonding between FTD and complementary adenine base occur more frequently. The double helix structural destabilization of DNA with FTD is resulted from autoagglutination caused by the bonding via halogen orbitals such as halogen bonding and the general van der Waals interactions such as CH-[Formula: see text], lone pair (LP)-[Formula: see text], and [Formula: see text]-[Formula: see text] interactions. Therefore, it is strongly speculated that such structural changes caused by trifluoromethyl group is important for the anti-tumor effect of FTD alone.

One-carbon metabolism for cancer diagnostic and therapeutic approaches.

Altered metabolism is critical for the rapid and unregulated proliferation of cancer cells; hence the requirement for an abundant source of nucleotides. One characteristic of this metabolic reprogramming is in one-carbon (1C) metabolism, which is particularly noteworthy for its role in DNA synthesis. Various forms of methylation are also noteworthy as they relate to cancer cell survival and proliferation. In recent years, 1C metabolism has received substantial attention for its role in cancer malignancy via these functions. Therefore, therapeutic inhibitors targeting 1C metabolism have been utilized as anticancer drugs. This review outlines the importance of 1C metabolism and its clinical application in cancer. Understanding 1C metabolism could aid the development of novel cancer diagnostic and therapeutic methods.

CD44/CD133-Positive Colorectal Cancer Stem Cells are Sensitive to Trifluridine Exposure.

Cancer stem cells (CSCs) are involved in metastatic colorectal cancer recurrence, but no effective therapy targeting these cells is currently available. Because trifluridine (FTD)/tipiracil therapy is used for refractory colorectal cancer, we sought to determine whether FTD is effective against CSC-like cells. CD44+CD133+ high-expressing and other populations of human DLD-1 colon cancer cells were separately isolated through fluorescence-activated cell sorting. The sphere-forming activity of each population and the anti-sphere-forming effects of FTD and fluorouracil (5-FU) on CD44+CD133+ cells were then measured. CD44+CD133+ DLD-1 cells formed substantially more spheres than other cells. Moreover, treating CD44+CD133+ DLD-1 cells with subtoxic concentrations of FTD (1 µM) inhibited sphere formation, and this was superior to the effect of subtoxic concentrations (1 µM) of 5-FU. The associated inhibition rates for FTD and 5-FU were 58.2% and 26.1%, respectively. Further, CD44+CD133+ DLD-1 cells expressed higher levels of thymidine kinase 1, which is responsible for FTD phosphorylation, than DLD-1 cells, and FTD was incorporated into the DNA of CD44+CD133+ DLD-1 cells. Thus, our data show that FTD treatment is effective against CSC-like cells and might be applied as CSC-targeting chemotherapy for tumor subtypes with high CD44 and CD133 expression.

Distinct methylation levels of mature microRNAs in gastrointestinal cancers.

The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology.

A Hydrogen Peroxide Activatable Gemcitabine Prodrug for the Selective Treatment of Pancreatic Ductal Adenocarcinoma.

The main concern in the use of anticancer chemotherapeutic drugs is host toxicity. Patients need to interrupt or change chemotherapy due to adverse effects. In this study, we aimed to decrease adverse events with gemcitabine (GEM) in the treatment of pancreatic ductal adenocarcinoma and focused on the difference of hydrogen peroxide levels in normal versus cancer cells. We designed and synthesized a novel boronate-ester-caged prodrug that is activated by the high H2 O2 concentrations found in cancer cells to release GEM. An H2 O2 -activatable GEM (A-GEM) has higher selectivity for H2 O2 over other reactive oxygen species (ROS) and cytotoxic effects corresponding to the H2 O2 concentration in vitro. A xenograft model of immunodeficient mice indicated that the effect of A-GEM was not inferior to that of GEM when administered in vivo. In particular, myelosuppression was significantly decreased following A-GEM treatment compared with that following GEM treatment.

Direct Analysis of Incorporation of an Anticancer Drug into DNA at Single-Molecule Resolution.

Identifying positions at which anticancer drug molecules incorporate into DNA is essential to define mechanisms underlying their activity, but current methodologies cannot yet achieve this. The thymidine fluorine substitution product trifluridine (FTD) is a DNA-damaging anticancer agent thought to incorporate into thymine positions in DNA. This mechanism, however, has not been directly confirmed. Here, we report a means to detect FTD in a single-stranded oligonucleotide using a method to distinguish single molecules by differences in electrical conductance. Entire sequences of 21-base single-stranded DNAs with and without incorporated drug were determined based on single-molecule conductances of the drug and four deoxynucleosides, the first direct observation of its kind. This methodology may foster rapid development of more effective anticancer drugs.

Mitochondrial pyruvate carrier 1 expression controls cancer epithelial-mesenchymal transition and radioresistance.

Mitochondrial pyruvate carrier (MPC) is known to cause different expressions in normal and cancer cells. We observed a change in phenotype with the suppression of MPC expression. We knocked down MPC1 and/or MPC2 using siRNA or shRNA. We observed its cell morphology and accompanying molecular marker. Furthermore, the radioresistance of the MPC knockdown cell line was examined using a colony formation assay. MPC1-suppressed cells changed their morphology to a spindle shape. Epithelial-mesenchymal transition (EMT) was suspected, and examination of the EMT marker by PCR showed a decrease in E-cadherin and an increase in fibronectin. Focusing on glutamine metabolism as the mechanism of this phenomenon, we knocked down the glutamine-metabolizing enzyme glutaminase (GLS). EMT was also observed in GLS-suppressed cells. Furthermore, when MPC1-suppressed cells were cultured in a glutamine-deficient medium, changes in EMT markers were suppressed. In addition, MPC1-suppressed cells also increased with a significant difference in radioresistance. Decreased MPC1 expression favorably affects EMT and radioresistance of cancer.

Disruption of Endolysosomal RAB5/7 Efficiently Eliminates Colorectal Cancer Stem Cells.

Given that cancer stem cells (CSC) play a key role in drug resistance and relapse, targeting CSCs remains promising in cancer therapy. Here we show that RAB5/7, which are involved in the endolysosomal pathway, play key roles in the maintenance of CSC survival via regulation of the mitophagic pathway. Inhibition of RAB5/7 efficiently eliminated colorectal CSCs and disrupted cancer foci. In addition, we identified mefloquine hydrochloride, an antimalarial drug, as a novel RAB5/7 inhibitor and promising colorectal CSC-targeting drug. Endolysosomal RAB5/7 and LAMP1/2 mediated parkin-dependent mitochondrial clearance and modulated mitophagy through lysosomal dynamics. In a patient-derived xenograft (PDX) model of colon cancer, treatment with mefloquine resulted in suppression of mitophagic PINK1/PARKIN and increased mitochondrial disorder and mitochondria-induced apoptosis without apparent side effects. These results suggest that the combination of mefloquine with chemotherapeutic agents in the PDX model potentially disrupts the hierarchy of colorectal cancer cells and identify endolysosomal RAB5/7 and LAMP1/2 as promising therapeutic targets in CSCs. SIGNIFICANCE: These findings show that endosomal/lysosomal RAB5 and RAB7, which regulate mitophagy, are essential for the survival of colon cancer stem cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1426/F1.large.jpg.

Poly(ethylene glycol)-poly(lysine) block copolymer-ubenimex conjugate targets aminopeptidase N and exerts an antitumor effect in hepatocellular carcinoma stem cells.

Previous studies highlighted that aminopeptidase N (APN)/CD13 acts as a scavenger in the survival of hepatocellular carcinoma (HCC) stem cells by reducing reactive oxygen species (ROS) levels. Hence, it has been proposed that APN/CD13 inhibition can increase cellular ROS levels and sensitize cells to chemotherapeutic agents. Although ubenimex, also known as bestatin, competitively inhibits proteases such as APN/CD13 on the cellular membrane and it is clinically used for patients with acute myeloid leukemia and lymphedema, research has demonstrated that higher concentrations of the agent induce the death of APN/CD13+ HCC stem cells. In this study, we developed a poly(ethylene glycol)-poly(lysine) block copolymer-ubenimex conjugate (PEG-b-PLys(Ube)) to increase the efficacy of reagents in APN/CD13+ cancer stem cells. Exposure to PEG-b-PLys(Ube) increased the intracellular ROS concentration by inhibiting APN enzyme activity, permitting the induction of apoptosis and attenuation of HCC cell proliferation. In addition, PEG-b-PLys(Ube) exhibited a relatively stronger antitumor effect in mice than PEG-b-PLys alone or phosphate-buffered saline. Moreover, an isobologram analysis revealed that combinations of fluorouracil, cisplatin, or doxorubicin with PEG-b-PLys(Ube) exhibited synergistic effects. This study demonstrated that PEG-b-PLys(Ube) does not impair the properties of ubenimex and exerts a potent antitumor effect.

Computational analyses for cancer biology based on exhaustive experimental backgrounds.

Antitumor drug therapy plays a very important role in cancer treatment. However, resistance to chemotherapy is a serious issue. Many studies have been conducted to understand and verify the cause of chemoresistance from multiple points of view such as oncogenes, tumor suppressor genes, DNA mutations and repairs, autophagy, cancer stemness, and mitochondrial metabolism and alteration. Nowadays, not only medical data from hospitals but also public big data exist on internet websites. Consequently, the importance of computational science has vastly increased in biological and medical sciences. Using statistical or mathematical analyses of these medical data with conventional experiments, many researchers have recently shown that there is a strong relationship between the biological metabolism and chemoresistance for cancer therapy. For example, folate metabolism that mediates one-carbon metabolism and polyamine metabolism have garnered attention regarding their association with cancer. It has been suggested that these metabolisms may be involved in causing resistance to chemotherapy.

Drug discovery of anticancer drugs targeting methylenetetrahydrofolate dehydrogenase 2.

Many anticancer drugs have serious adverse effects; therefore, it is necessary to target features specific to cancer cells to minimize the effects on healthy cells. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) was reported to be specifically enhanced in cancer. We confirmed the validity of MTHFD2 as a drug discovery target using clinical data. In addition, we performed in silico screening to design an anticancer drug specifically targeting MTHFD2. Analysis of the clinical data indicated that MTHFD2 was enhanced in most cancers compared with normal tissues, and affected the prognosis in cancer patients. Candidate compounds for MTHFD2 inhibitors were identified using in silico drug discovery techniques, and the important interactions for MTHFD2 binding were determined. In addition, these candidate compounds decreased levels of MTHFD2 metabolites in cancer cells. The findings of the present study may help to develop anticancer drugs targeting MTHFD2, with a view to minimizing the adverse effects of anticancer drugs.