The role of AMPK in cancer metabolism and its impact on the immunomodulation of the tumor microenvironment.

Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic sensor that is pivotal for the maintenance of cellular energy homeostasis. AMPK contributes to diverse metabolic and physiological effects besides its fundamental role in glucose and lipid metabolism. Aberrancy in AMPK signaling is one of the determining factors which lead to the development of chronic diseases such as obesity, inflammation, diabetes, and cancer. The activation of AMPK and its downstream signaling cascades orchestrate dynamic changes in the tumor cellular bioenergetics. It is well documented that AMPK possesses a suppressor role in the context of tumor development and progression by modulating the inflammatory and metabolic pathways. In addition, AMPK plays a central role in potentiating the phenotypic and functional reprogramming of various classes of immune cells which reside in the tumor microenvironment (TME). Furthermore, AMPK-mediated inflammatory responses facilitate the recruitment of certain types of immune cells to the TME, which impedes the development, progression, and metastasis of cancer. Thus, AMPK appears to play an important role in the regulation of anti-tumor immune response by regulating the metabolic plasticity of various immune cells. AMPK effectuates the metabolic modulation of anti-tumor immunity via nutrient regulation in the TME and by virtue of its molecular crosstalk with major immune checkpoints. Several studies including that from our lab emphasize on the role of AMPK in regulating the anticancer effects of several phytochemicals, which are potential anticancer drug candidates. The scope of this review encompasses the significance of the AMPK signaling in cancer metabolism and its influence on the key drivers of immune responses within the TME, with a special emphasis on the potential use of phytochemicals to target AMPK and combat cancer by modulating the tumor metabolism.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

Termination of TCR-mediated activation signals is regulated by CrkII-dependent Cbl-mediated ubiquitination and degradation of C3G.

Crk adaptor proteins are key players in signal transduction from multiple cell surface receptors, including the T cell antigen receptor (TCR). The involvement of CrkII in the early stages of T cell activation is well documented, but little is known about its role during the termination of the activation response. We substantiated findings showing that CrkII utilizes its SH3N and SH2 domains to constitutively associate with C3G and transiently with Cbl in resting and TCR/CD3-stimulated T cells, respectively. Association of CrkII with Cbl peaks within 1 min post-TCR/CD3 stimulation, and involves the formation of multiple CrkII-containing complexes of different molecular mass. Ubiquitination of C3G commences at ∼5 min post TCR/CD3 stimulation concomitantly with its degradation. This entire process conversely correlates with the levels of expression of CrkII and is dependent on the presence of the CrkII-bound Cbl protein. The data suggest that CrkII functions as a scaffold that brings Cbl into close proximity with C3G in TCR/CD3-stimulated T cells and that tyrosine phosphorylation and activation of Cbl promotes C3G ubiquitination and degradation. We suggest that this mechanism contributes to the termination of the TCR/CD3-induced activation signal and helps tune the length and intensity of T cell-mediated immune responses.

Corrigendum: Targeting thymidylate synthase enhances the chemosensitivity of triple-negative breast cancer towards 5-FU-based combinatorial therapy.

[This corrects the article DOI: 10.3389/fonc.2021.656804.].

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

Pharmacological attenuation of melanoma by tryptanthrin pertains to the suppression of MITF-M through MEK/ERK signaling axis.

Melanoma is the most aggressive among all types of skin cancers. The current strategies against melanoma utilize BRAFV600E, as a focal point for targeted therapy. However, therapy resistance developed in melanoma patients against the conventional anti-melanoma drugs hinders the ultimate benefits of targeted therapies. A major mechanism by which melanoma cells attain therapy resistance is via the activation of microphthalmia-associated transcription factor-M (MITF-M), the key transcription factor and oncogene aiding the survival of melanoma cells. We demonstrate that tryptanthrin (Tpn), an indole quinazoline alkaloid, which we isolated and characterized from Wrightia tinctoria, exhibits remarkable anti-tumor activity towards human melanoma through the down-regulation of MITF-M. Microarray analysis of Tpn-treated melanoma cells followed by a STRING protein association network analysis revealed that differential expression of genes in melanoma converges at MITF-M. Furthermore, in vitro and in vivo studies conducted using melanoma cells with differential MITF-M expression status, endogenously or ectopically, demonstrated that the anti-melanoma activity of Tpn is decisively contingent on its efficacy in down-regulating MITF-M expression. Tpn potentiates the degradation of MITF-M via the modulation of MEK1/2-ERK1/2-MITF-M signaling cascades. Murine models demonstrate the efficacy of Tpn in attenuating the migration and metastasis of melanoma cells, while remaining pharmacologically safe. In addition, Tpn suppresses the expression of mutated BRAFV600E and inhibits Casein Kinase 2α, a pro-survival enzyme that regulates ERK1/2 homeostasis in many tumor types, including melanoma. Together, we point to a promising anti-melanoma drug in Tpn, by virtue of its attributes to impede melanoma invasion and metastasis by attenuating MITF-M.

Corrigendum: Cucurbitacin B, purified and characterized from the rhizome of Corallocarpus epigaeus exhibits anti-melanoma potential.

[This corrects the article DOI: 10.3389/fonc.2022.903832.].

Cucurbitacin B, Purified and Characterized From the Rhizome of Corallocarpus epigaeus Exhibits Anti-Melanoma Potential.

The ethnomedicinal plant from the Cucurbitaceae family, Corallocarpus epigaeus, or its bioactive derivatives have been widely utilized in traditional medicine owing to their distinct applications against various human ailments and have lured the interest of ethnobotanists and biochemists. Here, we report for the first time, the anti-cancer potential of a bio-active fraction isolated from the dried rhizome of C. epigaeus, and the bioactive principle identified as cucurbitacin B (Cu-B). The purification processes involving the utilization of multiple organic extracts of C. epigaeus rhizome powder, yielded Cu-B from the Ethyl acetate Cytotoxic Fraction (ECF), obtained by the chromatographic separation of the ethyl acetate extract. Amongst the various cancer lines tested, melanoma cells exhibit maximal sensitivity towards the Cu-B-containing ECF fraction. Cu-B induces an apoptotic mode of cell death initiated intrinsically as well as extrinsically in A375 melanoma cells whilst remaining comparatively less toxic to normal skin fibroblasts. In vivo studies involving a NOD-SCID murine model of human melanoma demonstrate the ability of Cu-B to attenuate tumor growth, while being pharmacologically safe in vivo, as assessed in Swiss albino mice. Furthermore, Cu-B inhibits MEK 1/2 as well as the constitutive and EGF-induced ERK 1/2 activation, indicating a definitive involvement of MAPK signal transducers in regulating Cu-B-mediated anti-melanoma activity. Together, our study demonstrates the anti-melanoma potential of C. epigaeus-derived Cu-B, which indicates the Cucurbitaceae succulent as a prospective source for deriving potent and pharmacologically safe anti-cancer compounds.

Augmented Efficacy of Uttroside B over Sorafenib in a Murine Model of Human Hepatocellular Carcinoma.

We previously reported the remarkable potency of uttroside B (Utt-B), saponin-isolated and characterized in our lab from Solanum nigrum Linn, against HCC. Recently, the U.S. FDA approved Utt-B as an 'orphan drug' against HCC. The current study validates the superior anti-HCC efficacy of Utt-B over sorafenib, the first-line treatment option against HCC. The therapeutic efficacies of Utt-B vs. sorafenib against HCC were compared in vitro, using various liver cancer cell lines and in vivo, utilizing NOD.CB17-Prkdcscid/J mice bearing human HCC xenografts. Our data indicate that Utt-B holds an augmented anti-HCC efficacy over sorafenib. Our previous report demonstrated the pharmacological safety of Utt-B in Chang Liver, the normal immortalized hepatocytes, and in the acute and chronic toxicity murine models even at elevated Utt-B concentrations. Here, we show that higher concentrations of sorafenib induce severe toxicity, in Chang Liver, as well as in acute and chronic in vivo models, indicating that, apart from the superior therapeutic benefit over sorafenib, Utt-B is a pharmacologically safer molecule, and the drug-induced undesirable effects can, thus, be substantially alleviated in the context of HCC chemotherapy. Clinical studies in HCC patients utilizing Utt-B, is a contiguous key step to promote this drug to the clinic.

Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs.

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5' untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

Targeting Thymidylate Synthase Enhances the Chemosensitivity of Triple-Negative Breast Cancer Towards 5-FU-Based Combinatorial Therapy.

BACKGROUND: The ongoing treatment modalities for breast cancer (BC) primarily rely on the expression status of ER, PR and HER-2 receptors in BC tissues. Our strategy of chemosensitization provides new insights to counter chemoresistance, a major obstacle that limits the benefits of chemotherapy of mammary cancers. METHODS: By utilizing a murine breast cancer model employing NSG mice bearing orthotopic triple-negative breast cancer (TNBC) xenografts, we have evaluated the ability of phytochemical curcumin in chemosensitizing BC to 5-Fluorouracil (5-FU) chemotherapy and the differential modulations of cellular events in response to this strategy, independent of their receptor status. RESULTS: A significant synergistic antitumor potential was observed in the murine model with a sub-optimal dose treatment of 5-FU plus curcumin, as evaluated by a reduction in the tumor-related parameters. We authenticated the pivotal role of thymidylate synthase (TS) in regulating the 5-FU-curcumin synergism using the TNBC pre-clinical model. Our study also confirmed the pharmacological safety of this chemotherapeutic plus phytoactive combination using acute and chronic toxicity studies in Swiss albino mice. Subsequently, the molecular docking analysis of curcumin binding to TS demonstrated the affinity of curcumin towards the cofactor-binding site of TS, rather than the substrate-binding site, where 5-FU binds. Our concomitant in vivo and in silico evidence substantiates the superior therapeutic index of this combination. CONCLUSION: This is the first-ever pre-clinical study portraying TS as the critical target of combinatorial therapy for mammary carcinomas and therefore we recommend its clinical validation, especially in TNBC patients, who currently have limited therapeutic options.

Insights into an Immunotherapeutic Approach to Combat Multidrug Resistance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) has emerged as one of the most lethal cancers worldwide because of its high refractoriness and multi-drug resistance to existing chemotherapies, which leads to poor patient survival. Novel pharmacological strategies to tackle HCC are based on oral multi-kinase inhibitors like sorafenib; however, the clinical use of the drug is restricted due to the limited survival rate and significant side effects, suggesting the existence of a primary or/and acquired drug-resistance mechanism. Because of this hurdle, HCC patients are forced through incomplete therapy. Although multiple approaches have been employed in parallel to overcome multidrug resistance (MDR), the results are varying with insignificant outcomes. In the past decade, cancer immunotherapy has emerged as a breakthrough approach and has played a critical role in HCC treatment. The liver is the main immune organ of the lymphatic system. Researchers utilize immunotherapy because immune evasion is considered a major reason for rapid HCC progression. Moreover, the immune response can be augmented and sustained, thus preventing cancer relapse over the post-treatment period. In this review, we provide detailed insights into the immunotherapeutic approaches to combat MDR by focusing on HCC, together with challenges in clinical translation.

6,6'-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity.

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6'-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.

Targeting K-Ras and apoptosis-driven cellular transformation in cancer.

Cellular transformation is a major event that helps cells to evade apoptosis, genomic instability checkpoints, and immune surveillance to initiate tumorigenesis and to promote progression by cancer stem cell expansion. However, the key molecular players that govern cellular transformation and ways to target cellular transformation for therapy are poorly understood to date. Here we draw key evidences from the literature on K-Ras-driven cellular transformation in the context of apoptosis to shed light on the key players that are required for cellular transformation and explain how aiming p53 could be useful to target cellular transformation. The defects in key apoptosis regulators such as p53, Bax, and Bak lead to apoptosis evasion, cellular transformation, and genomic instability to further lead to stemness, tumorigenesis, and metastasis via c-Myc-dependent transcription. Therefore enabling key apoptotic checkpoints in combination with K-Ras inhibitors will be a promising therapeutic target in cancer therapy.

Protein kinase C eta is activated in reactive astrocytes of an Alzheimer's disease mouse model: Evidence for its immunoregulatory function in primary astrocytes.

Alzheimer's disease (AD) is the primary cause of age-related dementia. Pathologically, AD is characterized by synaptic loss, the accumulation of ß-amyloid peptides and neurofibrillary tangles, glial activation, and neuroinflammation. Whereas extensive studies focused on neurons and activation of microglia in AD, the role of astrocytes has not been well-characterized. Protein kinase C (PKC) was also implicated in AD; however, its role in astrocyte activation was not elucidated. Using the 5XFAD mouse model of AD, we show that PKC-eta (PKCη), an astrocyte-specific stress-activated and anti-apoptotic kinase, plays a role in reactive astrocytes. We demonstrate that PKCη staining is highly enriched in cortical astrocytes in a disease-dependent manner and in the vicinity of amyloid-ß peptides plaques. Moreover, activation of PKCη, as indicated by its increased phosphorylation levels, is exhibited mainly in cortical astrocytes derived from adult 5XFAD mice. PKCη activation was associated with elevated levels of reactive astrocytic markers and upregulation of the pro-inflammatory cytokine interleukin 6 (IL-6) compared to littermate controls. Notably, inhibiting the kinase activity of PKCη in 5XFAD astrocyte cultures markedly increased the levels of secreted IL-6-a phenomenon that was also observed in wild-type astrocytes stimulated by inflammatory cytokines (e.g., TNFα, IL-1). Similar increase in the release of IL-6 was also observed upon inhibition of either the mammalian target of rapamycin (mTOR) or the protein phosphatase 2A (PP2A). Our findings suggest that the mTOR-PKCη-PP2A signaling cascade functions as a negative feedback loop of NF-κB-induced IL-6 release in astrocytes. Thus, we identify PKCη as a regulator of neuroinflammation in AD.