The evolutionary legacy of immune checkpoint inhibitors.

Immune check point inhibitors (ICIs) have marked their existence in the field of cancer immunotherapy. Their existence dates to 2011 when the first anti-cytotoxic T lymphocyte-associated protein 4 (CTLA-4) got its FDA approval for the management of metastatic melanoma. The class of ICIs now also include antibodies against programmed cell death-1 (PD-1) and its ligand (PD-L1) which immediately gained FDA approval for use against multiple cancer types because of their effect on patient survival. These discoveries were followed by a significant rise in the identification of novel ICIs with potential anti-tumor response. Researchers have identified various novel checkpoint inhibitors which are currently under clinical trials. Despite the success of ICIs, only a small subset of patients with specific tumor types achieves a promising response. Not only efficient therapeutic response but also development of resistance, recurrence and other immune-related adverse effects limit the applicability of immune checkpoint inhibitors. These challenges can only be addressed when a directed approach is implemented at both basic and translational level. In this review, we have briefly discussed the history of ICIs, the next generation of inhibitors which are currently under clinical trial and mechanisms of resistance that can lead to treatment failure. Ultimately, by combining these insights researchers might be able to achieve a more durable and effective response in cancer patients.

Immune checkpoint proteins: Signaling mechanisms and molecular interactions in cancer immunotherapy.

Immune checkpoint proteins (ICP) are currently one of the most novel and promising areas of immune-oncology research. This novel way of targeting tumor cells has shown favorable success over the past few years with some FDA approvals such as Ipilimumab, Nivolumab, Pembrolizumab etc. Currently, more than 3000 clinical trials of immunotherapeutic agents are ongoing with majority being ICPs. However, as the number of trials increase so do the challenges. Some challenges such as adverse side effects, non-specific binding on healthy tissues and absence of response in some subset populations are critical obstacles. For a safe and effective further therapeutic development of molecules targeting ICPs, understanding their mechanism at molecular level is crucial. Since ICPs are mostly membrane bound receptors, a number of downstream signaling pathways divaricate following ligand-receptor binding. Most ICPs are expressed on more than one type of immune cell populations. Further, the expression varies within a cell type. This naturally varied expression pattern adds to the difficulty of targeting specific effector immune cell types against cancer. Hence, understanding the expression pattern and cellular mechanism helps lay out the possible effect of any immunotherapy. In this review, we discuss the signaling mechanism, expression pattern among various immune cells and molecular interactions derived using interaction database analysis (BioGRID).

GABAA receptor agonist suppresses pediatric medulloblastoma progression by inhibiting PKA-Gli1 signaling axis.

The Sonic hedgehog-activated subgroup of medulloblastoma (SHH-MB) is one of the most common malignant pediatric brain tumors. Recent clinical studies and genomic databases indicate that GABAA receptor holds significant clinical relevance as a therapeutic target for pediatric MB. Herein, we report that "moxidectin," a GABAA receptor agonist, inhibits the proliferation of Daoy, UW426, UW228, ONS76, and PFSK1 SHH-MB cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy demonstrated that moxidectin significantly induced GABAA receptor expression and inhibited cyclic AMP (cAMP)-mediated protein kinase A (PKA)-cAMP response element-binding protein (CREB)-Gli1 signaling in SHH-MB. Gli1 and the downstream effector cancer stem cell (CSC) molecules such as Pax6, Oct4, Sox2, and Nanog were also inhibited by moxidectin treatment. Interestingly, moxidectin also inhibited the expression of MDR1. Mechanistic studies using pharmacological or genetic inhibitors/activators of PKA and Gli1 confirmed that the anti-proliferative and apoptotic effects of moxidectin were mediated through inhibition of PKA-Gli1 signaling. Oral administration of 2.5 mg/kg moxidectin suppressed the growth of SHH-MB tumors by 55%-80% in subcutaneous and intracranial tumor models in mice. Ex vivo analysis of excised tumors confirmed the observations made in the in vitro studies. Moxidectin is an FDA-approved drug with an established safety record, therefore any positive findings from our studies will prompt its further clinical investigation for the treatment of MB patients.

Repurposing antipsychotics of the diphenylbutylpiperidine class for cancer therapy.

The recent development of high throughput compound screening has allowed drug repurposing to emerge as an effective avenue for discovering novel treatments for cancer. FDA-approved antipsychotic drugs fluspirilene, penfluridol, and pimozide are clinically used for the treatment of psychotic disorders, primarily schizophrenia. These compounds, belong to diphenylbutylpiperidine class of antipsychotic drugs, are the potent inhibitors of dopamine D2 receptor and calcium channel. A correlation has been found that patients treated for schizophrenia have lower incidences of certain types of cancer, such as respiratory, prostate, and bladder cancers. These compounds have also been shown to inhibit cancer proliferation in a variety of cancer cells, including melanoma, lung carcinoma, breast cancer, pancreatic cancer, glioma, and prostate cancer, among others. Antipsychotic drugs induce apoptosis and suppress metastasis in in vitro and in vivo models through mechanisms involving p53, STAT3, STAT5, protein phosphatase 2A, cholesterol homeostasis, integrins, autophagy, USP1, wnt/ß-catenin signaling, and DNA repair. Additionally, pre-clinical evidence suggests that penfluridol and pimozide act synergistically with existing chemotherapeutic agents, such as dasatinib, temozolomide, and cisplatin. Some studies have also reported that the cytotoxic activity of the antipsychotics is selective for dividing cells. Based on this growing body of evidence and the availability and previous FDA-approval of the drugs, the compounds appear to be promising anti-cancer agents.

Drug rechanneling: A novel paradigm for cancer treatment.

Cancer continues to be one of the leading contributors towards global disease burden. According to NIH, cancer incidence rate per year will increase to 23.6 million by 2030. Even though cancer continues to be a major proportion of the disease burden worldwide, it has the lowest clinical trial success rate amongst other diseases. Hence, there is an unmet need for novel, affordable and effective anti-neoplastic medications. As a result, a growing interest has sparkled amongst researchers towards drug repurposing. Drug repurposing follows the principle of polypharmacology, which states, "any drug with multiple targets or off targets can present several modes of action". Drug repurposing also known as drug rechanneling, or drug repositioning is an economic and reliable approach that identifies new disease treatment of already approved drugs. Repurposing guarantees expedited access of drugs to the patients as these drugs are already FDA approved and their safety and toxicity profile is completely established. Epidemiological studies have identified the decreased occurrence of oncological or non-oncological conditions in patients undergoing treatment with FDA approved drugs. Data from multiple experimental studies and clinical observations have depicted that several non-neoplastic drugs have potential anticancer activity. In this review, we have summarized the potential anti-cancer effects of anti-psychotic, anti-malarial, anti-viral and anti-emetic drugs with a brief overview on their mechanism and pathways in different cancer types. This review highlights promising evidences for the repurposing of drugs in oncology.

Therapeutic Targeting of Vasculature in the Premetastatic and Metastatic Niches Reduces Lung Metastasis.

In the metastasis-targeted organs, angiogenesis is essential for the progression of dormant micrometastases to rapidly growing and clinically overt lesions. However, we observed changes suggesting angiogenic switching in the mouse lungs prior to arrival of tumor cells (i.e., in the premetastatic niche) in the models of breast carcinoma. This angiogenic switching appears to be caused by myeloid-derived suppressor cells recruited to the premetastatic lungs through complement C5a receptor 1 signaling. These myeloid cells are known to secrete several proangiogenic factors in tumors, including IL-1ß and matrix metalloproteinase-9, and we found upregulation of these genes in the premetastatic lungs. Blockade of C5a receptor 1 synergized with antiangiogenic Listeria monocytogenes-based vaccines to decrease the lung metastatic burden by reducing vascular density and improving antitumor immunity in the lungs. This was mediated even when growth of primary breast tumors was not affected by these treatments. This work provides initial evidence that angiogenesis contributes to the premetastatic niche in rapidly progressing cancers and that inhibiting this process through immunotherapy is beneficial for reducing or even preventing metastasis.

CRISPR-Cas9: A multifaceted therapeutic strategy for cancer treatment.

CRISPR-Cas9 is an RNA guided endonuclease that has revolutionized the ability to edit genome and introduce desired manipulations in the target genomic sequence. It is a flexible methodology and is capable of targeting multiple loci simultaneously. Owing to the fact that cancer is an amalgamation of several genetic mutations, application of CRISPR-Cas9 technology is considered as a novel strategy to combat cancer. Genetic and epigenetic modulations in cancer leads to development of resistance to conventional therapy options. Given the abundance of transcriptomic and genomic alterations in cancer, developing a strategy to decipher these alterations is critical. CRISPR-Cas9 system has proven to be a promising tool in generating cellular and animal models to mimic the mutations and understand their role in tumorigenesis. CRISPR-Cas9 is an upheaval in the field of cancer immunotherapy. Furthermore, CRISPR-Cas9 plays an important role in the development of whole genome libraries for cancer patients. This approach will help understand the diversity in genome variation among the patients and also, will provide multiple variables to scientists to investigate and improvise cancer therapy. This review will focus on the discovery of CRISPR-Cas9 system, mechanisms behind CRISPR technique and its current status as a potential tool for investigating the genomic mutations associated with all cancer types.

Atovaquone Suppresses Triple-Negative Breast Tumor Growth by Reducing Immune-Suppressive Cells.

A major contributing factor in triple-negative breast cancer progression is its ability to evade immune surveillance. One mechanism for this immunosuppression is through ribosomal protein S19 (RPS19), which facilitates myeloid-derived suppressor cells (MDSCs) recruitment in tumors, which generate cytokines TGF-ß and IL-10 and induce regulatory T cells (Tregs), all of which are immunosuppressive and enhance tumor progression. Hence, enhancing the immune system in breast tumors could be a strategy for anticancer therapeutics. The present study evaluated the immune response of atovaquone, an antiprotozoal drug, in three independent breast-tumor models. Our results demonstrated that oral administration of atovaquone reduced HCC1806, CI66 and 4T1 paclitaxel-resistant (4T1-PR) breast-tumor growth by 45%, 70% and 42%, respectively. MDSCs, TGF-ß, IL-10 and Tregs of blood and tumors were analyzed from all of these in vivo models. Our results demonstrated that atovaquone treatment in mice bearing HCC1806 tumors reduced MDSCs from tumor and blood by 70% and 30%, respectively. We also observed a 25% reduction in tumor MDSCs in atovaquone-treated mice bearing CI66 and 4T1-PR tumors. In addition, a decrease in TGF-ß and IL-10 in tumor lysates was observed in atovaquone-treated mice with a reduction in tumor Tregs. Moreover, a significant reduction in the expression of RPS19 was found in tumors treated with atovaquone.

Targeting Glioblastoma Tumor Microenvironment.

Glioblastoma, also referred to as glioblastoma multiforme (GBM), is an aggressive type of brain cancer. The prognosis for GBM is poor with an average medium survival rate of 12-15 months. GBM is highly challenging to treat due to neural stem cells phenotypic variations. These variations are determined by the tumor microenvironment (TME), which refers to all the molecules, cells, and structures that encompass and support other cells and tissues. Along with these, other vital components of the TME are fibroblasts, immune and inflammatory cells, blood and lymphatic vascular networks, extracellular matrix, and signaling molecules. This chapter provides an in-depth review of the vital components that form the TME and methods currently under development attempting to target each key area.

Low Dose of Penfluridol Inhibits VEGF-Induced Angiogenesis.

: Metastasis is considered a major burden in cancer, being responsible for more than 90% of cancer-related deaths. Tumor angiogenesis is one of the main processes that lead to tumor metastasis. Penfluridol is a classic and commonly used antipsychotic drug, which has a great ability to cross the blood-brain barrier. Recent studies have revealed that penfluridol has significant anti-cancer activity in diverse tumors, such as metastatic breast cancer and glioblastoma. Here, we aim to identify the effect of low doses of penfluridol on tumor microenvironment and compare it with its effect on tumor cells. Although low concentration of penfluridol was not toxic for endothelial cells, it blocked angiogenesis in vitro and in vivo. In vitro, penfluridol inhibited VEGF-induced primary endothelial cell migration and tube formation, and in vivo, it blocked VEGF- and FGF-induced angiogenesis in the matrigel plug assay. VEGF-induced VEGFR2 phosphorylation and the downstream p38 and ERK signaling pathways were not affected in endothelial cells, although VEGF-induced Src and Akt activation were abrogated by penfluridol treatment. When cancer cells were treated with the same low concentration of penfluridol, basal Src activation levels were mildly impaired, thus impacting their cell migration and wound healing efficiency. The potential of cancer-induced paracrine effect on endothelial cells was explored, although that did not seem to be a player for angiogenesis. Overall, our data demonstrates that low penfluridol levels, similar to the ones clinically used for anti-psychotic conditions, suppress angiogenic efficiency in the tumor microenvironment.

Role of Forkhead Box Class O proteins in cancer progression and metastasis.

It is now widely accepted that several gene alterations including transcription factors are critically involved in cancer progression and metastasis. Forkhead Box Class O proteins (FoxOs) including FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX and FoxO6 transcription factors are known to play key roles in proliferation, apoptosis, metastasis, cell metabolism, aging and cancer biology through their phosphorylation, ubiquitination, acetylation and methylation. Though FoxOs are proved to be mainly regulated by upstream phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K)/Akt signaling pathway, the role of FoxOs in cancer progression and metastasis still remains unclear so far. Thus, with previous experimental evidences, the present review discussed the role of FoxOs in association with metastasis related molecules including cannabinoid receptor 1 (CNR1), Cdc25A/Cdk2, Src, serum and glucocorticoid inducible kinases (SGKs), CXCR4, E-cadherin, annexin A8 (ANXA8), Zinc finger E-box-binding homeobox 2 (ZEB2), human epidermal growth factor receptor 2 (HER2) and mRNAs such as miR-182, miR-135b, miR-499-5p, miR-1274a, miR-150, miR-34b/c and miR-622, subsequently analyzed the molecular mechanism of some natural compounds targeting FoxOs and finally suggested future research directions in cancer progression and metastasis.

Role of Phytochemicals in Cancer Prevention.

The use of synthetic, natural, or biological agents to minimize the occurrence of cancer in healthy individuals is defined as cancer chemoprevention. Chemopreventive agents inhibit the development of cancer either by impeding DNA damage, which leads to malignancy or by reversing or blocking the division of premalignant cells with DNA damage. The benefit of this approach has been demonstrated in clinical trials of breast, prostate, and colon cancer. The continuous increase in cancer cases, failure of conventional chemotherapies to control cancer, and excessive toxicity of chemotherapies clearly demand an alternative approach. The first trial to show benefit of chemoprevention was undertaken in breast cancer patients with the use of tamoxifen, which demonstrated a significant decrease in invasive breast cancer. The success of using chemopreventive agents for protecting the high risk populations from cancer indicates that the strategy is rational and promising. Dietary components such as capsaicin, cucurbitacin B, isoflavones, catechins, lycopenes, benzyl isothiocyanate, phenethyl isothiocyanate, and piperlongumine have demonstrated inhibitory effects on cancer cells indicating that they may serve as chemopreventive agents. In this review, we have addressed the mechanism of chemopreventive and anticancer effects of several natural agents.

Correction to: Regulation of SIRT1/AMPK axis is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in hepatocellular carcinoma cells.

This article contains an error.

Regulation of SIRT1/AMPK axis is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most fatal malignancies with high mortality worldwide. Here the underlying antitumor mechanism of gallotannin was elucidated in HCC cells. Gallotannin suppressed viability and colony formation, increased subG1 portion and also induced senescence via upregulation of p21, G0/G1 arrest and higher SA-ß-gal activity in HepG2 and SK-Hep1 cells. However, pan-caspase inhibitor Z-VAD-FMK reversed the ability of gallotannin to activate caspase 3 at 48 h after treatment in two HCC cells. Of note, gallotannin also induced autophagic features by increasing LC3 punctae, LC3B-II conversion, autophagic vacuoles and decreasing the expression of Beclin1 in two HCC cells. Furthermore, autophagy flux assay using GFP-mRFP-LC3 plasmid revealed increased yellowish color and late autophagy inhibitor CQ or NH4Cl enhanced cytotoxicity, LC3B-II conversion, and LC3 punctae in gallotannin-treated HepG2 and SK-Hep1 cells compared to early autophagy inhibitor 3-MA or wortmannin. Interestingly, gallotannin attenuated the expression of SIRT1 and mTOR and activated phosphorylation of AMPK in two HCC cells. Furthermore, AMPK activator AICAR significantly enhanced SA-ß-gal activity and antiproliferation induced by gallotannin, while AMPK inhibitor compound C did not in two HCC cells. Consistently, LC3B-II conversion by gallotannin was not shown in AMPKα1 -/- MEF cells compared to WT AMPK +/+ MEF cells. Consistently, gallotannin reduced in vivo growth of HepG2 cells implanted in NCr nude mice along with decreased expression of PCNA and SIRT1 and increased AMPKα1 and TUNEL. Overall, these findings highlight evidence that regulation of SIRT1/AMPK is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in HCC cells.

Penfluridol induces endoplasmic reticulum stress leading to autophagy in pancreatic cancer.

Pancreatic cancer is one of the most aggressive and difficult to treat cancers. Experimental and clinical evidence suggests that high basal state autophagy in pancreatic tumors could induce resistance to chemotherapy. Recently, we have demonstrated that penfluridol suppresses pancreatic tumor growth by autophagy-mediated apoptosis both in vitro and in vivo; however, the mechanism of autophagy induction by penfluridol was not clear. Several studies have established that endoplasmic reticulum stress could lead to autophagy and inhibit tumor progression. In this study, we demonstrated that penfluridol induced endoplasmic reticulum stress in BxPC-3, AsPC-1, and Panc-1 pancreatic cancer cell lines as indicated by upregulation of endoplasmic reticulum stress markers such as binding protein (BIP), C/EBP homologous protein (CHOP) and inositol requiring 1α (IRE1α) after treatment with penfluridol in a concentration-dependent manner. Inhibiting endoplasmic reticulum stress by pretreatment with pharmacological inhibitors such as sodium phenylbutyrate and mithramycin or by silencing CHOP using CHOP small interfering RNA, blocked penfluridol-induced autophagy. These results clearly indicate that penfluridol-induced endoplasmic reticulum stress lead to autophagy in our model. Western blot analysis of subcutaneously implanted AsPC-1 and BxPC-3 tumors as well as orthotopically implanted Panc-1 tumors demonstrated upregulation of BIP, CHOP, and IRE1α expression in the tumor lysates from penfluridol-treated mice as compared to tumors from control mice. Altogether, our study establishes that penfluridol-induced endoplasmic reticulum stress leads to autophagy resulting in reduced pancreatic tumor growth. Our study opens a new therapeutic target for advanced chemotherapies against pancreatic cancer.

STAT3 induces anoikis resistance, promotes cell invasion and metastatic potential in pancreatic cancer cells.

Tumor cells need to attain anoikis resistance to survive prior to metastasis making it a vital trait of malignancy. The mechanism by which pancreatic cancer cells resist anoikis and metastasize is not well established. Significant proportion of pancreatic cancer cells resisted anoikis when grown under anchorage-independent conditions. The cells that resisted anoikis showed higher migratory and invasive characteristics than the cells that were cultured under anchorage-dependent condition. Interestingly, anoikis-resistant cells exhibited significantly increased expression and phosphorylation of signal transducer and activation of transcription 3 (STAT3) at Tyr 705, as compared to adherent cells. AG 490 and piplartine (PL) induced significant anoikis in anoikis-resistant pancreatic cancer cells. Silencing STAT3 not only reduced the capacity of pancreatic cancer cells to resist anoikis but also reversed its invasive characteristics. Interleukin-6 treatment and overexpression of STAT3 enhanced anoikis resistance and protected the cells from PL-induced anoikis. PL-treated cells completely failed to develop tumors when injected subcutaneously in immune-compromised mice. Moreover, these cells also failed to metastasize when injected intravenously. On the other hand, untreated anoikis-resistant cells not only formed aggressive tumors but also metastasized substantially to lungs and liver when injected intravenously. Metastatic nodules formed by untreated anoikis-resistant cells in lungs exhibited significant phosphorylation of STAT3 at Tyr705. Taken together, our results established the critical involvement of STAT3 in conferring anoikis resistance to pancreatic cancer cells and increased metastasis.

Phenethyl isothiocyanate: a comprehensive review of anti-cancer mechanisms.

The epidemiological evidence suggests a strong inverse relationship between dietary intake of cruciferous vegetables and the incidence of cancer. Among other constituents of cruciferous vegetables, isothiocyanates (ITC) are the main bioactive chemicals present. Phenethyl isothiocyanate (PEITC) is present as gluconasturtiin in many cruciferous vegetables with remarkable anti-cancer effects. PEITC is known to not only prevent the initiation phase of carcinogenesis process but also to inhibit the progression of tumorigenesis. PEITC targets multiple proteins to suppress various cancer-promoting mechanisms such as cell proliferation, progression and metastasis. Pre-clinical evidence suggests that combination of PEITC with conventional anti-cancer agents is also highly effective in improving overall efficacy. Based on accumulating evidence, PEITC appears to be a promising agent for cancer therapy and is already under clinical trials for leukemia and lung cancer. This is the first review which provides a comprehensive analysis of known targets and mechanisms along with a critical evaluation of PEITC as a future anti-cancer agent.

Syntheses, neural protective activities, and inhibition of glycogen synthase kinase-3ß of substituted quinolines.

A new series of fifteen 5-, 6-, and 8-appended 4-methylquinolines were synthesized and evaluated for their neural protective activities. Selected compounds were further examined for their inhibition of glycogen synthase kinase-3ß (GSK-3ß) and protein kinase C (PKC). Two most potent analogs, compounds 3 and 10, show nanomolar protective activities in amyloid ß-induced MC65 cells and enzymatic inhibitory activities against GSK-3ß, but poor PKC inhibitory activities. Using normal mouse model, the distribution of the most potent analog 3 in various tissues and possible toxic effects in the locomotors and inhibition of liver transaminases activities were carried out. No apparent decline of locomotor activity and no inhibition of liver transaminases were found. The compound appears to be safe for long-term use in Alzheimer's disease mouse model.