Immune checkpoints consist of inhibitory and stimulatory pathways that maintain self-tolerance and assist with immune response. In cancer, immune checkpoint pathways are often activated to inhibit the nascent anti-tumor immune response. Immune checkpoint therapies act by blocking or stimulating these pathways and enhance the body’s immunological activity against tumors. Cytotoxic T lymphocyte-associated molecule-4 (CTLA-4), programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1(PD-L1) are the most widely studied and recognized inhibitory checkpoint pathways. Drugs blocking these pathways are currently utilized for a wide variety of malignancies and have demonstrated durable clinical activities in a subset of cancer patients. This approach is rapidly extending beyond CTLA-4 and PD-1/PD-L1. New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA, or B7/H3 are being investigated. Furthermore, agonists of stimulatory checkpoint pathways such as OX40, ICOS, GITR, 4-1BB, CD40, or molecules targeting tumor microenvironment components like IDO or TLR are under investigation. In this article, we have provided a comprehensive review of immune checkpoint pathways involved in cancer immunotherapy, and discuss their mechanisms and the therapeutic interventions currently under investigation in phase I/II clinical trials. We also reviewed the limitations, toxicities, and challenges and outline the possible future research directions.

Significance Using microfiltration as a liquid biopsy for the recovery of circulating tumor cells (CTCs) has revealed an accompanying macrophage subset that we use as a highly sensitive biomarker for solid tumors. We supply evidence that this circulating giant cell is a subset of disseminated tumor-associated macrophages capable of binding CTCs in peripheral blood of cancer patients. The presence of this cell expands the concept of using a liquid biopsy not only to indicate cancer presence but also to track cancer treatment effects sequentially using other circulating blood cells. Further, we supply observational evidence hypothesizing a metastasis pathway model in which CTCs migrate with pro-angiogenic macrophages, linking cancer cell intravasation, migration, and extravasation and the formation of metastatic microenvironments.

SIRT1 is a class III histone deacetylase and plays important roles in aging, obesity, and cancer (1, 2). Dramatic up-regulation of SIRT1 has been observed in various cancers including breast, prostate, and ovarian cancers, implicating a role for SIRT1 in tumorigenesis (3-5). SIRT1 functions by deacetylating histone (e.g. H3-Lys9 and H4-Lys16) and non-histone proteins (e.g. p300 and Ku70) in an NAD ϩ -dependent manner, thus modifying gene expression and modulating protein activity (1, 6). Previous studies have illustrated several mechanisms of SIRT1-dependent gene silencing in addition to histone deacetylation. It was shown that at sites of DNA damage, SIRT1 recruits DNA methyltransferases (DNMTs) 2 to promoter regions leading to hypermethylation and potential silencing of tumor suppressor genes (e.g. E-cadherin) (7). It is also known that SIRT1 facilitates transcriptional repression of tumor suppressor genes by modulating histone methyltransferase SUV39h1, the key enzyme responsible for histone H3 methylation (H3-Lys9-me3) in regions of heterochromatin (8). SIRT1 induction of tumor suppressor gene silencing promotes the initiation and progression of tumors as well as drug resistance (1, 9, 10). Studies from our laboratory and others show that inhibition of SIRT1 by pharmacological inhibitors or genetic depletion reduces estrogen-dependent signaling pathways in breast cancer cells (11,12). The inhibition of SIRT1 in breast and prostate cancer cell lines has resulted in acetylation of p53 and subsequent growth arrest and apoptosis, while not affecting viability of several non-cancer epithelial cell lines (13,14). Although several inhibitors of sirtuins have been described (reviewed in Ref. 15), and the potential value that SIRT1 inhibition may possess for cancer therapy has been recognized, there are no ongoing clinical trials of SIRT1 inhibitors for cancer therapy because of serious concerns, e.g. stability and toxicity. These deficiencies have lead to the search for new molecules that regulate SIRT1 expression. SIRT1 expression can be mediated at the transcriptional level and several mechanisms involved in dysregulation of SIRT1 in cancer cells have been proposed (16). Tumor suppressors p53 and HIC1 (hypermethylated in cancer 1) can bind to the SIRT1 promoter and form a complex with SIRT1, leading to inhibition of SIRT1 transcription (17,18). In cancer cells, inactivation of these tumor suppressor genes by genetic or epigenetic mechanisms leads to up-regulation of SIRT1 transcription. However, this is not the sole mechanism for overexpression of SIRT1 in tumors. For example, the RNA binding protein HuR, a potential oncoprotein, stabilizes SIRT1 mRNA through 3Ј-untranslated region (3Ј-UTR) interactions leading to elevated SIRT1 levels (19). This suggests that the 3Ј-UTR of SIRT1 mRNA may also be important in governing SIRT1 expression in tumors.

Estrogens are known to be important in the growth of breast cancers in both pre- and postmenopausal women. As the number of breast cancer patients increases with age, the majority of breast cancer patients are postmenopausal women. Although estrogens are no longer made in the ovaries after menopause, peripheral tissues produce sufficient concentrations to stimulate tumor growth. As aromatase catalyzes the final and rate-limiting step in the biosynthesis of estrogen, inhibitors of this enzyme are effective targeted therapy for breast cancer. Three aromatase inhibitors (AIs) are now FDA approved and have been shown to be more effective than the antiestrogen tamoxifen and are well tolerated. AIs are now a standard treatment for postmenopausal patients. AIs are effective in adjuvant and first-line metastatic setting. This review describes the development of AIs and their current use in breast cancer. Recent research focuses on elucidating mechanisms of acquired resistance that may develop in some patients with long term AI treatment and also on innate resistance. Preclinical data in resistance models demonstrated that the crosstalk between ER and other signaling pathways particularly MAPK and PI3K/Akt is an important resistant mechanism. Blockade of these other signaling pathways is an attractive strategy to circumvent the resistance to AI therapy in breast cancer. Several clinical trials are ongoing to evaluate the role of these novel targeted therapies to reverse resistance to AIs.