Modulatory Role of Phytochemicals/Natural Products in Cancer Immunotherapy.

Immunotherapy is a newly emerging and effective approach to treating cancer. However, there are many challenges associated with using checkpoint inhibitors in this treatment strategy. The component of the tumor microenvironment plays a crucial role in antitumor immune response, regulating tumor immune surveillance as immunological evasion. Natural products/phytochemicals can modulate the tumor microenvironment and function as immunomodulatory agents. In clinical settings, there is a strong need to develop synergistic combination regimens using natural products that can effectively enhance the therapeutic benefits of immune checkpoint inhibitors relative to their effectiveness as single therapies. The review discusses immunotherapy, its side effects, and a summary of evidence suggesting the use of natural products to modulate immune checkpoint pathways.

Beyond cyclopamine: Targeting Hedgehog signaling for cancer intervention.

Hedgehog (Hh) signaling plays a significant role in embryogenesis and several physiological processes, such as wound healing and organ homeostasis. In a pathological setting, it is associated with oncogenesis and is responsible for disease progression and poor clinical outcomes. Hedgehog signaling mediates downstream actions via Glioma Associated Oncogene Homolog (GLI) transcription factors. Inhibiting Hh signaling is an important oncological strategy in which inhibitors of the ligands SMO or GLI have been looked at. This review briefly narrates the Hh ligands, signal transduction, the target genes involved and comprehensively describes the numerous inhibitors that have been evaluated for use in various neoplastic settings.

Targeting acid ceramidase enhances antitumor immune response in colorectal cancer.

INTRODUCTION: Acid ceramidase (hereafter referred as ASAH1) is an enzyme in sphingolipid metabolism that converts pro-survival ceramide into sphingosine. ASAH1 has been shown to be overexpressed in certain cancers. However, the role of ASAH1 in colorectal cancer still remain elusive. OBJECTIVE: The present study is aimed to understand how ASAH1 regulates colorectal cancer (CRC) progression and resistance to checkpoint inhibitor therapy. METHODS: Both pharmacological and genetic silencing of ASAH1 was used in the study. In vitro experiments were done on human and mouse CRC cell lines. The in vivo studies were conducted in NOD-SCID and BALB/c mice models. The combination of ASAH1 inhibitor and checkpoint inhibitor was tested using a syngeneic tumor model of CRC. Transcriptomic and metabolomic analyses were done to understand the effect of ASAH1 silencing. RESULTS: ASAH1 is overexpressed in human CRC cases, and silencing the expression resulted in the induction of immunological cell death (ICD) and mitochondrial stress. The ASAH1 inhibitor (LCL-521), either as monotherapy or in combination with an anti-PD-1 antibody, resulted in reduction of tumors and, through induction of type I and II interferon response, activation of M1 macrophages and T cells, leading to enhanced infiltration of cytotoxic T cells. Our findings supported that the combination of LCL-521 and ICIs, which enhances the antitumor responses, and ASAH1 can be a druggable target in CRC.

Sphingosine 1-phosphate signaling during infection and immunity.

Sphingolipids are essential components of all eukaryotic membranes. The bioactive sphingolipid molecule, Sphingosine 1-Phosphate (S1P), regulates various important biological functions. This review aims to provide a comprehensive overview of the role of S1P signaling pathway in various immune cell functions under different pathophysiological conditions including bacterial and viral infections, autoimmune disorders, inflammation, and cancer. We covered the aspects of S1P pathways in NOD/TLR pathways, bacterial and viral infections, autoimmune disorders, and tumor immunology. This implies that targeting S1P signaling can be used as a strategy to block these pathologies. Our current understanding of targeting various components of S1P signaling for therapeutic purposes and the present status of S1P pathway inhibitors or modulators in disease conditions where the host immune system plays a pivotal role is the primary focus of this review.

Computational drug repurposing of Akt-1 allosteric inhibitors for non-small cell lung cancer.

Non-small cell lung carcinomas (NSCLC) are the predominant form of lung malignancy and the reason for the highest number of cancer-related deaths. Widespread deregulation of Akt, a serine/threonine kinase, has been reported in NSCLC. Allosteric Akt inhibitors bind in the space separating the Pleckstrin homology (PH) and catalytic domains, typically with tryptophan residue (Trp-80). This could decrease the regulatory site phosphorylation by stabilizing the PH-in conformation. Hence, in this study, a computational investigation was undertaken to identify allosteric Akt-1 inhibitors from FDA-approved drugs. The molecules were docked at standard precision (SP) and extra-precision (XP), followed by Prime molecular mechanics-generalized Born surface area (MM-GBSA), and molecular dynamics (MD) simulations on selected hits. Post XP-docking, fourteen best hits were identified from a library of 2115 optimized FDA-approved compounds, demonstrating several beneficial interactions such as pi-pi stacking, pi-cation, direct, and water-bridged hydrogen bonds with the crucial residues (Trp-80 and Tyr-272) and several amino acid residues in the allosteric ligand-binding pocket of Akt-1. Subsequent MD simulations to verify the stability of chosen drugs to the Akt-1 allosteric site showed valganciclovir, dasatinib, indacaterol, and novobiocin to have high stability. Further, predictions for possible biological interactions were performed using computational tools such as ProTox-II, CLC-Pred, and PASSOnline. The shortlisted drugs open a new class of allosteric Akt-1 inhibitors for the therapy of NSCLC.

Therapeutic options in hepatocellular carcinoma: a comprehensive review.

Hepatocellular carcinoma (HCC) is a chronic liver disease that is highly fatal if not detected and treated early. The incidence and death rate of HCC have been increasing in recent decades despite the measures taken for preventive screening and effective diagnostic and treatment strategies. The pathophysiology of HCC is multifactorial and highly complex owing to its molecular and immune heterogeneity, and thus the gap in knowledge still precludes making choices between viable therapeutic options and also the development of effective regimens. The treatment of HCC demands multidisciplinary approaches and primarily depends on tumor stage, hepatic functional reserve, and response to treatment by patients. Although curative treatments are limited but critical in the early stages of cancer, there are numerous palliative treatments available for patients with intermediate and advanced-stage HCC. In recent times, the use of combination therapy has succeeded over the use of monotherapy in the treatment of HCC by achieving effective tumor suppression, increasing survival rate, decreasing toxicity, and also aiding in overcoming drug resistance. This work focuses on reviewing the current and emerging treatment strategies for HCC.

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.

Development and Functional Characterization of a Versatile Radio-/Immunotheranostic Tool for Prostate Cancer Management.

Due to its overexpression on the surface of prostate cancer (PCa) cells, the prostate stem cell antigen (PSCA) is a potential target for PCa diagnosis and therapy. Here we describe the development and functional characterization of a novel IgG4-based anti-PSCA antibody (Ab) derivative (anti-PSCA IgG4-TM) that is conjugated with the chelator DOTAGA. The anti-PSCA IgG4-TM represents a multimodal immunotheranostic compound that can be used (i) as a target module (TM) for UniCAR T cell-based immunotherapy, (ii) for diagnostic positron emission tomography (PET) imaging, and (iii) targeted alpha therapy. Cross-linkage of UniCAR T cells and PSCA-positive tumor cells via the anti-PSCA IgG4-TM results in efficient tumor cell lysis both in vitro and in vivo. After radiolabeling with 64Cu2+, the anti-PSCA IgG4-TM was successfully applied for high contrast PET imaging. In a PCa mouse model, it showed specific accumulation in PSCA-expressing tumors, while no uptake in other organs was observed. Additionally, the DOTAGA-conjugated anti-PSCA IgG4-TM was radiolabeled with 225Ac3+ and applied for targeted alpha therapy. A single injection of the 225Ac-labeled anti-PSCA IgG4-TM was able to significantly control tumor growth in experimental mice. Overall, the novel anti-PSCA IgG4-TM represents an attractive first member of a novel group of radio-/immunotheranostics that allows diagnostic imaging, endoradiotherapy, and CAR T cell immunotherapy.

DOT1L Is a Novel Cancer Stem Cell Target for Triple-Negative Breast Cancer.

PURPOSE: Although chemotherapies kill most cancer cells, stem cell-enriched survivors seed metastasis, particularly in triple-negative breast cancers (TNBC). TNBCs arise from and are enriched for tumor stem cells. Here, we tested if inhibition of DOT1L, an epigenetic regulator of normal tissue stem/progenitor populations, would target TNBC stem cells. EXPERIMENTAL DESIGN: Effects of DOT1L inhibition by EPZ-5676 on stem cell properties were tested in three TNBC lines and four patient-derived xenograft (PDX) models and in isolated cancer stem cell (CSC)-enriched ALDH1+ and ALDH1- populations. RNA sequencing compared DOT1L regulated pathways in ALDH1+ and ALDH1- cells. To test if EPZ-5676 decreases CSC in vivo, limiting dilution assays of EPZ-5676/vehicle pretreated ALDH1+ and ALDH1- cells were performed. Tumor latency, growth, and metastasis were evaluated. Antitumor activity was also tested in TNBC PDX and PDX-derived organoids. RESULTS: ALDH1+ TNBC cells exhibit higher DOT1L and H3K79me2 than ALDH1-. DOT1L maintains MYC expression and self-renewal in ALDH1+ cells. Global profiling revealed that DOT1L governs oxidative phosphorylation, cMyc targets, DNA damage response, and WNT activation in ALDH1+ but not in ALDH1- cells. EPZ-5676 reduced tumorspheres and ALDH1+ cells in vitro and decreased tumor-initiating stem cells and metastasis in xenografts generated from ALDH1+ but not ALDH1- populations in vivo. EPZ-5676 significantly reduced growth in vivo of one of two TNBC PDX tested and decreased clonogenic 3D growth of two other PDX-derived organoid cultures. CONCLUSIONS: DOT1L emerges as a key CSC regulator in TNBC. Present data support further clinical investigation of DOT1L inhibitors to target stem cell-enriched TNBC.

Cardamonin Attenuates Experimental Colitis and Associated Colorectal Cancer.

Cardamonin is a naturally occurring chalcone, majorly from the Zingiberaceae family, which includes a wide range of spices from India. Herein, we investigated the anti-inflammatory property of cardamonin using different in vitro and in vivo systems. In RAW 264.7 cells, treatment with cardamonin showed a reduced nitrous oxide production without affecting the cell viability and decreased the expression of iNOS, TNF-α, and IL-6, and inhibited NF-kB signaling which emphasizes the role of cardamonin as an anti-inflammatory molecule. In a mouse model of dextran sodium sulfate (DSS)-induced colitis, cardamonin treatment protected the mice from colitis. Subsequently, we evaluated the therapeutic potential of this chalcone in a colitis-associated colon cancer model. We performed microRNA profiling in the different groups and observed that cardamonin modulates miRNA expression, thereby inhibiting tumor formation. Together, our findings indicate that cardamonin has the potential to be considered for future therapy against colorectal cancer.

Deletion or inhibition of SphK1 mitigates fulminant hepatic failure by suppressing TNFα-dependent inflammation and apoptosis.

Acute liver failure (ALF) causes severe liver dysfunction that can lead to multi-organ failure and death. Previous studies suggest that sphingosine kinase 1 (SphK1) protects against hepatocyte injury, yet not much is still known about its involvement in ALF. This study examines the role of SphK1 in D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF, which is a well-established experimental mouse model that mimics the fulminant hepatitis. Here we report that deletion of SphK1, but not SphK2, dramatically decreased GalN/LPS-induced liver damage, hepatic apoptosis, serum alanine aminotransferase levels, and mortality rate compared to wild-type mice. Whereas GalN/LPS treatment-induced hepatic activation of NF-κB and JNK in wild-type and SphK2-/- mice, these signaling pathways were reduced in SphK1-/- mice. Moreover, repression of ALF in SphK1-/- mice correlated with decreased expression of the pro-inflammatory cytokine TNFα. Adoptive transfer experiments indicated that SphK1 in bone marrow-derived infiltrating immune cells but not in host liver-resident cells, contribute to the development of ALF. Interestingly, LPS-induced TNFα production was drastically suppressed in SphK1-deleted macrophages, whereas IL-10 expression was markedly enhanced, suggesting a switch to the anti-inflammatory phenotype. Finally, treatment with a specific SphK1 inhibitor ameliorated inflammation and protected mice from ALF. Our findings suggest that SphK1 regulates TNFα secretion from macrophages and inhibition or deletion of SphK1 mitigated ALF. Thus, a potent inhibitor of SphK1 could potentially be a therapeutic agent for fulminant hepatitis.

p27 as a Transcriptional Regulator: New Roles in Development and Cancer.

p27 binds and inhibits cyclin-CDK to arrest the cell cycle. p27 also regulates other processes including cell migration and development independent of its cyclin-dependent kinase (CDK) inhibitory action. p27 is an atypical tumor suppressor-deletion or mutational inactivation of the gene encoding p27, CDKN1B, is rare in human cancers. p27 is rarely fully lost in cancers because it can play both tumor suppressive and oncogenic roles. Until recently, the paradigm was that oncogenic deregulation results from either loss of growth restraint due to excess p27 proteolysis or from an oncogenic gain of function through PI3K-mediated C-terminal p27 phosphorylation, which disrupts the cytoskeleton to increase cell motility and metastasis. In cancers, C-terminal phosphorylation alters p27 protein-protein interactions and shifts p27 from CDK inhibitor to oncogene. Recent data indicate p27 regulates transcription and acts as a transcriptional coregulator of cJun. C-terminal p27 phosphorylation increases p27-cJun recruitment to and action on target genes to drive oncogenic pathways and repress differentiation programs. This review focuses on noncanonical, CDK-independent functions of p27 in migration, invasion, development, and gene expression, with emphasis on how transcriptional regulation by p27 illuminates its actions in cancer. A better understanding of how p27-associated transcriptional complexes are regulated might identify new therapeutic targets at the interface between differentiation and growth control.

Regulatory role of SphK1 in TLR7/9-dependent type I interferon response and autoimmunity.

Plasmacytoid dendritic cells (pDCs) express Toll like receptors (TLRs) that modulate the immune response by production of type I interferons. Here, we report that sphingosine kinase 1 (SphK1) which produces the bioactive sphingolipid metabolite, sphingosine 1-phosphate (S1P), plays a critical role in the pDC functions and interferon production. Although dispensable for the pDC development, SphK1 is essential for the pDC activation and production of type I IFN and pro-inflammatory cytokines stimulated by TLR7/9 ligands. SphK1 interacts with TLRs and specific inhibition or deletion of SphK1 in pDCs mitigates uptake of CpG oligonucleotide ligands by TLR9 ligand. In the pristane-induced murine lupus model, pharmacological inhibition of SphK1 or its genetic deletion markedly decreased the IFN signature, pDC activation, and glomerulonephritis. Moreover, increases in the SphK1 expression and S1P levels were observed in human lupus patients. Taken together, our results indicate a pivotal regulatory role for the SphK1/S1P axis in maintaining the balance between immunosurveillance and immunopathology and suggest that specific SphK1 inhibitors might be a new therapeutic avenue for the treatment of type I IFN-linked autoimmune disorders.

TAMing pancreatic cancer: combat with a double edged sword.

Among all the deadly cancers, pancreatic cancer ranks seventh in mortality. The absence of any grave symptoms coupled with the unavailability of early prognostic and diagnostic markers make the disease incurable in most of the cases. This leads to a late diagnosis, where the disease would have aggravated and thus, incurable. Only around 20% of the cases present the early disease diagnosis. Surgical resection is the prime option available for curative local disease but in the case of advanced cancer, chemotherapy is the standard treatment modality although the patients end up with drug resistance and severe side effects. Desmoplasia plays a very important role in chemoresistance associated with pancreatic cancer and consists of a thick scar tissue around the tumor comprised of different cell populations. The interplay between this heterogenous population in the tumor microenvironment results in sustained tumor growth and metastasis. Accumulating evidences expose the crucial role played by the tumor-associated macrophages in pancreatic cancer and this review briefly presents the origin from their parent lineage and the importance in maintaining tumor hallmarks. Finally we have tried to address their role in imparting chemoresistance and the therapeutic interventions leading to reduced tumor burden.

Acid Ceramidase: A Novel Therapeutic Target in Cancer.

Sphingolipids are important constituents of the eukaryotic cell membrane which govern various signaling pathways related to different aspects of cell survival. Ceramide and Sphingosine are interconvertible sphingolipid metabolites, out of which Ceramide is pro-apoptotic and sphingosine is anti-apoptotic in nature. The conversion of ceramide to sphingosine is mediated by Acid Ceramidase (ASAH1) thus maintaining a rheostat between a tumor suppressor and a tumor promoter. This rheostat is completely altered in many tumors leading to uncontrolled proliferation. This intriguing property of ASAH1 can be used by cancer cells to their advantage, by increasing the expression of the tumor promoter, sphingosine inside cells, thus creating a favorable environment for cancer growth. The different possibilities through which this enzyme serves its role in formation, progression and resistance of different types of cancers will lead to the possibility of making Acid Ceramidase a promising drug target. This review discusses the current understanding of the role of acid ceramidase in cancer progression, metastasis and resistance, strategies to develop novel natural and synthetic inhibitors of ASAH1 and their usefulness in cancer therapy.

Examination of the role of sphingosine kinase 2 in a murine model of systemic lupus erythematosus.

Systemic lupus erythematosus is an autoimmune disease characterized by overproduction of type 1 IFN that causes multiple organ dysfunctions. Plasmacytoid dendritic cells (pDCs) that secrete large amounts of IFN have recently been implicated in the initiation of the disease in preclinical mouse models. Sphingosine-1-phosphate, a bioactive sphingolipid metabolite, is produced by 2 highly conserved isoenzymes, sphingosine kinase (SphK) 1 and SphK2, and regulates diverse processes important for immune responses and autoimmunity. However, not much is known about the role of SphK2 in autoimmune disorders. In this work, we examined the role of SphK2 in pDC development and activation and in the pristane-induced lupus model in mice that mimics the hallmarks of the human disease. Increases in pDC-specific markers were observed in peripheral blood of SphK2 knockout mice. In agreement, the absence of SphK2 increased the differentiation of FMS-like tyrosine kinase 3 ligand dendritic cells as well as expression of endosomal TLRs, TLR7 and TLR9, that modulate production of IFN. Surprisingly, however, SphK2 deficiency did not affect the initiation or progression of pristane-induced lupus. Moreover, although absence of SphK2 increased pDC frequency in pristane-induced lupus, there were no major changes in their activation status. Additionally, SphK2 expression was unaltered in lupus patients. Taken together, our results suggest that SphK2 may play a role in dendritic cell development. Yet, because its deletion had no effect on the clinical lupus parameters in this preclinical model, inhibitors of SphK2 might not be useful for treatment of this devastating disease.-Mohammed, S., Vineetha, N. S., James, S., Aparna, J. S., Lankadasari, M. B., Allegood, J. C., Li, Q.-Z., Spiegel, S., Harikumar, K. B. Examination of the role of sphingosine kinase 2 in a murine model of systemic lupus erythematosus.

Targeting S1PR1/STAT3 loop abrogates desmoplasia and chemosensitizes pancreatic cancer to gemcitabine.

Rationale: Pancreatic cancer is associated with poor prognosis with a 5-year survival rate of less than 6%. Approximately 90% of pancreatic cancer patients harbor somatic mutations in the KRAS gene. Multiple lines of evidence suggest a persistent activation of STAT3 in KRAS-driven oncogenesis contributing to desmoplasia and gemcitabine resistance. Sphingosine 1-phosphate receptor 1 (S1PR1) is an integral component of tumor progression and maintains an activated state of STAT3. FTY720 is an approved drug for multiple sclerosis and acts as a functional antagonist for S1PR1. Here we explored the potential utility of FTY720 to target S1PR1/STAT3 and other major signaling pathways in pancreatic cancer, and sought proof-of-principle for repurposing FTY720 for the treatment of pancreatic cancer. Methods: We examined the activity of FTY720 in the proliferation, apoptosis, and cell cycle assays in human and mouse pancreatic cancer model systems. Further, we studied the efficacy of using a combination of FTY720 and gemcitabine as opposed to individual agents in vitro as well as in vivoResults: Treatment of human and mouse pancreatic cancer cells with FTY720 resulted in inhibition of growth, increased apoptosis, and cell cycle arrest. FTY720 in combination with gemcitabine breached the mitochondrial membrane potential, altered the S1PR1-STAT3 loop, and inhibited epithelial to mesenchymal (EMT) transition. Data from murine models exhibited a marked reduction in the tumor size, increased apoptosis, inhibited NF-κB, S1PR1/STAT3, Shh signaling and desmoplasia, modulated the expression of gemcitabine-metabolizing transport enzymes, and restored the expression of tumor suppressor gene PP2A. Conclusion: Taken together, our results established FTY720 as a propitious molecule, which increases the efficacy of gemcitabine and represents a promising agent in the management of pancreatic cancer.

Enhanced expression of histone chaperone APLF associate with breast cancer.

DNA damage-specific histone chaperone Aprataxin PNK-like factor (APLF) regulates mesenchymal-to-epithelial transition (MET) during cellular reprogramming. We investigated the role of APLF in epithelial-to-mesenchymal transition (EMT) linked to breast cancer invasiveness and metastasis. Here, we show that a significant manifestation of APLF is present in tumor sections of patients with invasive ductal carcinoma when compared to their normal adjacent tissues. APLF was significantly induced in triple negative breast cancer (TNBC) cells, MDAMB-231, in comparison to invasive MCF7 or normal MCF10A breast cells and supported by studies on invasive breast carcinoma in The Cancer Genome Atlas (TCGA). Functionally, APLF downregulation inhibited proliferative capacity, altered cell cycle behavior, induced apoptosis and impaired DNA repair ability of MDAMB-231 cells. Reduction in APLF level impeded invasive, migratory, tumorigenic and metastatic potential of TNBC cells with loss in expression of genes associated with EMT while upregulation of MET-specific gene E-cadherin (CDH1). So, here we provided novel evidence for enrichment of APLF in breast tumors, which could regulate metastasis-associated EMT in invasive breast cancer. We anticipate that APLF could be exploited as a biomarker for breast tumors and additionally could be targeted in sensitizing cancer cells towards DNA damaging agents.

Cardamonin inhibits colonic neoplasia through modulation of MicroRNA expression.

Colorectal cancer is currently the third leading cause of cancer related deaths. There is considerable interest in using dietary intervention strategies to prevent chronic diseases including cancer. Cardamonin is a spice derived nutraceutical and herein, for the first time we evaluated the therapeutic benefits of cardamonin in Azoxymethane (AOM) induced mouse model of colorectal cancer. Mice were divided into 4 groups of which three groups were given six weekly injections of AOM. One group served as untreated control and remaining groups were treated with either vehicle or Cardamonin starting from the same day or 16 weeks after the first AOM injection. Cardamonin treatment inhibited the tumor incidence, tumor multiplicity, Ki-67 and ß-catenin positive cells. The activation of NF-kB signaling was also abrogated after cardamonin treatment. To elucidate the mechanism of action a global microRNA profiling of colon samples was performed. Computational analysis revealed that there is a differential expression of miRNAs between these groups. Subsequently, we extend our findings to human colorectal cancer and found that cardamonin inhibited the growth, induces cell cycle arrest and apoptosis in human colorectal cancer cell lines. Taken together, our study provides a better understanding of chemopreventive potential of cardamonin in colorectal cancer.