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.

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.

First-in-Human Evaluation of 18F-PF-06445974, a PET Radioligand That Preferentially Labels Phosphodiesterase-4B.

Phosphodiesterase-4 (PDE4), which metabolizes the second messenger cyclic adenosine monophosphate (cAMP), has 4 isozymes: PDE4A, PDE4B, PDE4C, and PDE4D. PDE4B and PDE4D have the highest expression in the brain and may play a role in the pathophysiology and treatment of depression and dementia. This study evaluated the properties of the newly developed PDE4B-selective radioligand 18F-PF-06445974 in the brains of rodents, monkeys, and humans. Methods: Three monkeys and 5 healthy human volunteers underwent PET scans after intravenous injection of 18F-PF-06445974. Brain uptake was quantified as total distribution volume (V T) using the standard 2-tissue-compartment model and serial concentrations of parent radioligand in arterial plasma. Results: 18F-PF-06445974 readily distributed throughout monkey and human brain and had the highest binding in the thalamus. The value of V T was well identified by a 2-tissue-compartment model but increased by 10% during the terminal portions (40 and 60 min) of the monkey and human scans, respectively, consistent with radiometabolite accumulation in the brain. The average human V T values for the whole brain were 9.5 ± 2.4 mL ⋅ cm-3 Radiochromatographic analyses in knockout mice showed that 2 efflux transporters-permeability glycoprotein (P-gp) and breast cancer resistance protein (BCRP)-completely cleared the problematic radiometabolite but also partially cleared the parent radioligand from the brain. In vitro studies with the human transporters suggest that the parent radioligand was a partial substrate for BCRP and, to a lesser extent, for P-gp. Conclusion: 18F-PF-06445974 quantified PDE4B in the human brain with reasonable, but not complete, success. The gold standard compartmental method of analyzing brain and plasma data successfully identified the regional densities of PDE4B, which were widespread and highest in the thalamus, as expected. Because the radiometabolite-induced error was only about 10%, the radioligand is, in the opinion of the authors, suitable to extend to clinical studies.

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.

PET Imaging of Phosphodiesterase-4 Identifies Affected Dysplastic Bone in McCune-Albright Syndrome, a Genetic Mosaic Disorder.

McCune-Albright syndrome (MAS) is a mosaic disorder arising from gain-of-function mutations in the GNAS gene, which encodes the 3',5'-cyclic adenosine monophosphate (cAMP) pathway-associated G-protein, Gsα. Clinical manifestations of MAS in a given individual, including fibrous dysplasia, are determined by the timing and location of the GNAS mutation during embryogenesis, the tissues involved, and the role of Gsα in the affected tissues. The Gsα mutation results in dysregulation of the cAMP signaling cascade, leading to upregulation of phosphodiesterase type 4 (PDE4), which catalyzes the hydrolysis of cAMP. Increased cAMP levels have been found in vitro in both animal models of fibrous dysplasia and in cultured cells from individuals with MAS but not in humans with fibrous dysplasia. PET imaging of PDE4 with 11C-(R)-rolipram has been used successfully to study the in vivo activity of the cAMP cascade. To date, it remains unknown whether fibrous dysplasia and other symptoms of MAS, including neuropsychiatric impairments, are associated with increased PDE4 activity in humans. Methods:11C-(R)-rolipram whole-body and brain PET scans were performed on 6 individuals with MAS (3 for brain scans and 6 for whole-body scans) and 9 healthy controls (7 for brain scans and 6 for whole-body scans). Results:11C-(R)-rolipram binding correlated with known locations of fibrous dysplasia in the periphery of individuals with MAS; no uptake was observed in the bones of healthy controls. In peripheral organs and the brain, no difference in 11C-(R)-rolipram uptake was noted between participants with MAS and healthy controls. Conclusion: This study is the first to find evidence for increased cAMP activity in areas of fibrous dysplasia in vivo. No differences in brain uptake between MAS participants and controls were detected-a finding that could be due to several reasons, including the limited anatomic resolution of PET. Nevertheless, the results confirm the usefulness of PET scans with 11C-(R)-rolipram to indirectly measure increased cAMP pathway activation in human disease.

Discovery, Radiolabeling, and Evaluation of Subtype-Selective Inhibitors for Positron Emission Tomography Imaging of Brain Phosphodiesterase-4D.

We aimed to develop radioligands for PET imaging of brain phosphodiesterase subtype 4D (PDE4D), a potential target for developing cognition enhancing or antidepressive drugs. Exploration of several chemical series gave four leads with high PDE4D inhibitory potency and selectivity, optimal lipophilicity, and good brain uptake. These leads featured alkoxypyridinyl cores. They were successfully labeled with carbon-11 (t1/2 = 20.4 min) for evaluation with PET in monkey. Whereas two of these radioligands did not provide PDE4D-specific signal in monkey brain, two others, [11C]T1660 and [11C]T1650, provided sizable specific signal, as judged by pharmacological challenge using rolipram or a selective PDE4D inhibitor (BPN14770) and subsequent biomathematical analysis. Specific binding was highest in prefrontal cortex, temporal cortex, and hippocampus, regions that are important for cognitive function. [11C]T1650 was progressed to evaluation in humans with PET, but the output measure of brain enzyme density (VT) increased with scan duration. This instability over time suggests that radiometabolite(s) were accumulating in the brain. BPN14770 blocked PDE4D uptake in human brain after a single dose, but the percentage occupancy was difficult to estimate because of the unreliability of measuring VT. Overall, these results show that imaging of PDE4D in primate brain is feasible but that further radioligand refinement is needed, most likely to avoid problematic radiometabolites.

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.

Time-Series Analysis of Tumorigenesis in a Murine Skin Carcinogenesis Model.

Recent years have witnessed substantial progress in understanding tumor heterogeneity and the process of tumor progression; however, the entire process of the transition of tumors from a benign to metastatic state remains poorly understood. In the present study, we performed a prospective cancer genome-sequencing analysis by employing an experimental carcinogenesis mouse model of squamous cell carcinoma to systematically understand the evolutionary process of tumors. We surgically collected a part of a lesion of each tumor and followed the progression of these tumors in vivo over time. Comparative time-series analysis of the genomes of tumors with different fates, i.e., those that eventually metastasized and regressed, suggested that these tumors acquired and inherited different mutations. These findings suggest that despite the occurrence of an intra-tumor selection event for malignant alteration during the transformation from early- to late-stage papilloma, the fate determination of tumors might be determined at an even earlier stage.

Imaging findings in radiation therapy complications of the central nervous system.

Radiation therapy is a useful treatment for tumors and vascular malformations of the central nervous system. Radiation therapy is associated with complications, including leukoencephalopathy, radiation necrosis, vasculopathy, and optic neuropathy. Secondary tumors are also often seen long after radiation therapy. Secondary tumors are often benign tumors, such as hemangiomas and meningiomas, but sometimes malignant gliomas and soft tissue sarcomas emerge. We review the imaging findings of complications that may occur after brain radiation therapy.

Semi-quantitative dopamine transporter standardized uptake value in comparison with conventional specific binding ratio in [123I] FP-CIT single-photon emission computed tomography (DaTscan).

PURPOSE: We developed a new analytical method to quantify the dopamine transporter (DAT) radiation dose in the striatum on [123I] FP-CIT single-photon emission computed tomography (SPECT). This method is based on the dopamine transporter standardized uptake value (DaTSUV). The purpose of this study was to compare DaTSUV with the classical specific binding ratio (SBR) in the discrimination of dopaminergic neurodegenerative diseases (dNDD) from non-dNDD. METHOD: Seventy-seven consecutive patients who underwent DaTscan were included. Patients were divided into a dNDD group (n = 44; 24 men, 20 women; median age 73 years) and a non-dNDD group (n = 33; 14 men, 19 women; median age 75 years) based on their clinical diagnoses. The relationship between each method was evaluated by Pearson's correlation coefficient. Differences in SBR and DaTSUV in each group were evaluated by t test. Pairwise comparison of receiver operating characteristic (ROC) curve analysis was performed to compare the discriminating abilities of each method according to the standard error of the area under the curve (AUC). A value of p < 0.05 was considered statistically significant. RESULT: There was a significant strong correlation between DaTSUV and SBR (r = 0.910 [95% CI = 0.862-0.942], p < 0.001). The dNDD group showed significantly lower SBR (3.48 [1.25-7.91] vs 6.58 [3.81-11.1], p < 0.001) and DaTSUV (4.91 [1.59-13.6] vs 8.61 [2.29-15.6], p < 0.001) than the non-dNDD group. The discriminating ability of SBR (AUC = 0.918) was significantly higher than that of DaTSUV (AUC = 0.838, p = 0.0176). CONCLUSION: DaTSUV has a good correlation with SBR, but it could not exceed SBR for discriminating dNDD from non-dNDD.

DEclust: A statistical approach for obtaining differential expression profiles of multiple conditions.

High-throughput RNA sequencing technology is widely used to comprehensively detect and quantify cellular gene expression. Thus, numerous analytical methods have been proposed for identifying differentially expressed genes (DEGs) between paired samples such as tumor and control specimens, but few studies have reported methods for analyzing differential expression under multiple conditions. We propose a novel method, DEclust, for differential expression analysis among more than two matched samples from distinct tissues or conditions. As compared to conventional clustering methods, DEclust more accurately extracts statistically significant gene clusters from multi-conditional transcriptome data, particularly when replicates of quantitative experiments are available. DEclust can be used for any multi-conditional transcriptome data, as well as for extending any DEG detection tool for paired samples to multiple samples. Accordingly, DEclust can be used for a wide range of applications for transcriptome data analysis. DEclust is freely available at http://www.dna.bio.keio.ac.jp/software/DEclust.

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.

Increased Pittsburgh Compound-B Accumulation in the Subcortical White Matter of Alzheimer's Disease Brain.

Using 11C-Pittsburgh compound B (PiB)-PET and MRI volume data, we investigated whether white matter (WM) PiB uptake in Alzheimer's disease (AD) brain is larger than that of cortical PiB uptake-negative (PiB-negative) brain. Forty-five subjects who underwent both PiB-PET and MRI were included in the study (32 AD patients with cortical PiB-positive and 13 cortical amyloid -negative patients). Individual areas of gray matter (GM) and WM were segmented, then regional GM and WM standard uptake value ratio (SUVR) normalized to cerebellar GM with partial volume effects correction was calculated. Three regional SUVRs except WM in the centrum semiovale in the AD group were significantly larger than those in the PiB-negative groups. Frontal WM SUVR in the AD group vs frontal WM SUVR in the PiB-negative group was 2.57 ± 0.55 vs 1.64 ± 0.22; parietal, 2.50 ± 0.52 vs 1.74 ± 0.22; posterior cingulate, 2.84 ± 0.59 vs 1.73 ± 0.22; and WM in the centrum semiovale, 2.21 ± 0.53 vs 2.42 ± 0.36, respectively. We found that PiB uptake in AD brain is significantly larger than that in PiB-negative brain in the frontal, parietal and posterior cingulate subcortical WM, except in the centrum semiovale.