The PI3K-Akt-mTOR and Associated Signaling Pathways as Molecular Drivers of Immune-Mediated Inflammatory Skin Diseases: Update on Therapeutic Strategy Using Natural and Synthetic Compounds.

The dysregulated phosphatidylinositol-3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway has been implicated in various immune-mediated inflammatory and hyperproliferative dermatoses such as acne, atopic dermatitis, alopecia, psoriasis, wounds, and vitiligo, and is associated with poor treatment outcomes. Improved comprehension of the consequences of the dysregulated PI3K/Akt/mTOR pathway in patients with inflammatory dermatoses has resulted in the development of novel therapeutic approaches. Nonetheless, more studies are necessary to validate the regulatory role of this pathway and to create more effective preventive and treatment methods for a wide range of inflammatory skin diseases. Several studies have revealed that certain natural products and synthetic compounds can obstruct the expression/activity of PI3K/Akt/mTOR, underscoring their potential in managing common and persistent skin inflammatory disorders. This review summarizes recent advances in understanding the role of the activated PI3K/Akt/mTOR pathway and associated components in immune-mediated inflammatory dermatoses and discusses the potential of bioactive natural products, synthetic scaffolds, and biologic agents in their prevention and treatment. However, further research is necessary to validate the regulatory role of this pathway and develop more effective therapies for inflammatory skin disorders.

Polarization and ß-Glucan Reprogram Immunomodulatory Metabolism in Human Macrophages and Ex Vivo in Human Lung Cancer Tissues.

Immunomodulatory (IM) metabolic reprogramming in macrophages (Mϕs) is fundamental to immune function. However, limited information is available for human Mϕs, particularly in response plasticity, which is critical to understanding the variable efficacy of immunotherapies in cancer patients. We carried out an in-depth analysis by combining multiplex stable isotope-resolved metabolomics with reversed phase protein array to map the dynamic changes of the IM metabolic network and key protein regulators in four human donors' Mϕs in response to differential polarization and M1 repolarizer ß-glucan (whole glucan particles [WGPs]). These responses were compared with those of WGP-treated ex vivo organotypic tissue cultures (OTCs) of human non-small cell lung cancer. We found consistently enhanced tryptophan catabolism with blocked NAD+ and UTP synthesis in M1-type Mϕs (M1-Mϕs), which was associated with immune activation evidenced by increased release of IL-1ß/CXCL10/IFN-γ/TNF-α and reduced phagocytosis. In M2a-Mϕs, WGP treatment of M2a-Mϕs robustly increased glucose utilization via the glycolysis/oxidative branch of the pentose phosphate pathway while enhancing UDP-N-acetyl-glucosamine turnover and glutamine-fueled gluconeogenesis, which was accompanied by the release of proinflammatory IL-1ß/TNF-α to above M1-Mϕ's levels, anti-inflammatory IL-10 to above M2a-Mϕ's levels, and attenuated phagocytosis. These IM metabolic responses could underlie the opposing effects of WGP, i.e., reverting M2- to M1-type immune functions but also boosting anti-inflammation. Variable reprogrammed Krebs cycle and glutamine-fueled synthesis of UTP in WGP-treated OTCs of human non-small cell lung cancer were observed, reflecting variable M1 repolarization of tumor-associated Mϕs. This was supported by correlation with IL-1ß/TNF-α release and compromised tumor status, making patient-derived OTCs unique models for studying variable immunotherapeutic efficacy in cancer patients.

p53 missense mutant G242A subverts natural killer cells in sheltering mouse breast cancer cells against immune rejection.

Cancer cells acquire immunoediting ability to evade immune surveillance and thus escape eradication. It is widely known that mutant proteins encoded from tumor suppressor TP53 exhibit gain-of-function in cancer cells, thereby promoting progression; however, how mutant p53 contributes to the sheltering of cancer cells from host anticancer immunity remains unclear. Herein, we report that murine p53 missense mutation G242A (corresponding to human G245A) suppresses the activation of host natural killer (NK) cells, thereby enabling breast cancer cells to avoid immune assault. We found that serial injection of EMT6 breast cancer cells that carry wild-type (wt) Trp53, like normal fibroblasts, promoted NK activity in mice, while SVTneg2 cells carrying Trp53 G242A+/+ mutation decreased NK cell numbers and increased CD8+ T lymphocyte numbers in spleen. Innate immunity based on NK cells and CD8 T cells was reduced in p53 mutant-carrying transgenic mice (Trp53 R172H/+, corresponding to human R175H/+). Further, upon co-culture with isolated NK cells, EMT6 cells substantively activated NK cells and proliferation thereof, increasing interferon-gamma (IFN-γ) production; however, SVTneg2 cells suppressed NK cell activation. Further mechanistic study elucidated that p53 can modulate expression by cancer cells of Mult-1 and H60a, which are activating and inhibitory ligands for NKG2D receptors of NK cells, respectively, to enhance immune surveillance against cancer. Our findings demonstrate that wt p53 is requisite for NK cell-based immune recognition and elimination of cancerous cells, and perhaps more importantly, that p53 missense mutant presence in cancer cells impairs NK cell-attributable responses, thus veiling cancerous cells from host immunity and enabling cancer progression.

Gb3-cSrc complex in glycosphingolipid-enriched microdomains contributes to the expression of p53 mutant protein and cancer drug resistance via ß-catenin-activated RNA methylation.

Glucosylceramide synthase (GCS) is a key enzyme catalyzing ceramide glycosylation to generate glucosylceramide (GlcCer), which in turn serves as the precursor for cells to produce glycosphingolipids (GSLs). In cell membranes, GSLs serve as essential components of GSL-enriched microdomains (GEMs) and mediate membrane functions and cell behaviors. Previous studies showed that ceramide glycosylation correlates with upregulated expression of p53 hotspot mutant R273H and cancer drug resistance. Yet, the underlying mechanisms remain elusive. We report herewith that globotriaosylceramide (Gb3) is associated with cSrc kinase in GEMs and plays a crucial role in modulating expression of p53 R273H mutant and drug resistance. Colon cancer cell lines, either WiDr homozygous for missense-mutated TP53 (R273H+/+) or SW48/TP53-Dox bearing heterozygous TP53 mutant (R273H/+), display drug resistance with increased ceramide glycosylation. Inhibition of GCS with Genz-161 (GENZ 667161) resensitized cells to apoptosis in these p53 mutant-carrying cancer cells. Genz-161 effectively inhibited GCS activity, and substantially suppressed the elevated Gb3 levels seen in GEMs of p53-mutant cells exposed to doxorubicin. Complex formation between Gb3 and cSrc in GEMs to activate ß-catenin was detected in both cultured cells and xenograft tumors. Suppression of ceramide glycosylation significantly decreased Gb3-cSrc in GEMs, ß-catenin, and methyltransferase-like 3 for m6A RNA methylation, thus altering pre-mRNA splicing, resulting in upregulated expression of wild-type p53 protein, but not mutants, in cells carrying p53 R273H. Altogether, increased Gb3-cSrc complex in GEMs of membranes in response to anticancer drug induced cell stress promotes expression of p53 mutant proteins and accordant cancer drug resistance.

Ceramide-Rubusoside Nanomicelles, a Potential Therapeutic Approach to Target Cancers Carrying p53 Missense Mutations.

Ceramide (Cer) is an active cellular sphingolipid that can induce apoptosis or proliferation-arrest of cancer cells. Nanoparticle-based delivery offers an effective approach for overcoming bioavailability and biopharmaceutics issues attributable to the pronounced hydrophobicity of Cer. Missense mutations of the protein p53, which have been detected in approximately 42% of cancer cases, not only lose the tumor suppression activity of wild-type p53, but also gain oncogenic functions promoting tumor progression and drug resistance. Our previous works showed that cellular Cer can eradicate cancer cells that carry a p53 deletion-mutation by modulating alternative pre-mRNA splicing, restoring wild-type p53 protein expression. Here, we report that new ceramide-rubusoside (Cer-RUB) nanomicelles considerably enhance Cer in vivo bioavailability and restore p53-dependent tumor suppression in cancer cells carrying a p53 missense mutation. Natural RUB encapsulated short-chain C6-Cer so as to form Cer-RUB nanomicelles (∼32 nm in diameter) that substantially enhanced Cer solubility and its levels in tissues and tumors of mice dosed intraperitoneally. Intriguingly, Cer-RUB nanomicelle treatments restored p53-dependent tumor suppression and sensitivity to cisplatin in OVCAR-3 ovarian cancer cells and xenograft tumors carrying p53 R248Q mutation. Moreover, Cer-RUB nanomicelles showed no signs of significant nonspecific toxicity to noncancerous cells or normal tissues, including bone marrow. Furthermore, Cer-RUB nanomicelles restored p53 phosphorylated protein and downstream function to wild-type levels in p53 R172H/+ transgenic mice. Altogether, this study, for the first time, indicates that natural Cer-RUB nanomicelles offer a feasible approach for efficaciously and safely targeting cancers carrying p53 missense mutations.

An N6-methyladenosine at the transited codon 273 of p53 pre-mRNA promotes the expression of R273H mutant protein and drug resistance of cancer cells.

Mutant p53 proteins that promote cancer cell invasive growth, metastasis and drug resistance emerge as therapeutic targets. Previously, we reported that suppression of ceramide glycosylation restored wild-type p53 protein and tumor suppressing function in cancer cells heterozygously carrying p53 R273H, a hot-spot missense mutation; however, the mechanisms underlying the control of mutant protein expression remain elusive. Herein, we report that an N6-methyladenosine (m6A) at the point-mutated codon 273 (G > A) of p53 pre-mRNA determines the mutant protein expression. Methylation of the transited adenosine was catalyzed by methyltransferase like 3 (METTL3), and this m6A-RNA promoted a preferential pre-mRNA splicing; consequently, the produced p53 R273H mutant protein resulted in acquired multidrug resistance in colon cancer cells. Furthermore, glycosphingolipids (particularly globotriaosylceramide) generated from serial ceramide glycosylation were seen to activate cSrc and ß-catenin signaling so as to upregulate METTL3 expression, in turn promoting expression of p53 R273H mutant protein, with consequent drug resistance. Conversely, either silencing METTL3 expression by using small interfering RNA (siRNA) or inhibiting RNA methylation with neplanocin A suppressed m6A formation in p53 pre-mRNA, and substantially increased the level of phosphorylated p53 protein (Ser15) and its function in cells heterozygously carrying the R273H mutation, thereby re-sensitizing these cells to anticancer drugs. Concordantly, suppression of ceramide glycosylation repressed METTL3 expression and m6A formation in p53 pre-mRNA, thus sensitizing cells carrying R273H to anticancer drugs. This study uncovers a novel function of pre-mRNA m6A as a determinant of mutant protein expression in cancer cells heterozygously carrying the TP53 R273H mutation. Suppressing both RNA methylation and ceramide glycosylation might constitute an efficacious and specific approach for targeting TP53 missense mutations coding for a G > A transition, thereby improving cancer treatments.

Graviola (Annona muricata) Exerts Anti-Proliferative, Anti-Clonogenic and Pro-Apoptotic Effects in Human Non-Melanoma Skin Cancer UW-BCC1 and A431 Cells In Vitro: Involvement of Hedgehog Signaling.

Non-melanoma skin cancers (NMSCs) are the leading cause of skin cancer-related morbidity and mortality. Effective strategies are needed to control NMSC occurrence and progression. Non-toxic, plant-derived extracts have been shown to exert multiple anti-cancer effects. Graviola (Annona muricata), a tropical fruit-bearing plant, has been used in traditional medicine against multiple human diseases including cancer. The current study investigated the effects of graviola leaf and stem extract (GLSE) and its solvent-extracted fractions on two human NMSC cell lines, UW-BCC1 and A431. GLSE was found to: (i) dose-dependently suppress UW-BCC1 and A431 cell growth, motility, wound closure, and clonogenicity; (ii) induce G0/G1 cell cycle arrest by downregulating cyclin/cdk factors while upregulating cdk inhibitors, and (iii) induce apoptosis as evidenced by cleavage of caspases-3, -8 and PARP. Further, GLSE suppressed levels of activated hedgehog (Hh) pathway components Smo, Gli 1/2, and Shh while inducing SuFu. GLSE also decreased the expression of pro-apoptotic protein Bax while decreasing the expression of the anti-apoptotic protein Bcl-2. We determined that these activities were concentrated in an acetogenin/alkaloid-rich dichloromethane subfraction of GLSE. Our data identify graviola extracts and their constituents as promising sources for new chemopreventive and therapeutic agent(s) to be further developed for the control of NMSCs.

Incorporation of Fluorescence Ceramide-Based HPLC Assay for Rapidly and Efficiently Assessing Glucosylceramide Synthase In Vivo.

Glucosylceramide synthase (GCS) is a rate-limiting enzyme catalyzing ceramide glycosylation, thereby regulating cellular ceramide levels and the synthesis of glycosphingolipids (GSLs) in cellular membranes. Alterations of GCS not only affect membrane integrity, but also closely correlate with stem cell pluripotency, cancer drug resistance, GSL storage disorders and other diseases. Enzyme activities measured conventionally with currently available ex-vivo methods do not enable reliable assessment of the roles played by GCS in vivo. We report herein a substrate-incorporation method enabling rapid and efficient assessment of GCS in-vivo activity. Upon nanoparticle-based delivery, fluorescent NBD C6-ceramide was efficiently converted to NBD C6-glucosylceramide in live cells or in mouse tissues, whereupon an HPLC assay enabled detection and quantification of NBD C6-glucosylceramide in the low-femtomolar range. The enzyme kinetics of GCS in live cells and mouse liver were well-described by the Michaelis-Menten model. GCS activities were significantly higher in drug-resistant cancer cells and in tumors overexpressing GCS, but reduced after silencing GCS expression or inhibiting this enzyme. Our studies indicate that this rapid and efficient method provides a valuable means for accurately assessing the roles played by GCS in normal vs. pathological states, including ones involving cancer drug resistance.

Inhibition of glucosylceramide synthase eliminates the oncogenic function of p53 R273H mutant in the epithelial-mesenchymal transition and induced pluripotency of colon cancer cells.

Missense mutation of tumor suppressor p53, which exhibits oncogenic gain-of-function (GOF), not only promotes tumor progression, but also diminishes therapeutic efficacies of cancer treatments. However, it remains unclear how a p53 missense mutant contributes to induced pluripotency of cancer stem cells (CSCs) in tumors exposed to chemotherapeutic agents. More importantly, it may be possible to abrogate the GOF by restoring wild-type p53 activity, thereby overcoming the deleterious effects resulting from heterotetramer formation, which often compromises the efficacies of current approaches being used to reactivate p53 function. Herewith, we report that p53 R273H missense mutant urges cancer cells to spawn CSCs. SW48/TP53 cells, which heterozygously carry the p53 R273H hot-spot mutant (R273H/+, introduced by a CRISPR/Casp9 system), were subchronically exposed to doxorubicin in cell culture and in tumor-bearing mice. We found that p53-R273H (TP53-Dox) cells were drug-resistant and exhibited epithelial-mesenchymal transition (EMT) and increased numbers of CSCs (CD44v6+/CD133+), which resulted in enhanced wound healing and tumor formation. Inhibition of glucosylceramide synthase with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) sensitized p53-R273H cancer cells and tumor xenografts to doxorubicin treatments. Intriguingly, PDMP treatments restored wild-type p53 expression in heterozygous R273H mutant cells and in tumors, decreasing CSCs and sensitizing cells and tumors to treatments. This study demonstrated that p53-R273H promotes EMT and induced pluripotency of CSCs in cancer cells exposed to doxorubicin, mainly through Zeb1 and ß-catenin transcription factors. Our results further indicate that restoration of p53 through inhibition of ceramide glycosylation might be an effective treatment approach for targeting cancers heterozygously harboring TP53 missense mutations.