Targeting PAK1 is effective against cutaneous squamous cell carcinoma in a syngenic mouse model.

By taking advantage of forward genetic analysis in mice, we have demonstrated that Pak1 plays a crucial role during DMBA/TPA skin carcinogenesis. Although Pak1 has been considered to promote cancer development, its overall function remains poorly understood. To clarify the functional significance of Pak1 in detail, we sought to evaluate the possible effect of an allosteric inhibitor against PAK1 (NVS-PAK1-1) on a syngeneic mouse model. To this end, we established two cell lines, 9AS1 and 19AS1, derived from DMBA/TPA-induced squamous cell carcinoma (SCC) that engrafted in FVB mice. Based on our present results, NVS-PAK1-1 treatment significantly inhibited the growth of tumors derived from 9AS1 and 19AS1 cells in vitro and in vivo. RNA-sequencing analysis on the engrafted tumors indicates that NVS-PAK1-1 markedly potentiates the epidermal cell differentiation and enhances the immune response in the engrafted tumors. Consistent with these observations, we found an expansion of Pan-keratin-positive regions and potentially elevated infiltration of CD8-positive immune cells in NVS-PAK1-1-treated tumors as examined by immunohistochemical analyses. Together, our present findings strongly suggest that PAK1 is tightly linked to the development of SCC, and that its inhibition is a promising therapeutic strategy against SCC.

Investigation of Prescription Status and Exploration of Risk Factors Related to Denosumab-Induced Hypocalcemia in Combination Therapy with 1α,25-Dihydroxy-vitamin D3.

To prevent denosumab-induced hypocalcemia in patients with renal dysfunction, combination therapy with 1α,25-dihydroxy-vitamin D3 (active vitamin D) is recommended. We previously developed a risk prediction model for hypocalcemia in patients with cholecalciferol/calcium (natural vitamin D). However, the prescription status and the risk factors of patients with active vitamin D have not been identified, so we designed this retrospective observational study using a large practice database covering June 2013 to May 2020 to analyze prescription status and risk factors. Patients were classified according to vitamin D type. After that, factors associated with development of hypocalcemia in patients with active vitamin D were explored. Univariate analysis was conducted to compare patient backgrounds between the hypocalcemia and non-hypocalcemia groups. Receiver operating characteristic analysis was conducted to evaluate the predictive potential of the extracted factors. Of the 33442 patients who received denosumab, 22347 and 3560 patients were co-administered natural and active vitamin D, respectively. Patients with active vitamin D had significantly lower renal function (estimated glomerular filtration rate (eGFR) median: 74.0 vs. 69.7 mL/min/1.73 m2), but some patients (23.6%) with sufficient renal function (eGFR ≥90) were also receiving active vitamin D. Of the 3560 patients with active vitamin D, non-hypocalcemia (n = 166) and hypocalcemia (n = 17) groups who met the study criteria were analyzed. Renal function was lower in the hypocalcemia group, and alkaline phosphatase gave the best discrimination. High aspartate aminotransferase (AST), renal dysfunction, high alkaline phosphatase (ALP), and low hemoglobin may be significant factors in risk prediction for hypocalcemia in patients with active vitamin D.

Meis1 plays roles in cortical development through regulation of cellular proliferative capacity in the embryonic cerebrum.

Meis1 (myeloid ecotropic insertion site 1) is known to be related to embryonic development and cancer. In this study, to analyze the function of Meis1 in neural stem cells, we crossed Meis1fl/fl (Meis1 floxed) mice with Nestin-Cre mice. The results showed that Meis1-conditional knockout mice showed cerebral cortex malformation. The mice had a significantly thinner cortex than wildtype mice. At E14.5, BrdU incorporation and Pax6-positive radial glial cells were significantly decreased in the cerebral cortex of Meis1 knockout embryos as compared with wild-type embryos, whereas Tbr2-positive intermediate progenitors and NeuN-positive differentiated neurons were not. Cell death detected by immunostaining with cleaved caspase3 antibody showed no difference in the cortex between knockout and wild-type embryos. Furthermore, knockout of Meis1 in embryo by in utero electroporation showed that cellular migration was disturbed during cortical development. Therefore, Meis1 could play important roles during cortical development through the regulation of cell proliferation and migration in the embryonic cerebral cortex.

Functional Polymorphism in Pak1-3' Untranslated Region Alters Skin Tumor Susceptibility by Alternative Polyadenylation.

We identified a functional SNP in the 3' untranslated region of Pak1 that is responsible for the skin tumor modifier of MSM 1a locus. Candidate SNPs in the 3' untranslated region of Pak1 from resistance strain MSM/Ms were introduced into susceptible strain FVB/N using CRISPR/Cas9. The 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate skin carcinogenesis experiments revealed an SNP (Pak1-3' untranslated region-6C>T: rs31627325) that strongly suppressed skin tumors. Furthermore, MBNL1 bound more strongly to FVB-allele (6C/C) and regulated the transcript length in the 3' untranslated region of Pak1 and tumorigenesis through polyadenylation. Therefore, the alternative polyadenylation of Pak1 is cis-regulated by rs31627325.

PHLDA3 Is an Important Downstream Mediator of p53 in Squamous Cell Carcinogenesis.

Squamous cell carcinomas (SCCs) are one of the most frequent solid cancer types in humans and are derived from stratified epithelial cells found in various organs. SCCs derived from various organs share common important properties, including genomic abnormalities in the tumor suppressor gene p53. There is a carcinogen-induced mouse model of SCC that produces benign papilloma, some of which progress to advanced carcinoma and metastatic SCCs. These SCCs undergo key genetic alterations that are conserved between humans and mice, including alterations in the genomic p53 sequence, and are therefore an ideal system to study the mechanisms of SCC tumorigenesis. Using this SCC model, we show that the PHLDA3 gene, a p53-target gene encoding a protein kinase B repressor, is involved in the suppression of benign and metastatic tumor development. Loss of PHLDA3 induces an epithelial‒mesenchymal transition and can complement p53 loss in the formation of metastatic tumors. We also show that in human patients with SCC, low PHLDA3 expression is associated with a poorer prognosis. Collectively, this study identifies PHLDA3 as an important downstream molecule of p53 involved in SCC development and progression.

The Japanese Wild-Derived Inbred Mouse Strain, MSM/Ms in Cancer Research.

MSM/Ms is a unique inbred mouse strain derived from the Japanese wild mouse, Mus musculus molossinus, which has been approximately 1 million years genetically distant from standard inbred mouse strains mainly derived from M. m. domesticus. Due to its genetic divergence, MSM/Ms has been broadly used in linkage studies. A bacterial artificial chromosome (BAC) library was constructed for the MSM/Ms genome, and sequence analysis of the MSM/Ms genome showed approximately 1% of nucleotides differed from those in the commonly used inbred mouse strain, C57BL/6J. Therefore, MSM/Ms mice are thought to be useful for functional genome studies. MSM/Ms mice show unique characteristics of phenotypes, including its smaller body size, resistance to high-fat-diet-induced diabetes, high locomotive activity, and resistance to age-onset hearing loss, inflammation, and tumorigenesis, which are distinct from those of common inbred mouse strains. Furthermore, ES (Embryonic Stem) cell lines established from MSM/Ms allow the MSM/Ms genome to be genetically manipulated. Therefore, genomic and phenotypic analyses of MSM/Ms reveal novel insights into gene functions that were previously not obtained from research on common laboratory strains. Tumorigenesis-related MSM/Ms-specific genetic traits have been intensively investigated in Japan. Furthermore, radiation-induced thymic lymphomas and chemically-induced skin tumors have been extensively examined using MSM/Ms.

A wild-derived inbred mouse strain, MSM/Ms, provides insights into novel skin tumor susceptibility genes.

Cancer is one of the most catastrophic human genetic diseases. Experimental animal cancer models are essential for gaining insights into the complex interactions of different cells and genes in tumor initiation, promotion, and progression. Mouse models have been extensively used to analyze the genetic basis of cancer susceptibility. They have led to the identification of multiple loci that confer, either alone or in specific combinations, an increased susceptibility to cancer, some of which have direct translatability to human cancer. Additionally, wild-derived inbred mouse strains are an advantageous reservoir of novel genetic polymorphisms of cancer susceptibility genes, because of the evolutionary divergence between wild and classical inbred strains. Here, we review mapped Stmm (skin tumor modifier of MSM) loci using a Japanese wild-derived inbred mouse strain, MSM/Ms, and describe recent advances in our knowledge of the genes responsible for Stmm loci in the 7,12-dimethylbenz(a)anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) two-stage skin carcinogenesis model.

CENP-50 is required for papilloma development in the two-stage skin carcinogenesis model.

CENP-50/U is a component of the CENP-O complex (CENP-O/P/Q/R/U) and localizes to the centromere throughout the cell cycle. Aberrant expression of CENP-50/U has been reported in many types of cancers. However, as Cenp-50/U-deficient mice die during early embryogenesis, its functions remain poorly understood in vivo. To investigate the role of Cenp-50/U in skin carcinogenesis, we generated Cenp-50/U conditional knockout (K14CreER -Cenp-50/Ufl/fl ) mice and subjected them to the 7,12-dimethylbenz(a)anthracene (DMBA)/terephthalic acid (TPA) chemical carcinogenesis protocol. As a result, early-stage papillomas decreased in Cenp-50/U-deficient mice. In contrast, Cenp-50/U-deficient mice demonstrated almost the same carcinoma incidence as control mice. Furthermore, mRNA expression analysis using DMBA/TPA-induced papillomas and carcinomas revealed that Cenp-50/U expression levels in papillomas were significantly higher than in carcinomas. These results suggest that Cenp-50/U functions mainly in early papilloma development and it has little effect on malignant conversion.

Pak1 maintains epidermal stem cells by regulating Langerhans cells and is required for skin carcinogenesis.

Pak1 (serine/threonine p21-activated kinases) was previously reported to have oncogenic activity in several cancers. However, its roles in the cancer microenvironment are poorly understood. We demonstrated that Pak1 expression in Langerhans cells (LCs) is essential for the maintenance of epidermal stem cells and skin tumor development. We found that PAK1 is localized in LCs by immunohistochemistry. Furthermore, the number of LCs significantly decreased in MSM/Ms Pak1 homozygous knockout mice (MSM/Ms-Pak1-/-). F1 hybrid (FVB/N×MSM/Ms) Pak1 heterozygous knockout mice (F1-Pak1+/-) had increased numbers of Th17 cells in the skin. Therefore, Pak1 knockdown cells were prepared using LC-derived XS52 cells (XS52-Pak1KD) and co-cultured with keratinocyte-derived C5N cells. As a result, XS52-Pak1KD cell supernatants promoted C5N cell proliferation. We then carried out DMBA/TPA skin carcinogenesis experiments using F1-Pak1+/- mice. Of note, F1-Pak1+/- mice exhibited stronger resistance to skin tumors than control mice. F1-Pak1+/- mice had fewer epidermal stem cells in the skin bulge. Our study suggested that Pak1 regulates the epidermal stem cell number by changing the properties of LCs and functions in skin carcinogenesis. We clarified a novel role of Pak1 in regulating LCs as a potential therapeutic target in skin immune disease and carcinogenesis.

First Japanese case of maternal phenylketonuria treated with sapropterin dihydrochloride and the normal growth and development of the child.

Sapropterin dihydrochloride (SD) may be a new treatment option for women with phenylketonuria (PKU) who plan to become pregnant. We report the first Japanese case of maternal PKU treated with SD. The patient was administered SD at 10-20 mg/kg/day, which increased phenylalanine tolerance during the pregnancy and lactation. No adverse events occurred, and she delivered a healthy neonate. Normal growth and development of the child confirms the efficacy and safety of SD.

Genetic analysis of Streptococcus equi subsp. equi isolated from horses imported into Japan.

Strangles is a commonly diagnosed and important infectious disease of equids worldwide, caused by Streptococcus equi subsp. equi. We determined the SeM genotypes of S. equi isolated from imported horses at the Japanese border within the past 8 years, which allowed us to classify 12 strains isolated from these horses from each exporter into four allelic groups. These alleles were different from the alleles of past isolates found in Japan. Furthermore, four strains classified into the same allele were isolated from horses from one exporter over several years. In this study, S. equi isolates from different exporters had different SeM alleles. Attention to the hygiene status of farms will be necessary to prevent the incursion of strangles.

A Polymorphic Variant in p19Arf Confers Resistance to Chemically Induced Skin Tumors by Activating the p53 Pathway.

Identification of the specific genetic variants responsible for the increased susceptibility to familial or sporadic cancers is important. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified a strong genetic locus, Stmm3, conferring resistance to chemically induced skin papillomas on chromosome 4. Here, we report the cyclin-dependent kinase inhibitor gene Cdkn2a/p19Arf as a major responsible gene for the Stmm3 locus. We provide evidence that the function of Stmm3 is dependent on p53 and that p19ArfMSM confers stronger resistance to papillomas than p16Ink4aMSMin vivo. In addition, we found that genetic polymorphism in p19Arf between a resistant strain, MSM/Ms (Val), and a susceptible strain, FVB/N (Leu), alters the susceptibility to papilloma development, malignant conversion, and the epithelial-mesenchymal transition. Moreover, we demonstrated that the p19ArfMSM allele more efficiently activates the p53 pathway than the p19ArfFVB allele in vitro and in vivo. Furthermore, we found polymorphisms in CDKN2A in the vicinity of a polymorphism in mouse Cdkn2a associated with the risk of human cancers in the Japanese population. Genetic polymorphisms in Cdkn2a and CDKN2A may affect the cancer risk in both mice and humans.

Regulation of the stability and activity of CDC25A and CDC25B by protein phosphatase PP2A and 14-3-3 binding.

Cyclin-dependent kinase (CDK)-activating phosphatases, CDC25A and CDC25B, are labile proteins, and their levels vary in a cell cycle-dependent manner. Immediate-early response IER5 protein negatively regulates the cellular CDC25B levels, and stress-induced IER5 expression potentiates G2/M arrest. IER5 binds to protein phosphatase PP2A and regulates the PP2A substrate specificity. We show that IER5 binds to CDC25B and assists PP2A to convert CDC25B to hypophosphorylated forms. Hypophosphorylation at Ser323 results in the dissociation of CDC25B from 14-3-3 phospho-binding proteins. In IER5 expressing cells, CDC25B dissociated from 14-3-3 is unstable but slightly activated, because 14-3-3 inhibits CDC25B polyubiquitination and CDC25B binding to CDK1. The 14-3-3 binding to CDC25A also impedes CDC25A degradation and CDC25A-CDK2 interaction. We propose that 14-3-3 is an important regulator of CDC25A and CDC25B and that PP2A/IER5 controls the stability and activity of CDC25B through regulating the interaction of CDC25B and 14-3-3.

The parathyroid hormone regulates skin tumour susceptibility in mice.

Using a forward genetics approach to map loci in a mouse skin cancer model, we previously identified a genetic locus, Skin tumour modifier of MSM 1 (Stmm1) on chromosome 7, conferring strong tumour resistance. Sub-congenic mapping localized Parathyroid hormone (Pth) in Stmm1b. Here, we report that serum intact-PTH (iPTH) and a genetic polymorphism in Pth are important for skin tumour resistance. We identified higher iPTH levels in sera from cancer-resistant MSM/Ms mice compared with susceptible FVB/NJ mice. Therefore, we performed skin carcinogenesis experiments with MSM-BAC transgenic mice (Pth MSM-Tg) and Pth knockout heterozygous mice (Pth +/-). As a result, the higher amounts of iPTH in sera conferred stronger resistance to skin tumours. Furthermore, we found that the coding SNP (rs51104087, Val28Met) localizes in the mouse Pro-PTH encoding region, which is linked to processing efficacy and increased PTH secretion. Finally, we report that PTH increases intracellular calcium in keratinocytes and promotes their terminal differentiation. Taken together, our data suggest that Pth is one of the genes responsible for Stmm1, and serum iPTH could serve as a prevention marker of skin cancer and a target for new therapies.

CENP-R acts bilaterally as a tumor suppressor and as an oncogene in the two-stage skin carcinogenesis model.

CENP-R is a component of the CENP-O complex, including CENP-O, CENP-P, CENP-Q, CENP-R, and CENP-U and is constitutively localized to kinetochores throughout the cell cycle in vertebrates. CENP-R-deficient chicken DT40 cells are viable and show a very minor effect on mitosis. To investigate the functional roles of CENP-R in vivo, we generated CENP-R-deficient mice (Cenp-r-/- ). Mice heterozygous or homozygous for Cenp-r null mutation are viable and healthy, with no apparent defect in growth and morphology, indicating Cenp-r is not essential for normal development. Accordingly, to investigate the role of the Cenp-r gene in skin carcinogenesis, we subjected Cenp-r-/- mice to the 7,12-dimethylbenz(a)anthracene (DMBA)/TPA chemical carcinogenesis protocol and monitored tumor development. As a result, Cenp-r-/- mice initially developed significantly more papillomas than control wild-type mice. However, papillomas in Cenp-r-/- mice showed a decrease of proliferative cells and an increase of apoptotic cells. As a result, they did not grow bigger and some papillomas showed substantial regression. Furthermore, papillomas in Cenp-r-/- mice showed lower frequency of malignant conversion to squamous cell carcinomas. These results indicate Cenp-r functions bilaterally in cancer development: during early developmental stages, Cenp-r functions as a tumor suppressor, but during the expansion and progression of papillomas it functions as a tumor-promoting factor.

Factors associated with medication amounts considered excessive among university students: a questionnaire survey of pharmacy students and those in non-medical schools.

BACKGROUND: Better insight and knowledge on factors associated with perception of medication numbers and amounts would contribute greatly to our current understanding of patient psychological response regarding taking medications, and would allow us to improve drug administration support and adherence. This study explored associations between attitudes toward medication dosage in a questionnaire survey that examined demographic characteristics, the number of tablets and types of prescription medications considered excessive by participants, current medication and supplement use, personal experiences with medications, and perceptions surrounding medications. METHODS: An original anonymous questionnaire was used for this survey. A total of 934 university students completed and returned surveys with no missing data. RESULTS: Mean values ± standard deviation for excessive thresholds for tablets and types of medications reported by all participants were 4.21 ± 1.63 tablets and 4.00 ± 1.25 medications, respectively. The number of tablets considered excessive was analyzed using a multiple regression model, which accounted for the variance (model-adjusted R 2 = 0.095, p < 0.001) between statistically significant factors, including personal experience with a major illness, supplement use, aversion to taking medications, gender, university departmental affiliation, and experience with family members or acquaintances who took excessive amounts of medications (|beta| > 0.094, p < 0.01). The number of medications considered excessive was subject to a multiple regression analysis (model-adjusted R 2 = 0.087 p < 0.01), which revealed statistically significant factors, including personal experience with a major illness, prescription medication use, aversion to taking medications, gender, university departmental affiliation, and experience with family members or acquaintances who took excessive amounts of medications (|beta| > 0.084, p < 0.01). CONCLUSIONS: Individual attitudes toward medication dosage are influenced by individual factors. Thus, patients should be provided with personalized advice when they receive medication instructions.

Association between rapid force production by the plantar flexors and balance performance in elderly men and women.

Plantar flexion strength and balance ability are considered to be crucial for avoiding falls. However, no clear relationship has been established between these two factors in elderly population. This study aimed to examine the association between plantar flexion strength and balance performance in elderly men and women. Forty-three men and 35 women aged over 65 years performed isometric plantar flexion as fast and hard as possible. From the time-torque curve, the rate of torque development in time intervals of 30, 50, 100, 150, and 200 ms from the onset of contraction was determined and normalized to peak torque. In addition, the center of pressure displacement during single-leg standing was calculated and normalized to height. When the data were collapsed over sexes, the normalized rate of torque development was negatively correlated with the normalized center of pressure displacement, except for the time interval of 200 ms. By sex, regardless of the time interval, there was a negative correlation between the normalized rate of torque development and the normalized center of pressure displacement in the elderly men but not in the elderly women. No correlation was seen between the peak torque and normalized center of pressure displacement in either pooled or separated data. The findings suggest that the capability of rapid force production rather than maximal force production of the plantar flexion is important for balance ability in elderly men, but this capability may not be relevant in elderly women.

Mechanoporation of living cells for delivery of macromolecules using nanoneedle array.

Efficient and rapid delivery of macromolecule probes, such as quenchbodies and other large biomarkers that cannot readily pass through the plasma membrane, is necessary for live-cell imaging and other intracellular analyses. We present here an alternative, simple method for delivery of macromolecules into live cells. In this method, which we term here mechanoporation, a nanoneedle array is used for making transient pores in the plasma membrane to allow access of desired macromolecules into thousands of live cells, simultaneously. This rapid, 3-step method facilitates an efficient delivery by adding macromolecules into the medium, inserting nanoneedles into the cells and oscillating the nanoneedle array, a process that takes no more than 5 min in total. In addition, we demonstrate here how this method can repeatedly and reproducibly deliver molecules into specifically-selected locations on a given cell culture dish. The results presented here show how this unique mechanoporation method enables rapid and high-throughput bio-macromolecule delivery and live-cell imaging.

Trial application of oxygen and carbon isotope analysis in tooth enamel for identification of past-war victims for discriminating between Japanese and US soldiers.

Stable isotope analysis has undergone rapid development in recent years and yielded significant results in the field of forensic sciences. In particular, carbon and oxygen isotopic ratios in tooth enamel obtained from human remains can provide useful information for the crosschecking of morphological and DNA analyses and facilitate rapid on-site prescreening for the identification of remains. This study analyzes carbon and oxygen isotopic ratios in the tooth enamel of Japanese people born between 1878 and 1930, in order to obtain data for methodological differentiation of Japanese and American remains from the Second World War. The carbon and oxygen isotopic ratios in the tooth enamel of the examined Japanese individuals are compared to previously reported data for American individuals (born post WWII), and statistical analysis is conducted using a discrimination method based on a logistic regression analysis. The discrimination between the Japanese and US populations, including Alaska and Hawaii, is found to be highly accurate. Thus, the present method has potential as a discrimination technique for both populations for use in the examination of mixed remains comprising Japanese and American fallen soldiers.