Research in my laboratory uses biochemical, cellular and molecular genetic approaches to understand questions in cell function, cancer and immunity. Recently, our work has focused on the following 4 topics. A common theme linking these topics is the role of cellular stress in pathology and therapeutics
1) Activation Enhanced Cell Death
Calcium ions (Ca2+) are usually kept at very low levels inside a cell. However in response to many different signals, cells will release Ca2+ from special stores inside the cell. This process, called 'mobilization' also triggers a rush of Ca2+ from the outside to the inside of the cell through specific channels. The result is a very rapid increase in cellular Ca2+. This activates many Ca2+-dependent proteins inside the cell, with short and long term consequences including activation, changes in gene expression and cell growth. While studying the role of calcium signaling in response to a growth factor1 2, we identified a novel mechanism of cell death that we termed 'Activation Enhanced Cell Death' (AECD)3. AECD involves induction of cellular stress by blocking Ca2+ influx and emptying intracellular Ca2+stores 4, 5. We found that cancer cells are more sensitive to the Ca2+influx blocker Econazole (Ec; an anti-fungal agent) than normal cells.For instance, we observed that human blast cells obtained from patients with Acute Myelogenous Leukemia displayed 4 orders of magnitude greater sensitivity to Ec than normal human blood cells 4. Human breast cancer cells also displayed 2 to 3 orders of magnitude more sensitivity to Ec than normal cells 6. Direct injection of Ec into a human breast cancer growing in mice was found to suppress tumor growth 7. We demonstrated that Ec depletes intracellular Ca2+ stores by blocking Ca2+influx AND stimulating Ca2+release directly. We showed that this latter effect was through the generation of reactive oxygen ('Free radicals') 8. Recently, we demonstrated that the oncogene c-myc sensitizes cells to Ec by regulating Ec's ability to generate reactive oxygen 9. Thus Ec targets c-myc-transformed cells. Translational work with this compound includes the generation of a unique liposomal formulation of Ec using a novel method of drug encapsulation called Micelle Transfer 7. Micelle Transfer is the subject of a patent application <10. We also showed that liposomal Ec is well tolerated in mice 7 and has anti-tumor activity using human breast cancer and lung cancer xenografts and a mouse model of metastatic breast cancer.
These experiments provide strong proof of concept for the use of Ec or an improved variant as an anti-cancer agent. However Ec has both practical and commercial limitations as a chemotherapeutic, including the requirement for liposomal encapsulation, some undesirable drug metabolic activity, and its status as an off-patent compound. Our elucidation of its mechanism of action however, makes possible a medicinal chemistry approach to improving its pharmacology while maintaining it's specific anti-cancer properties. I am interested in pursuing opportunities based on this approach.
2) ABC50
In an effort to identify additional cellular factors governing sensitivity and resistance to Ec, we generated cells that were resistant to Ec. Using stepwise selection in increasing amounts of the drug, we isolated a series of clones of HL60 human myelomonocytic leukemia cells that had variable levels of resistance to the drug 11. Characterization of these cells demonstrated: 1) Although selected for resistance to Ec, the cells were also resistant to other stress agents. The cells however remained sensitive to drugs that kill by other mechanisms, indicating that sensitivity was specific for cellular stress. 2)Resistant cells had increased Ca2+ influx capacity compared to the unselected cells and did not deplete Ca2+ stores in response to Ec. 3) Resistant cells contained increased protein synthesis machinery and 4) Resistant cells maintained protein synthesis in response to cellular stress agents. These studies therefore defined a novel phenotype of multi-drug resistance associated specifically with cellular stress.
To further characterize these cells, we performed a technique (called Differential Display) that identifies genes that are expressed at different levels in different cell types. From this analysis, we identified ABC50 (aka ABCF1) as a gene overexpressed in Ec-resistant cells. Biochemically, ABC50 is known to be involved in the initiation of protein synthesis.
Given that the phenotype of Ec-resistant cells involves the maintenance of protein synthesis in response to cellular stress, we investigated the contribution of ABC50 to stress resistance. We observed that knockdown of ABC50 gene expression in Ec-resistant cells partially reversed this resistance. Furthermore, we observed that knockdown of ABC50 in HL60 parental cells enhanced sensitivity to cellular stress while overexpression of the gene partially protected cells. Knockdown and overexpression was associated with altered global protein synthesis levels. Thus knockdown reduced protein synthesis while overexpression enhanced it. This latter effect was of particular interest since it suggested that overexpression of ABC50 might be of general use in boosting expression of proteins, particularly in stressed cells. To test this possibility, we overexpressed ABC50 in a cell line that produces a monoclonal antibody and measured its effect on antibody production. We observed a significant boost in antibody production in these cells. Therefore, ABC50 overexpression may be generally useful in the boosting of protein expression for antibodies or other useful proteins.
A manuscript describing this work has been published 12. A patent claiming the use of ABC50 for boosting protein expression was filed in May 2009 13. Our development strategy is to evaluate the effect of ABC50 overexpressionfor recombinant protein expression under scale-up conditions. This technology may be exceedingly valuable for the production of monoclonal antibodies and recombinant proteins of various kinds.
3) Fibroblast-like synoviocytes in Rheumatoid Arthritis
Statins (i.e. Lipitor and others) are a class of drugs that lower cholesterol production. Statins are exceedingly successful and are one of the most popular drugs of all time. Increasingly, it has been recognized that statins have effects that go beyond cholesterol reduction. These so-called 'pleiotropic' effects include anti-inflammatory activity.Rheumatoid Arthritis (RA) is a chronic inflammatory disease causing progressive joint destruction, deformity and disability. In RA, the joint space fills up with cells of the immune system. These cells stimulate increases in a cell type called fibroblast-like synoviocytes or FLS. FLS are found in normal joints however they are dramatically increased in number and activated in RA. FLS contribute to joint damage by stimulating immune cells and releasing cartilage-damaging enzymes. In RA, FLS expand in number, in part, due to resistance to a normal process of cell death. We have shown that statins can reverse the resistance of the FLS to cell death and 'normalize' the response of these cells 14. Our studies indicate that statins target intracellular signaling pathways that are stimulated by the cytokine TNF-á. These observations suggest that if statins are delivered appropriately in arthritic joints, they will not only exert anti-inflammatory actions but may also act as disease modifying agents with the potential for preventing joint damage caused by activated synovial fibroblasts. Current work is directed towards better understanding the mechanism of action of statins in RA and in developing appropriate drug delivery and therapeutic strategies for treating RA with statins. We have used Micelle Transfer to generate a unique liposomal statin formulation and would like to test it in both arthritis and cancer.
4) Calpain and calpastatin in cancer
Calpains are intracellular proteases (enzymes that break down other proteins) implicated in a wide variety of biological processes. While investigating the role of c-myc turnover in sensitivity to Ec (see above), we found that calpain (but not proteasome) inhibition preferentially killed c-myc-transformed cells. We went on to show that c-myc regulates calpain activity by suppressing expression of the endogenous inhibitor calpastatin 15. Knockdown of calpastatin in c-myc-negative cells restored cell growth and tumorigenicity in these cells implicating calpain activity as an important part of how c-myc transforms cells. We have also observed that calpain activity is highly elevated and varies widely in AML patient samples. This activity variation correlates inversely with calpastatin levels suggesting that calpastatin (possibly through c-myc) is responsible for the calpain variation. Sensitivity of AML blast cells to calpain inhibition also correlates with calpain activity. Human lymphoma cell lines also display variable calpain activity that correlates with sensitivity to inhibition. Current work is directed towards determining if calpain activity16 can be a useful biomarker or prognostic factor in AML, whether calpastatin is a tumor suppressor and determining the therapeutic potential of the calpain inhibition in models of AML and lymphoma.
References
1. Gommerman JL, Berger SA. Protection from apoptosis by steel factor but not interleukin-3 is reversed through blockade of calcium influx. Blood. 1998;91:1891.
2. Gommerman JL, Sittaro D, Klebasz NZ, Williams DA, Berger SA. Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel Factor correlates with leukemic potential. Blood. 2000;96:3734.
3. Berger SA. Signaling pathways influencing SLF and c-kit-mediated survival and proliferation. Immunol Res. 2006;35:1.
4. Soboloff J, Zhang Y, Minden M, Berger S. Sensitivity of myeloid leukemia cells to calcium influx blockade. Application to bone marrow purging. Exp Hematol. 2002;30:1219.
5. Soboloff J, Berger SA. Sustained ER Ca2+ depletion suppresses protein synthesis and induces activation-enhanced cell death in mast cells. J Biol Chem. 2002;277:13812.
6. Zhang Y, Crump M, Berger SA. Purging of contaminating breast cancer cells from hematopoietic progenitor cell preparations using activation enhanced cell death. Breast Cancer Res Treat. 2002;72:265.
7. Cogswell S, Berger S, Waterhouse D, Bally MB, Wasan EK. A parenteral econazole formulation using a novel micelle-to-liposome transfer method: in vitro characterization and tumor growth delay in a breast cancer xenograft model. Pharm Res. 2006;23:2575.
8. Zhang Y, Soboloff J, Zhu Z, Berger SA. Inhibition of Ca2+ influx is required for mitochondrial reactive oxygen species-induced endoplasmic reticulum Ca2+ depletion and cell death in leukemia cells. Mol Pharmacol. 2006;70:1424.
9. Yu Y, Niapour M, Zhang Y, Berger SA. Mitochondrial regulation by c-Myc and hypoxia-inducible factor-1 alpha controls sensitivity to econazole. Mol Cancer Ther. 2008;7:483.
10. Wasan EK, Bally M, Cogswell S, Berger S. Liposomal compositions for parenteral delivery of agents. BC Cancer Agency and University Health Network. 2009;US2009/0028931 A1: filed.
11. Zhang Y, Berger SA. Increased calcium influx and ribosomal content correlate with resistance to endoplasmic reticulum stress-induced cell death in mutant leukemia cell lines. J Biol Chem. 2004;279:6507.
12. Yu Y, Zhang Y, Zhu, Z. Berger SA. ABC50 modulates sensitivity of HL60 leukemic cells to ER stress. Biochem. Pharmacol.2011;81:488.
13. Berger SA. Methods and Compositions for increasing protein production. University Health Network. 2009;US61/175,642: Provisional.
14. Connor AM, Berger S, Narendran A, Keystone EC. Inhibition of protein geranylgeranylation induces apoptosis in synovial fibroblasts. Arthritis Res Ther. 2006;8:R94.
15. Niapour M, Yu Y, Berger SA. Regulation of calpain activity by c-Myc through calpastatin and promotion of transformation in c-Myc-negative cells by calpastatin suppression. J Biol Chem. 2008;283:21371.
16. Niapour M, Berger S. Flow cytometric measurement of calpain activity in living cells. Cytometry A. 2007;71:475.
