Abstract
Cancer is the uncontrolled growth of abnormal cells which may lead to the formation of malignant tumors. This disease is the second leading cause of death in the United States, and nearly 600,000 deaths are attributed to cancer each year. Approximately 20% of the US gross domestic product is spent on health care, and the cost of cancer care accounts for 5% of total health care spending.
Cancer stem cells are pluripotent cancer cells that represent a small proportion of malignant cells that can differentiate into different cell types found in tumors, self-renew and divide slowly. These abilities known as “stemness” are responsible for the growth of many primary cancer tumors, and the formation of new tumors. Recent studies have shown that cancer stem cells are resistant to current cytotoxic and antiproliferative therapies, and trigger metastasis and tumor relapse. Cancer stem cell subpopulations have been found in almost every human cancer type and have been shown to have tumorigenic potential in breast cancer, pancreatic cancer, and sarcomas.
An innovative immunotherapy targeting those resistant cancer stem cells involves natural killer (NK) cells. NK cells are lymphocytes critical to the innate immune system. They can quickly and efficiently induce lysis of infected cells and tumor cells without initial sensitization. Those NK cells display several activating and inhibitory receptors on their surface which regulate their activity. Human leukocyte antigen (HLA) complex can bind to inhibitory receptors on NK cells, and trigger an inhibitory signal. This complex serves as a marker to distinguish self from non-self. As a result, cells with low expression of self-HLA on their surface are more susceptible to being targeted and lysed by NK cells. On the other hand, NKG2D (natural-killer group-2, member D) are activating receptors found on NK cells whose ligands are proteins belonging to the MIC family: MICA (Major histocompatibility complex (MHC) class I chain-related protein A) and MICB (MHC class I chain-related protein B). In general, inhibitory signals dominate activating signals. However, NK cells activated through NKG2D signals are capable of inducing apoptosis, even if inhibitory signals are received simultaneously. As a result, NKG2D signaling is described as the “master-switch” in NK cells activation and represents an attractive field of research for cancer therapy.
Additionally, bortezomib is a FDA approved proteasome inhibitor that is utilized as a molecular targeting agent for the treatment of multiple myeloma and mantle cell lymphoma. Proteasomes play a vital role in regulating degradation of unnecessary proteins. As a result, inhibiting proteasomes activity leads to an accumulation of unwanted proteins resulting in programmed cell death. Interestingly, a recent study has shown that bortezomib might promote NK cell-mediated response by reducing HLA class I expression on multiple myeloma cells.
Our primary goal was to use natural killer cells to eliminate residual CSCs as part of cancer treatment with bortezomib. We hypothesized that NKG2D ligands are upregulated after bortezomib treatment which enhances NK cell mediated killing.
To evaluate the effect of bortezomib, we treated several cancer cells with different concentrations and incubation periods. Cell counts, and viability assessments showed a dose and time dependent susceptibility, and flow cytometry revealed an enrichment for cancer stem cells after treatment. We then determined by PCR and flow cytometry that bortezomib increases expression of stress and NK cell ligands. We discovered that MICA/B, as well as DR5, and Fas which are two death receptors are upregulated in a dose dependent manner post treatment with bortezomib. To further evaluate the effect of this molecular targeting agent on cancer stem cells, we sorted U87 glioblastoma cancer cells based on their expression of ALDH, and by PCR we showed that ALDHbright cells displayed an increase in cancer stem cell genes. Furthermore, we also performed ex-vivo experiments by co-culturing activated NK cells with U87 xenografts that revealed a decrease in ALDHbright cells.
Treatment of cancer using molecular targets and NK cells activated in a NKG2D dependent manner could greatly complement and improve our current treatment therapies. It would not only reduce cancer relapse and ultimately cancer mortality, but also cut health care expenses and the increasing economic burden.