The epithelial polarity axis controls the resting membrane potential and Cl- co-transport in breast glandular structures.

The membrane potential (MP) controls cell homeostasis by directing molecule transport and gene expression. How the MP is set upon epithelial differentiation is unknown. Given that tissue architecture also controls homeostasis, we investigated the relationship between basoapical polarity and resting MP in three-dimensional culture of the HMT-3522 breast cancer progression. A microelectrode technique to measure MP and input resistance reveals that the MP is raised by gap junction intercellular communication (GJIC), which directs tight-junction mediated apical polarity, and is decreased by the Na+/K+/2Cl- (NKCC, encoded by SLC12A1 and SLC12A2) co-transporter, active in multicellular structures displaying basal polarity. In the tumor counterpart, the MP is reduced. Cancer cells display diminished GJIC and do not respond to furosemide, implying loss of NKCC activity. Induced differentiation of cancer cells into basally polarized multicellular structures restores widespread GJIC and NKCC responses, but these structures display the lowest MP. The absence of apical polarity, necessary for cancer onset, in the non-neoplastic epithelium is also associated with the lowest MP under active Cl- transport. We propose that the loss of apical polarity in the breast epithelium destabilizes cellular homeostasis in part by lowering the MP.

Corrigendum: High-throughput discovery of novel developmental phenotypes.

This corrects the article DOI: 10.1038/nature19356.

High-throughput discovery of novel developmental phenotypes.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Exclusion of Expert Contributors From Authorship Limits the Quality of Scientific Articles.

Veterinary pathologists are key contributors to multidisciplinary biomedical research. However, they are occasionally excluded from authorship in published articles despite their substantial intellectual and data contributions. To better understand the potential origins and implications of this practice, we identified and analyzed 29 scientific publications where the contributing pathologist was excluded as an author. The amount of pathologist-generated data contributions were similar to the calculated average contributions for authors, suggesting that the amount of data contributed by the pathologist was not a valid factor for their exclusion from authorship. We then studied publications with pathologist-generated contributions to compare the effects of inclusion or exclusion of the pathologist as an author. Exclusion of the pathologist from authorship was associated with significantly lower markers of rigor and reproducibility compared to articles in which the pathologist was included as author. Although this study did not find justification for the exclusion of pathologists from authorship, potential consequences of their exclusion on data quality were readily detectable.

Naturally Occurring Canine Melanoma as a Predictive Comparative Oncology Model for Human Mucosal and Other Triple Wild-Type Melanomas.

Melanoma remains mostly an untreatable fatal disease despite advances in decoding cancer genomics and developing new therapeutic modalities. Progress in patient care would benefit from additional predictive models germane for human disease mechanisms, tumor heterogeneity, and therapeutic responses. Toward this aim, this review documents comparative aspects of human and naturally occurring canine melanomas. Clinical presentation, pathology, therapies, and genetic alterations are highlighted in the context of current basic and translational research in comparative oncology. Somewhat distinct from sun exposure-related human cutaneous melanomas, there is growing evidence that a variety of gene copy number alterations and protein structure/function mutations play roles in canine melanomas, in circumstances more analogous to human mucosal melanomas and to some extent other melanomas with murine sarcoma viral oncogene homolog B (BRAF), Neuroblastoma RAS Viral (V-Ras) Oncogene Homolog (NRAS), and neurofibromin 1 tumor suppressor NF1 triple wild-type genotype. Gaps in canine genome annotation, as well as an insufficient number and depth of sequences covered, remain considerable barriers to progress and should be collectively addressed. Preclinical approaches can be designed to include canine clinical trials addressing immune modulation as well as combined-targeted inhibition of Rat Sarcoma Superfamily/Mitogen-activated protein kinase (RAS/MAPK) and/or Phosphatidylinositol-3-Kinase/Protein Kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal transduction, pathways frequently activated in both human and canine melanomas. Future investment should be aimed towards improving understanding of canine melanoma as a predictive preclinical surrogate for human melanoma and for mutually benefiting these uniquely co-dependent species.

Cancer incidence profile in sub-Saharan African-born blacks in the United States: Similarities and differences with US-born non-Hispanic blacks.

BACKGROUND: Sub-Saharan African-born blacks (ABs) are one of the fastest-growing populations in the United States. However, to the authors' knowledge, data regarding the cancer burden in this group are lacking, which would inform targeted cancer prevention and control. METHODS: The authors calculated age-standardized proportional incidence ratios (PIRs) comparing the frequency of the top 15 cancers in ABs with that of US-born non-Hispanic blacks (USBs) by sex and region of birth using incidence data for 2000 through 2012 from the Surveillance, Epidemiology, and End Results (SEER 17) program. RESULTS: Compared with USBs, ABs had significantly higher PIRs of infection-related cancers (liver, stomach, and Kaposi sarcoma), blood cancers (leukemia and non-Hodgkin lymphoma), prostate cancer, and thyroid cancers (females only). For example, the PIR for Kaposi sarcoma in AB versus USB women was 12.06 (95% confidence interval [95% CI], 5.23-18.90). In contrast, ABs had lower PIRs for smoking-related and colorectal cancers (eg, for lung cancer among men, the PIR was 0.30 [95% CI, 0.27-0.34]). Furthermore, cancer occurrence in ABs versus USBs varied by region of birth. For example, the higher PIRs for liver cancer noted among male ABs (PIR, 3.57; 95% CI, 1.79-5.35) and for thyroid cancer in female ABs (PIR, 3.03; 95% CI, 2.03-4.02) were confined to Eastern African-born blacks, whereas the higher PIR for prostate cancer (PIR, 1.90; 95% CI, 1.78, 2.02) was confined to Western African-born blacks. CONCLUSIONS: The cancer incidence profile of ABs is different from that of USBs and varies by region of birth, suggesting differences in environmental, cultural, social, and genetic factors. The findings of the current study could stimulate etiologic research and help to inform targeted interventions. Cancer 2017;123:3116-24. © 2017 American Cancer Society.

MacroH2A1 isoforms are associated with epigenetic markers for activation of lipogenic genes in fat-induced steatosis.

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variant macroH2A1 under alternative splicing gives rise to macroH2A1.1 and macroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histone marks on their promoters. This study underlines the importance of the replacement of noncanonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis.

Polymer-lipid hybrid nanoparticles synchronize pharmacokinetics of co-encapsulated doxorubicin-mitomycin C and enable their spatiotemporal co-delivery and local bioavailability in breast tumor.

UNLABELLED: Effective combination chemotherapy requires the delivery of drugs of synergism to tumor sites while sparing normal tissues. Herein we investigated whether coencapsulation of doxorubicin and mitomycin C within polymer-lipid hybrid nanoparticles (DMPLN) achieved this goal via ratiometric drugs in an orthotopic murine breast tumor model with nanocarrier-modified biodistribution, pharmacokinetics, local bioavailability and toxicity. Fluorescence imaging revealed quickened and extended tumor uptake but reduced cardiac accumulation of DMPLN. Quantitative drug analysis demonstrated prolonged systemic circulation, increased tumor accumulation and sustained synergistic ratios of doxorubicin and mitomycin C delivered by DMPLN over 24h. Higher levels of tumor cell apoptosis and reduced organ toxicity were obtained with DMPLN compared to free drug cocktails. DMPLN released DOX in tumors more efficiently than that from liposomal doxorubicin, as evidenced by a higher extent of the metabolite, doxorubicinol. These findings substantiate the importance of rational design of nanoparticles for synergistic drug combination therapy. FROM THE CLINICAL EDITOR: The treatment of cancer usually involves using combination chemotherapeutic agents. In adopting a nanomedicine approach, one can in theory design combination therapy consisting of drugs of synergistic activities, with the aim to target tumor specifically while minimizing systemic toxicity. The authors in this study provided evidence for this rational design by co-encapsulation of doxorubicin and mitomycin C within polymer-lipid hybrid nanoparticles (DMPLN) in a breast cancer model.

Lnk adaptor suppresses radiation resistance and radiation-induced B-cell malignancies by inhibiting IL-11 signaling.

The Lnk (Sh2b3) adaptor protein dampens the response of hematopoietic stem cells and progenitors (HSPCs) to a variety of cytokines by inhibiting JAK2 signaling. As a consequence, Lnk(-/-) mice develop hematopoietic hyperplasia, which progresses to a phenotype resembling the nonacute phase of myeloproliferative neoplasm. In addition, Lnk mutations have been identified in human myeloproliferative neoplasms and acute leukemia. We find that Lnk suppresses the development of radiation-induced acute B-cell malignancies in mice. Lnk-deficient HSPCs recover more effectively from irradiation than their wild-type counterparts, and this resistance of Lnk(-/-) HSPCs to radiation underlies the subsequent emergence of leukemia. A search for the mechanism responsible for radiation resistance identified the cytokine IL-11 as being critical for the ability of Lnk(-/-) HSPCs to recover from irradiation and subsequently become leukemic. In IL-11 signaling, wild-type Lnk suppresses tyrosine phosphorylation of the Src homology region 2 domain-containing phosphatase-2/protein tyrosine phosphatase nonreceptor type 11 and its association with the growth factor receptor-bound protein 2, as well as activation of the Erk MAP kinase pathway. Indeed, Src homology region 2 domain-containing phosphatase-2 has a binding motif for the Lnk Src Homology 2 domain that is phosphorylated in response to IL-11 stimulation. IL-11 therefore drives a pathway that enhances HSPC radioresistance and radiation-induced B-cell malignancies, but is normally attenuated by the inhibitory adaptor Lnk.

Timp3 loss accelerates tumour invasion and increases prostate inflammation in a mouse model of prostate cancer.

BACKGROUND: Altered expression and activity of proteases is implicated in inflammation and cancer progression. An important negative regulator of protease activity is TIMP3 (tissue inhibitor of metalloproteinase 3). TIMP3 expression is lacking in many cancers including advanced prostate cancer, and this may facilitate invasion and metastasis by allowing unrestrained protease activity. METHODS: To investigate the role of TIMP3 in prostate cancer progression, we crossed TIMP3-deficient mice (Timp3(-/-)) to mice with prostate-specific deletion of the tumor suppressor Pten (Pten(-/-)), a well-established mouse model of prostate cancer. Tumor growth and progression were compared between Pten(-/-), Timp3(-/-) and control (Pten(-/-), Timp3(+/+)) mice at 16 weeks of age by histopathology and markers of proliferation, vascularity, and tumor invasion. Metalloproteinase activity within the tumors was assessed by gelatin zymography. Inflammatory infiltrates were assessed by immunohistochemistry for macrophages and lymphocytes whereas expression of cytokines and other inflammatory mediators was assessed by quantitative real time PCR and multiplex ELISA. RESULTS: Increased tumor growth, proliferation index, increased microvascular density, and invasion was observed in Pten(-/-), Timp3(-/-) prostate tumors compared to Pten(-/-), Timp3(+/+) tumors. Tumor cell invasion in Pten(-/-), Timp3(-/-) mice was associated with increased expression of matrix metalloprotease (MMP)-9 and activation of MMP-2. There was markedly increased inflammatory cell infiltration into the TIMP3-deficient prostate tumors along with increased expression of monocyte chemoattractant protein-1, cyclooxygenase-2, TNF-α, and interleukin-1ß; all of which are implicated in inflammation and cancer. CONCLUSIONS: This study provides important insights into the role of altered protease activity in promoting prostate cancer invasion and implicates prostate inflammation as an important promoting factor in prostate cancer progression.

The role of sphingosine-1-phosphate transporter Spns2 in immune system function.

Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm1a(KOMP)Wtsi) allele (Spns2(tm1a)). The Spns2(tm1a/tm1a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm1a/tm1a) mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm1a/tm1a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

Biocompatible and bioactivable terpolymer-lipid-MnO2 Nanoparticle-based MRI contrast agent for improving tumor detection and delineation.

Early and precise detection of solid tumor cancers is critical for improving therapeutic outcomes. In this regard, magnetic resonance imaging (MRI) has become a useful tool for tumor diagnosis and image-guided therapy. However, its effectiveness is limited by the shortcomings of clinically available gadolinium-based contrast agents (GBCAs), i.e. poor tumor penetration and retention, and safety concerns. Thus, we have developed a novel nanoparticulate contrast agent using a biocompatible terpolymer and lipids to encapsulate manganese dioxide nanoparticles (TPL-MDNP). The TPL-MDNP accumulated in tumor tissue and produced paramagnetic Mn2+ ions, enhancing T1-weight MRI contrast via the reaction with H2O2 rich in the acidic tumor microenvironment. Compared to the clinically used GBCA, Gadovist®1.0, TPL-MDNP generated stronger T1-weighted MR signals by over 2.0-fold at 30 % less of the recommended clinical dose with well-defined tumor delineation in preclinical orthotopic tumor models of brain, breast, prostate, and pancreas. Importantly, the MRI signals were retained for 60 min by TPL-MDNP, much longer than Gadovist®1.0. Biocompatibility of TPL-MDNP was evaluated and found to be safe up to 4-fold of the dose used for MRI. A robust large-scale manufacturing process was developed with batch-to-batch consistency. A lyophilization formulation was designed to maintain the nanostructure and storage stability of the new contrast agent.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Tankyrase represses autoinflammation through the attenuation of TLR2 signaling.

Dysregulation of Toll-like receptor (TLR) signaling contributes to the pathogenesis of autoimmune diseases. Here, we provide genetic evidence that tankyrase, a member of the poly(ADP-ribose) polymerase (PARP) family, negatively regulates TLR2 signaling. We show that mice lacking tankyrase in myeloid cells developed severe systemic inflammation with high serum inflammatory cytokine levels. We provide mechanistic evidence that tankyrase deficiency resulted in tyrosine phosphorylation and activation of TLR2 and show that phosphorylation of tyrosine 647 within the TIR domain by SRC and SYK kinases was critical for TLR2 stabilization and signaling. Last, we show that the elevated cytokine production and inflammation observed in mice lacking tankyrase in myeloid cells were dependent on the adaptor protein 3BP2, which is required for SRC and SYK activation. These data demonstrate that tankyrase provides a checkpoint on the TLR-mediated innate immune response.

Analysis of genome-wide knockout mouse database identifies candidate ciliopathy genes.

We searched a database of single-gene knockout (KO) mice produced by the International Mouse Phenotyping Consortium (IMPC) to identify candidate ciliopathy genes. We first screened for phenotypes in mouse lines with both ocular and renal or reproductive trait abnormalities. The STRING protein interaction tool was used to identify interactions between known cilia gene products and those encoded by the genes in individual knockout mouse strains in order to generate a list of "candidate ciliopathy genes." From this list, 32 genes encoded proteins predicted to interact with known ciliopathy proteins. Of these, 25 had no previously described roles in ciliary pathobiology. Histological and morphological evidence of phenotypes found in ciliopathies in knockout mouse lines are presented as examples (genes Abi2, Wdr62, Ap4e1, Dync1li1, and Prkab1). Phenotyping data and descriptions generated on IMPC mouse line are useful for mechanistic studies, target discovery, rare disease diagnosis, and preclinical therapeutic development trials. Here we demonstrate the effective use of the IMPC phenotype data to uncover genes with no previous role in ciliary biology, which may be clinically relevant for identification of novel disease genes implicated in ciliopathies.

Proteotyping of knockout mouse strains reveals sex- and strain-specific signatures in blood plasma.

We proteotyped blood plasma from 30 mouse knockout strains and corresponding wild-type mice from the International Mouse Phenotyping Consortium. We used targeted proteomics with internal standards to quantify 375 proteins in 218 samples. Our results provide insights into the manifested effects of each gene knockout at the plasma proteome level. We first investigated possible contamination by erythrocytes during sample preparation and labeled, in one case, up to 11 differential proteins as erythrocyte originated. Second, we showed that differences in baseline protein abundance between female and male mice were evident in all mice, emphasizing the necessity to include both sexes in basic research, target discovery, and preclinical effect and safety studies. Next, we identified the protein signature of each gene knockout and performed functional analyses for all knockout strains. Further, to demonstrate how proteome analysis identifies the effect of gene deficiency beyond traditional phenotyping tests, we provide in-depth analysis of two strains, C8a-/- and Npc2+/-. The proteins encoded by these genes are well-characterized providing good validation of our method in homozygous and heterozygous knockout mice. Ig alpha chain C region, a poorly characterized protein, was among the differentiating proteins in C8a-/-. In Npc2+/- mice, where histopathology and traditional tests failed to differentiate heterozygous from wild-type mice, our data showed significant difference in various lysosomal storage disease-related proteins. Our results demonstrate how to combine absolute quantitative proteomics with mouse gene knockout strategies to systematically study the effect of protein absence. The approach used here for blood plasma is applicable to all tissue protein extracts.

Factors necessary to produce basoapical polarity in human glandular epithelium formed in conventional and high-throughput three-dimensional culture: example of the breast epithelium.

BACKGROUND: Basoapical polarity in epithelia is critical for proper tissue function, and control of proliferation and survival. Cell culture models that recapitulate epithelial tissue architecture are invaluable to unravel developmental and disease mechanisms. Although factors important for the establishment of basal polarity have been identified, requirements for the formation of apical polarity in three-dimensional tissue structures have not been thoroughly investigated. RESULTS: We demonstrate that the human mammary epithelial cell line-3522 S1, provides a resilient model for studying the formation of basoapical polarity in glandular structures. Testing three-dimensional culture systems that differ in composition and origin of substrata reveals that apical polarity is more sensitive to culture conditions than basal polarity. Using a new high-throughput culture method that produces basoapical polarity in glandular structures without a gel coat, we show that basal polarity-mediated signaling and collagen IV are both necessary for the development of apical polarity. CONCLUSION: These results provide new insights into the role of the basement membrane, and especially collagen IV, in the development of the apical pole, a critical element of the architecture of glandular epithelia. Also, the high-throughput culture method developed in this study should open new avenues for high-content screening of agents that act on mammary tissue homeostasis and thus, on architectural changes involved in cancer development.

Efficacy, Tolerability, and Pharmacokinetics of Combined Targeted MEK and Dual mTORC1/2 Inhibition in a Preclinical Model of Mucosal Melanoma.

Melanomas arising in the mucous membranes are a rare and aggressive subtype. New treatment approaches are needed, yet accumulating sufficient evidence to improve patient outcomes is difficult. Clinical and pathological correlates between human and canine mucosal melanomas are substantial, and the relatively greater incidence of spontaneous naturally occurring mucosal melanoma in dogs represents a promising opportunity for predictive modeling. The genomic landscapes of human and canine mucosal melanoma appear highly diverse and generally lack recurring hotspot mutations associated with cutaneous melanomas. Although much remains to be determined, evidence indicates that Ras/MAPK and/or PI3K/AKT/mTOR signaling pathway activations are common in both species and may represent targets for therapeutic intervention. Sapanisertib, an mTORC1/2 inhibitor, was selected from a PI3K/mTOR inhibitor library to collaborate with MEK inhibition; the latter preclinical efficacy was demonstrated previously for canine mucosal melanoma. Combined inhibition of MEK and mTORC1/2, using trametinib and sapanisertib, produced apoptosis and cell-cycle alteration, synergistically reducing cell survival in canine mucosal melanoma cell lines with varying basal signaling activation levels. Compared with individual inhibitors, a staggered sapanisertib dose, coupled with daily trametinib, was optimal for limiting primary mucosal melanoma xenograft growth in mice, and tumor dissemination in a metastasis model, while minimizing hematologic and renal side effects. Inhibitors downmodulated respective signaling targets and the combination additionally suppressed pathway reciprocal crosstalk. The combination did not significantly change plasma sapanisertib pharmacokinetics; however, trametinib area under the curve was increased in the presence of sapanisertib. Targeting Ras/MAPK and PI3K/AKT/mTOR signal transduction pathways appear rational therapies for canine and human mucosal melanoma.