Deletion of MyD88 in T Cells Improves Antitumor Activity in Melanoma.

Cytotoxic CD8+ T cells are central to the antitumor immune response by releasing cytotoxic granules that kill tumor cells. They are activated by antigen-presenting cells, which become activated by DAMPs (damage associated molecular patterns) through MyD88. However, the suppressive tumor microenvironment promotes T-cell tolerance to tumor antigens in part by enhancing the activity of immune checkpoint molecules that prevent CD8+ T-cell activation and cytotoxicity. The authors recently reported that MyD88 limits CD4+ T-cell activation during cardiac adaptation to stress and hypothesized that a similar mechanism exists in CD8+ T cells that could be modulated to improve antitumor immunity. The authors found that adoptive transfer of MyD88-/- CD8+ T cells in melanoma-bearing T-cell-deficient mice resulted in slower tumor growth, greater intratumoral T-cell accumulation, and higher melanoma cell death compared with transfer of wild-type CD8+ T cells. These findings were also observed in T-cell-specific MyD88-/- mice compared with wild-type littermates implanted with melanoma. Mechanistically, deletion of MyD88 enhances CD8+ T-cell activation and survival, and T-cell receptor induced degranulation of cytotoxic molecules, overall improving their killing of melanoma cells. This enhanced cytotoxicity was retained in mice bearing tumors expressing the specific antigen for which cytotoxic T-cells were restricted. This study's results demonstrate a conserved mechanism for MyD88 in modulating CD8+ T-cell activation and represent a novel target in improving cancer immunotherapy.

AKT2 Loss Impairs BRAF-Mutant Melanoma Metastasis.

Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients.

AKT2 Loss Impairs BRAF-Mutant Melanoma Metastasis.

Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer, due to its highly metastatic nature. Melanomas harboring oncogenic BRAF V600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT-isoform specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo , while AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that elucidation of AKT2-specific functions in metastasis could inform therapeutic strategies to improve treatment options for melanoma patients.

A kinase-independent function of cyclin-dependent kinase 6 promotes outer radial glia expansion and neocortical folding.

The neocortex, the center for higher brain function, first emerged in mammals and has become massively expanded and folded in humans, constituting almost half the volume of the human brain. Primary microcephaly, a developmental disorder in which the brain is smaller than normal at birth, results mainly from there being fewer neurons in the neocortex because of defects in neural progenitor cells (NPCs). Outer radial glia (oRGs), NPCs that are abundant in gyrencephalic species but rare in lissencephalic species, are thought to play key roles in the expansion and folding of the neocortex. However, how oRGs expand, whether they are necessary for neocortical folding, and whether defects in oRGs cause microcephaly remain important questions in the study of brain development, evolution, and disease. Here, we show that oRG expansion in mice, ferrets, and human cerebral organoids requires cyclin-dependent kinase 6 (CDK6), the mutation of which causes primary microcephaly via an unknown mechanism. In a mouse model in which increased Hedgehog signaling expands oRGs and intermediate progenitor cells and induces neocortical folding, CDK6 loss selectively decreased oRGs and abolished neocortical folding. Remarkably, this function of CDK6 in oRG expansion did not require its kinase activity, was not shared by the highly similar CDK4 and CDK2, and was disrupted by the mutation causing microcephaly. Therefore, our results indicate that CDK6 is conserved to promote oRG expansion, that oRGs are necessary for neocortical folding, and that defects in oRG expansion may cause primary microcephaly.

AKT1 Is Required for a Complete Palbociclib-Induced Senescence Phenotype in BRAF-V600E-Driven Human Melanoma.

Cellular senescence is a carefully regulated process of proliferative arrest accompanied by functional and morphologic changes. Senescence allows damaged cells to avoid neoplastic proliferation; however, the induction of the senescence-associated secretory phenotype (SASP) can promote tumor growth. The complexity of senescence may limit the efficacy of anti-neoplastic agents, such as CDK4/6 inhibitors (Cdk4/6i), that induce a senescence-like state in tumor cells. The AKT kinase family, which contains three isoforms that play both unique and redundant roles in cancer progression, is commonly hyperactive in many cancers including melanoma and has been implicated in the regulation of senescence. To interrogate the role of AKT isoforms in Cdk4/6i-induced cellular senescence, we generated isoform-specific AKT knockout human melanoma cell lines. We found that the CDK4/6i Palbociclib induced a form of senescence in these cells that was dependent on AKT1. We then evaluated the activity of the cGAS-STING pathway, recently implicated in cellular senescence, finding that cGAS-STING function was dependent on AKT1, and pharmacologic inhibition of cGAS had little effect on senescence. However, we found SASP factors to require NF-κB function, in part dependent on a stimulatory phosphorylation of IKKα by AKT1. In summary, we provide the first evidence of a novel, isoform-specific role for AKT1 in therapy-induced senescence in human melanoma cells acting through NF-κB but independent of cGAS.

Hyperlipidemia-induced metabolic changes in regulatory T cells result in altered function.

Regulatory T cells (Tregs) play a critical role in maintaining self-tolerance and controlling inflammation. However, physiologically relevant conditions that alter Treg function and drive disease pathogenesis are poorly understood and few have been defined. We have previously shown that induction of hyperlipidemia in mice results in changes in Tregs that reduce their function. Here, we set out to examine mechanisms by which hyperlipidemia alters Tregs. Using live-cell metabolic assays, we observed that induction of hyperlipidemia increases metabolism in Tregs but not conventional T cells. Increased metabolism resulted from preferential activation of the serine/threonine kinase Akt2 (PKB-ß). Expression of a constitutively activated form of Akt2 in CD4 T cells was sufficient to increase glycolysis in Tregs and drive changes in Treg subsets. Induction of hyperlipidemia did not alter Treg metabolism in mice lacking Akt2. Activation of Akt2 was sufficient to drive the production of inflammatory cytokines by Tregs. We suggest that hyperlipidemia alters Treg function through effects on metabolism via Akt2 activation thereby promoting plasticity and decreased function of FoxP3+ T cells.

Akt3 induces oxidative stress and DNA damage by activating the NADPH oxidase via phosphorylation of p47phox.

Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant N-acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in p53-/- and INK4a-/-/Arf-/- mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47phox, which results in NADPH oxidase activation. Expression of Akt3 in p47phox-/- MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47phox, but not by the phosphorylation site mutant of p47phox In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, Akt3 alterations correlate with a higher frequency of mutation of p53, suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.

Knockdown of CD44 expression decreases valve interstitial cell calcification in vitro.

The lack of pharmaceutical targets available to treat patients with calcific aortic valve disease (CAVD) necessitates further research into the specific mechanisms of the disease. The significant changes that occur to the aortic valves extracellular matrix (ECM) during the progression of CAVD suggests that these proteins may play an important role in calcification. Exploring the relationship between valve interstitial cells (VICs) and the ECM may lead to a better understand of CAVD mechanisms and potential pharmaceutical targets. In this study, we look at the effect of two ECM components, collagen and hyaluronic acid (HA), on the mineralization of VICs within the context of a two-dimensional, polyacrylamide (PAAM) model system. Using a novel, nondestructive imaging technique, we were able to track calcific nodule development in culture systems over a 3-wk time frame. We saw a significant increase in the size of the nodules grown on HA PAAM gels as compared with collagen PAAM gels, suggesting that HA has a direct effect on mineralization. Directly looking at the two known receptors of HA, CD44 and receptor for HA-mediated motility (RHAMM), and using siRNA knockdown revealed that a decrease in CD44 expression resulted in a reduction of calcification. A decrease in CD44, through siRNA knockdown, reduces mineralization on HA PAAM gels, suggesting a potential new target for CAVD treatment. NEW & NOTEWORTHY Our in vitro model of calcific aortic valve disease shows an interaction between the hyaluronic acid binding protein CD44 with the osteogenic factor OPN as a potential mechanism of aortic valve calcification. Using siRNA knockdown of CD44, we show an upregulation of OPN expression with a decrease in overall mineralization.

CDK6 Antagonizes p53-Induced Responses during Tumorigenesis.

Tumor formation is a multistep process during which cells acquire genetic and epigenetic changes until they reach a fully transformed state. We show that CDK6 contributes to tumor formation by regulating transcriptional responses in a stage-specific manner. In early stages, the CDK6 kinase induces a complex transcriptional program to block p53 in hematopoietic cells. Cells lacking CDK6 kinase function are required to mutate TP53 (encoding p53) to achieve a fully transformed immortalized state. CDK6 binds to the promoters of genes including the p53 antagonists Prmt5, Ppm1d, and Mdm4 The findings are relevant to human patients: Tumors with low levels of CDK6 have mutations in TP53 significantly more often than expected.Significance: CDK6 acts at the interface of p53 and RB by driving cell-cycle progression and antagonizing stress responses. While sensitizing cells to p53-induced cell death, specific inhibition of CDK6 kinase activity may provoke the outgrowth of p53-mutant clones from premalignant cells. Cancer Discov; 8(7); 884-97. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 781.

Proliferation of hippocampal progenitors relies on p27-dependent regulation of Cdk6 kinase activity.

Neural stem cells give rise to granule dentate neurons throughout life in the hippocampus. Upon activation, these stem cells generate fast proliferating progenitors that complete several rounds of divisions before differentiating into neurons. Although the mechanisms regulating the activation of stem cells have been intensively studied, little attention has been given so far to the intrinsic machinery allowing the expansion of the progenitor pool. The cell cycle protein Cdk6 positively regulates the proliferation of hippocampal progenitors, but the mechanism involved remains elusive. Whereas Cdk6 functions primarily as a cell cycle kinase, it can also act as transcriptional regulator in cancer cells and hematopoietic stem cells. Using mouse genetics, we show here that the function of Cdk6 in hippocampal neurogenesis relies specifically on its kinase activity. The present study also reveals a specific regulatory mechanism for Cdk6 in hippocampal progenitors. In contrast to the classical model of the cell cycle, we observe that the Cip/Kip family member p27, rather than the Ink4 family, negatively regulates Cdk6 in the adult hippocampus. Altogether, our data uncover a unique, cell type-specific regulatory mechanism controlling the expansion of hippocampal progenitors, where Cdk6 kinase activity is modulated by p27.

Conditional deletion of RB1 in the Tie2 lineage leads to aortic valve regurgitation.

OBJECTIVE: Aortic valve disease is a complex process characterized by valve interstitial cell activation, disruption of the extracellular matrix culminating in valve mineralization occurring over many years. We explored the function of the retinoblastoma protein (pRb) in aortic valve disease, given its critical role in mesenchymal cell differentiation including bone development and mineralization. APPROACH AND RESULTS: We generated a mouse model of conditional pRb knockout (cKO) in the aortic valve regulated by Tie2-Cre-mediated excision of floxed RB1 alleles. Aged pRb cKO animals showed significantly more aortic valve regurgitation by echocardiography compared to pRb het control animals. The pRb cKO aortic valves had increased leaflet thickness without increased cellular proliferation. Histologic studies demonstrated intense α-SMA expression in pRb cKO leaflets associated with disorganized extracellular matrix and increased leaflet stiffness. The pRb cKO mice also showed increased circulating cytokine levels. CONCLUSIONS: Our studies demonstrate that pRb loss in the Tie2-lineage that includes aortic valve interstitial cells is sufficient to cause age-dependent aortic valve dysfunction.

Senescence and tumor suppression.

Cellular senescence has emerged as a potent tumor suppression mechanism that restrains proliferation of cells at risk for malignant transformation. Although senescent cells have permanently exited the cell cycle, their presence can have detrimental effects on the surrounding tissue, largely due to the development of the senescence-associated secretory phenotype (SASP). Here, we review the tumor-suppressive and tumor-promoting consequences of the senescence response, focusing on the SASP as a key mediator of this dichotomy. Accumulating evidence suggests that the persistence of senescent cells can exacerbate the development of a pro-inflammatory, immunosuppressive microenvironment that can favor tumorigenesis. Given that senescence of tumor and stromal cells is a frequent outcome of anti-cancer therapy, approaches that harness the growth inhibitory effects of senescence while limiting its detrimental effects are likely to have great clinical potential.

Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps.

Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21Cip. CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.

Cdk6 contributes to cytoskeletal stability in erythroid cells.

Mice lacking Cdk6 kinase activity suffer from mild anemia accompanied by elevated numbers of Ter119+ cells in the bone marrow. The animals show hardly any alterations in erythroid development, indicating that Cdk6 is not required for proliferation and maturation of erythroid cells. There is also no difference in stress erythropoiesis following hemolysis in vivo However, Cdk6-/- erythrocytes have a shortened lifespan and are more sensitive to mechanical stress in vitro, suggesting differences in cytoskeletal architecture. Erythroblasts contain both Cdk4 and Cdk6, while mature erythrocytes apparently lack Cdk4 and their Cdk6 is partly associated with the cytoskeleton. We used mass spectrometry to show that Cdk6 interacts with a number of proteins involved in cytoskeleton organization. Cdk6-/- erythroblasts show impaired F-actin formation and lower levels of gelsolin, which interacts with Cdk6. We also found that Cdk6 regulates the transcription of a panel of genes involved in actin (de-)polymerization. Cdk6-deficient cells are sensitive to drugs that interfere with the cytoskeleton, suggesting that our findings are relevant to the treatment of patients with anemia - and may be relevant to cancer patients treated with the new generation of CDK6 inhibitors.

Non-destructive two-photon excited fluorescence imaging identifies early nodules in calcific aortic-valve disease.

Calcifications occur during the development of healthy bone, and at the onset of calcific aortic-valve disease (CAVD) and many other pathologies. Although the mechanisms regulating early calcium deposition are not fully understood, they may provide targets for new treatments and for early interventions. Here, we show that two-photon excited fluorescence (TPEF) can provide quantitative and sensitive readouts of calcific nodule formation, in particular in the context of CAVD. Specifically, by means of the decomposition of TPEF spectral images from excised human CAVD valves and from rat bone prior to and following demineralization, as well as from calcific nodules formed within engineered gels, we identified an endogenous fluorophore that correlates with the level of mineralization in the samples. We then developed a ratiometric imaging approach that provides a quantitative readout of the presence of mineral deposits in early calcifications. TPEF should enable non-destructive, high-resolution imaging of three-dimensional tissue specimens for the assessment of the presence of calcification.

Osteosarcoma: prognosis plateau warrants retinoblastoma pathway targeted therapy.

Osteosarcoma (OS) is the most common primary bone cancer in children and adolescents, affecting ~560 young patients in the United States annually. The term OS describes a diverse array of subtypes with varying prognoses, but the majority of tumors are high grade and aggressive. Perhaps because the true etiology of these aggressive tumors remains unknown, advances in OS treatment have reached a discouraging plateau, with only incremental improvements over the past 40 years. Thus, research surrounding the pathogenesis of OS is essential, as it promises to unveil novel therapeutic targets that can attack tumor cells with greater specificity and lower toxicity. Among the candidate molecular targets in OS, the retinoblastoma (RB) pathway demonstrates the highest frequency of inactivation and thus represents a particularly promising avenue for molecular targeted therapy. This review examines the present thinking and practices in OS treatment and specifically highlights the relevance of the RB pathway in osteosarcomagenesis. Through further investigation into RB pathway-related novel therapeutic targets, we believe that a near-term breakthrough in improved OS prognosis is possible.

The E3 ligase APC/C(Cdh1) promotes ubiquitylation-mediated proteolysis of PAX3 to suppress melanocyte proliferation and melanoma growth.

The anaphase-promoting complex or cyclosome with the subunit Cdh1 (APC/C(Cdh1)) is an E3 ubiquitin ligase involved in the control of the cell cycle. Here, we identified sporadic mutations occurring in the genes encoding APC components, including Cdh1, in human melanoma samples and found that loss of APC/C(Cdh1) may promote melanoma development and progression, but not by affecting cell cycle regulatory targets of APC/C. Most of the mutations we found in CDH1 were those associated with ultraviolet light (UV)-induced melanomagenesis. Compared with normal human skin tissue and human or mouse melanocytes, the abundance of Cdh1 was decreased and that of the transcription factor PAX3 was increased in human melanoma tissue and human or mouse melanoma cell lines, respectively; Cdh1 abundance was further decreased with advanced stages of human melanoma. PAX3 was a substrate of APC/C(Cdh1) in melanocytes, and APC/C(Cdh1)-mediated ubiquitylation marked PAX3 for proteolytic degradation in a manner dependent on the D-box motif in PAX3. Either mutating the D-box in PAX3 or knocking down Cdh1 prevented the ubiquitylation and degradation of PAX3 and increased proliferation and melanin production in melanocytes. Knocking down Cdh1 in melanoma cells in culture or before implantation in mice promoted doxorubicin resistance, whereas reexpressing wild-type Cdh1, but not E3 ligase-deficient Cdh1 or a mutant that could not interact with PAX3, restored doxorubicin sensitivity in melanoma cells both in culture and in xenografts. Thus, our findings suggest a tumor suppressor role for APC/C(Cdh1) in melanocytes and that targeting PAX3 may be a strategy for treating melanoma.

Haploinsufficiency for BRCA1 leads to cell-type-specific genomic instability and premature senescence.

Although BRCA1 function is essential for maintaining genomic integrity in all cell types, it is unclear why increased risk of cancer in individuals harbouring deleterious mutations in BRCA1 is restricted to only a select few tissues. Here we show that human mammary epithelial cells (HMECs) from BRCA1-mutation carriers (BRCA1(mut/+)) exhibit increased genomic instability and rapid telomere erosion in the absence of tumour-suppressor loss. Furthermore, we uncover a novel form of haploinsufficiency-induced senescence (HIS) specific to epithelial cells, which is triggered by pRb pathway activation rather than p53 induction. HIS and telomere erosion in HMECs correlate with misregulation of SIRT1 leading to increased levels of acetylated pRb as well as acetylated H4K16 both globally and at telomeric regions. These results identify a novel form of cellular senescence and provide a potential molecular basis for the rapid cell- and tissue- specific predisposition of breast cancer development associated with BRCA1 haploinsufficiency.