14-3-3σ/Stratifin and p21 limit AKT-related malignant progression in skin carcinogenesis following MDM2-associated p53 loss.

To study mechanisms driving/inhibiting skin carcinogenesis, stage-specific expression of 14-3-3σ (Stratifin) was analyzed in skin carcinogenesis driven by activated rasHa/fos expression (HK1.ras/fos) and ablation of PTEN-mediated AKT regulation (K14.creP/Δ5PTENflx/flx). Consistent with 14-3-3σ roles in epidermal differentiation, HK1.ras hyperplasia and papillomas displayed elevated 14-3-3σ expression in supra-basal keratinocytes, paralleled by supra-basal p-MDM2166 activation and sporadic p-AKT473 expression. In bi-genic HK1.fos/Δ5PTENflx/flx hyperplasia, basal-layer 14-3-3σ expression appeared, and alongside p53/p21, was associated with keratinocyte differentiation and keratoacanthoma etiology. Tri-genic HK1.ras/fos-Δ5PTENflx/flx hyperplasia/papillomas initially displayed increased basal-layer 14-3-3σ, suggesting attempts to maintain supra-basal p-MDM2166 and protect basal-layer p53. However, HK1.ras/fos-Δ5PTENflx/flx papillomas exhibited increasing basal-layer p-MDM2166 activation that reduced p53, which coincided with malignant conversion. Despite p53 loss, 14-3-3σ expression persisted in well-differentiated squamous cell carcinomas (wdSCCs) and alongside elevated p21, limited malignant progression via inhibiting p-AKT1473 expression; until 14-3-3σ/p21 loss facilitated progression to aggressive SCC exhibiting uniform p-AKT1473. Analysis of TPA-promoted HK1.ras-Δ5PTENflx/flx mouse skin, demonstrated early loss of 14-3-3σ/p53/p21 in hyperplasia and papillomas, with increased p-MDM2166/p-AKT1473 that resulted in rapid malignant conversion and progression to poorly differentiated SCC. In 2D/3D cultures, membranous 14-3-3σ expression observed in normal HaCaT and SP1ras61 papilloma keratinocytes was unexpectedly detected in malignant T52ras61/v-fos SCC cells cultured in monolayers, but not invasive 3D-cells. Collectively, these data suggest 14-3-3σ/Stratifin exerts suppressive roles in papillomatogenesis via MDM2/p53-dependent mechanisms; while persistent p53-independent expression in early wdSCC may involve p21-mediated AKT1 inhibition to limit malignant progression.

Invasion-Block and S-MARVEL: A high-content screening and image analysis platform identifies ATM kinase as a modulator of melanoma invasion and metastasis.

Robust high-throughput assays are crucial for the effective functioning of a drug discovery pipeline. Herein, we report the development of Invasion-Block, an automated high-content screening platform for measuring invadopodia-mediated matrix degradation as a readout for the invasive capacity of cancer cells. Combined with Smoothen-Mask and Reveal, a custom-designed, automated image analysis pipeline, this platform allowed us to evaluate melanoma cell invasion capacity posttreatment with two libraries of compounds comprising 3840 U.S. Food and Drug Administration (FDA)-approved drugs with well-characterized safety and bioavailability profiles in humans as well as a kinase inhibitor library comprising 210 biologically active compounds. We found that Abl/Src, PKC, PI3K, and Ataxia-telangiectasia mutated (ATM) kinase inhibitors significantly reduced melanoma cell invadopodia formation and cell invasion. Abrogation of ATM expression in melanoma cells via CRISPR-mediated gene knockout reduced 3D invasion in vitro as well as spontaneous lymph node metastasis in vivo. Together, this study established a rapid screening assay coupled with a customized image-analysis pipeline for the identification of antimetastatic drugs. Our study implicates that ATM may serve as a potent therapeutic target for the treatment of melanoma cell spread in patients.

Ultrafine PdCo bimetallic nanoclusters confined in N-doped porous carbon for the efficient semi-hydrogenation of alkynes.

Semi-hydrogenation of alkynes to prepare alkenes is an important reaction in the petrochemical and fine chemical industries. The use of conventional Pd nanoparticle-based catalysts is limited by alkyne over-hydrogenation and low Pd utilization. In this study, a nitrogen-doped mesoporous carbon material (m-NC), which was rich in defect sites after Zn volatilization, was fabricated by the carbonization of ZIF-8. Ultrafine PdCo bimetallic nanoclusters with Co atom-modified Pd active site electronic and compositional structure were highly dispersed and confined in m-NC. As-obtained Pd0.43Co1/m-NC was used for the semi-hydrogenation of alkynes and it exhibited high selectivity with high conversion under mild reaction conditions. Pd0.43Co1/m-NC also exhibited excellent stability in leaching tests and maintained its catalytic activity for at least nine reaction cycles. The highly dispersed active sites in Pd0.43Co1/m-NC served as the active sites for the catalytic semi-hydrogenation of alkynes; as a regulator, the second metal Co effectively improved selectivity, and m-NC endowed the catalyst with excellent stability. The research work presented here may provide a foundation for the design of highly active, selective, and stable Pd-based bimetallic catalysts for selective hydrogenation.

The SETDB1-TRIM28 Complex Suppresses Antitumor Immunity.

The tumor immune microenvironment is influenced by the epigenetic landscape of the tumor. Here, we have identified the SETDB1-TRIM28 complex as a critical suppressor of antitumor immunity. An epigenetic CRISPR-Cas9 screen of 1,218 chromatin regulators identified TRIM28 as a suppressor of PD-L1 expression. We then revealed that expression of the SETDB1-TRIM28 complex negatively correlated with infiltration of effector CD8+ T cells. Inhibition of SETDB1-TRIM28 simultaneously upregulated PD-L1 and activated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) innate immune response pathway to increase infiltration of CD8+ T cells. Mechanistically, SETDB1-TRIM28 inhibition led to micronuclei formation in the cytoplasm, which is known to activate the cGAS-STING pathway. Thus, SETDB1-TRIM28 inhibition bridges innate and adaptive immunity. Indeed, SETDB1 knockout enhanced the antitumor effects of immune checkpoint blockade with anti-PD-L1 in a mouse model of ovarian cancer in a cGAS-dependent manner. Our findings establish the SETDB1-TRIM28 complex as a regulator of antitumor immunity and demonstrate that its loss activates cGAS-STING innate immunity to boost the antitumor effects of immune checkpoint blockade.

Targeting the IRE1α/XBP1s pathway suppresses CARM1-expressing ovarian cancer.

CARM1 is often overexpressed in human cancers including in ovarian cancer. However, therapeutic approaches based on CARM1 expression remain to be an unmet need. Cancer cells exploit adaptive responses such as the endoplasmic reticulum (ER) stress response for their survival through activating pathways such as the IRE1α/XBP1s pathway. Here, we report that CARM1-expressing ovarian cancer cells are selectively sensitive to inhibition of the IRE1α/XBP1s pathway. CARM1 regulates XBP1s target gene expression and directly interacts with XBP1s during ER stress response. Inhibition of the IRE1α/XBP1s pathway was effective against ovarian cancer in a CARM1-dependent manner both in vitro and in vivo in orthotopic and patient-derived xenograft models. In addition, IRE1α inhibitor B-I09 synergizes with immune checkpoint blockade anti-PD1 antibody in an immunocompetent CARM1-expressing ovarian cancer model. Our data show that pharmacological inhibition of the IRE1α/XBP1s pathway alone or in combination with immune checkpoint blockade represents a therapeutic strategy for CARM1-expressing cancers.

Abrogation of RAB27A expression transiently affects melanoma cell proliferation.

The role of the small GTPase RAB27A as an essential melanosome trafficking regulator in melanocytes is well-accepted. A decade ago, RAB27A was identified as a tumor dependency gene that promotes melanoma cell proliferation. RAB27A has since been linked to another propeller of cancer progression: exosome secretion. We have recently demonstrated that RAB27A is overexpressed in a subset of melanomas. High RAB27A gene and protein expression correlate with poor prognosis in melanoma patients. Mechanistic investigations revealed that the generation of pro-invasive exosomes was RAB27A-dependent and, therefore, silencing RAB27A reduced melanoma cell invasion in vitro and in vivo. However, previous studies have implicated RAB27A to be involved in both proliferation and invasion of melanoma cells. Employing four human cell lines, stratified by RAB27A expression, and one RAB27A-high mouse cell line, we demonstrate in this study that the effects of abrogating RAB27A expression on proliferation are only temporary, in contrast to our previously reported persistent effects on tumor invasion and metastasis. Therefore, we assist in the dissection of the short-term effects of RAB27A knockdown on melanoma cell proliferation versus long-term effects on melanoma invasion and metastasis. We believe that our findings provide novel insights into the effects of RAB27A blockade.

RAB27A promotes melanoma cell invasion and metastasis via regulation of pro-invasive exosomes.

Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.

Single-Cell Transcriptome Analysis Reveals Estrogen Signaling Coordinately Augments One-Carbon, Polyamine, and Purine Synthesis in Breast Cancer.

Estrogen drives breast cancer (BCa) progression by directly activating estrogen receptor α (ERα). However, because of the stochastic nature of gene transcription, it is important to study the estrogen signaling pathway at the single-cell level to fully understand how ERα regulates transcription. Here, we performed single-cell transcriptome analysis on ERα-positive BCa cells following 17ß-estradiol stimulation and reconstructed the dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Notably, differentially expressed genes show an estrogen-stimulated metabolic switch that favors biosynthesis but reduces estrogen degradation. Moreover, folate-mediated one-carbon metabolism is reprogrammed through the mitochondrial folate pathway and polyamine and purine synthesis are upregulated coordinately. Finally, we show AZIN1 and PPAT are direct ERα targets that are essential for BCa cell survival and growth. In summary, our study highlights the dynamic transcriptional heterogeneity in ERα-positive BCa cells upon estrogen stimulation and uncovers a mechanism of estrogen-mediated metabolic switch.