Ovarian & Pancreatic Cancer Research – Dr. Batchu (Surgical Oncology & Developmental Therapeutics Lab)
Our research focuses on the use of Adeno-associated virus (AAV) as the gene transfer vehicle. First project is the knockdown of Enhance of Zeste Homolog 2 (EZH2) by microRNA-101 (miR-101) and microRNA-26a (miR-26a) targeting multiple cancers. Second project is the development of therapeutic cancer vaccine for pancreatic and ovarian cancers using cell fusion proteins of cell penetrating domain (CPD) with tumor specific antigens (TSA) or with AAV delivery of TSA. We are working to develop mutant AAV serotype that can efficiently transducer various target cells in both projects.
- miR-101 and miR-26a mediated inhibition of EzH2.
- Development of therapeutic cancer vaccine.
- Cell penetrating domain delivery o f tumor specific protein.
- Adeno-associated mediated delivery of tumor specific genes.
1. Targeting Pancreatic and Ovarian Cancers by RNAi Mediated Inhibition of EzH2
Enhancer of Zeste homolog 2 (EzH2) is the catalytic subunit of the PRC2 complex which trimethylates histone H3 at lysine residuce 27 (H3K27me3), often at the sites of tumor suppressor genes. This epigenetic modification leads to dense packaging of chromatin, making it less accessible to the transcriptional machinery, eventually resulting in silencing of these genes. Although EzH2 is not over-expressed in normal adult differentiated tissues, it is often over-expressed in human cancers. High levels of EzH2 have been associated with aggressive forms of prostate, breast, hepatocellular, and gastric cancers, as well as melanoma making it a potential marker of solid tumors. RNA interference (RNAi)-mediated reduction in EzH2 expression significantly suppressed tumor growth and proliferation in multiple cancers.Inhibition of tumor suppressor genes is a key event in carcinogenesis. Recently, an inverse correlation between the expression of EzH2 and that of tumor suppressor genes such as E-cadherin in gastric cancer, p27kip (CDKN1B) in pancreatic cancer, p57kip2 (CDKI) in breast cancer and RAS associated domain family 1 (RASSF1) in ovarian cancer has been described. The loss ofp21waf1/cip1, a G1 phase cell cycle inhibitor is frequently observed in ovarian cancers and is known to confer a growth advantage. In fact p21waf1/cip1 acts downstream of p53, which allows cells to repair damaged DNA and eventually inhibit carcinogenesis.MicroRNAs (miRNAs) are a group of short non-coding RNAs classified as RNA interference (RNAi), known to regulate cellular processes such as proliferation, differentiation, and apoptosis. Emerging evidence strongly suggests that some miRNAs are consistently deregulated in human cancers, underlining their important role in tumorigenesis. Comparison between human cancers and their respective normal tissue counterparts have revealed distinct miRNA expression profiles. Evidence suggests that the expression of mirRNAs is remarkably deregulated in PDAC implicating their involvement in the initiation and progreassion of this disease. A significant association between the expression of miRNAs and disease status in pancreatic cancer has also been established in the recent past. MiR-101 and miR-26a are known to target EzH2, thus inhibiting tumor progression.
The main difficulty thus far in extending the powers of RNAi to clinical practice has been the development of safe vectors coding for shRNAs or microRNAs in vivo. Adeno-associated viral (AAV) vectors are different from other vectors as the only gene expressed from recombinant vector is trans-gene itself, a naturally gutless by design, thus avoiding any cytotoxic cellular immune responses in the host adenoviral vectors. Furthermore, AAV vectors show only a modest frequency of integration into the host genome thus avoiding insertional mutagenesis, which has been a stumbling block for the clinical use of retroviral or lentiviral vectors. Although RNAi molecules can be introduces into cells as double stranded or expressed from a plasmid to inhibit abnormally elevated genes, transfections of these purchased molecules from commercial sources is impractical and has little value for translational work. Our lab has been working to modify AAV vectors for efficient entry of RNAi molecules to target cells and with robust expression in the cell.
Following is the schematic representation of signaling mechanisms by miR-101 and miR-26a in tumor cells.
2. Therapeutic cancer vaccines
The clinical course of cancer is often marked by periods of relapse and remission until chemotherapeutic resistance develops. Immune-based strategies may be able to eliminate minimal disease burden of patients who are in remission. Immunotherapy with Dendritic Cells (DCs) is a relatively non-toxic alternative that may overcome elements of host immune incompetence to generate Cytotoxic T Lymphocytes (CTLs) that lyse tumor cells. We are further exploring possibility of immunotherapy with B celsl that are more readily available and easy to expand compared with DCs. MAGE-A3 and NY-ESO-1 belong to a family of tumor-specific cancer/testes (C/T) antigens which are highly expressed in a significant proportion of Epithelial Ovarian Cancers (EOCs)but absent in normal tissue except for immune-privileged sites. C/T antigen expression in the cytoplasm of DCs is necessary to access the HLA class I pathway of antigen processing in order to prime CD8+T cells leading to the generation of CTLs. However, most first-generation vaccines are formulated by exogenous pulsing of DCs with tumor specific antigen (TSA)/lysate/RNA resulting in compartmentalization of TSAs in endosomes with only limited entry into the cytoplasm. We are addressing the problem of inadequate cytoplasmic TSA expression via two different approaches: (1) Use of synthetic cell-penetrating domains (CPDs) to create fusion proteins that penetrate through the plasma membrane or alternatively, (2) use of non-pathogenic recombinant adeno-associated viral (rAAV) vectors which, by virtue of novel capsid mutations, will avoid proteasome degradation and allow for increased TSA expression in the cytosol.
Therapeutic Cancer Vaccines Cell Penetrating Domain (CPD)-Mediated Delivery
Various CPDs are known to ferry covalently-linked heterologous antigens to the intracellular compartment by penetrating through the plasma membrane. We hypothesize that MAGE-A3/NY-ESO-1 antigens generated as fusion proteins in-frame with CPDs will gain access to the HLAC class I pathway in the cytosol, eventually leading to robust CTL responses.
We intend to demonstrate that DC vaccination with protein-modified CPD-MAGE-A3 will generate robust CTL responses that increase tumor cell killing. Verification of both hypotheses would pave the way for development of a strategy to boost a patient’s own natural resistance to tumor cells and achieve durable tumor regression.
Following data demonstrates cloning of full-length MAGE-A3 in an expression vector in-frame with unique CPD that we designed and efficient delivery of recombinant CPD-MAGE-A3 fusion protein into the cytosol of DCs.
1. Protein expression vectors for MAGE-A3 and CPD-MAGE-A3 were constructed as follows: MAGE-A3 was PCR amplified from kidney tissue cDNA and sub-cloned into a pDRIVE T vector (Fig. 1A) (Qiagen, Inc., Valencia, CA) and further cloned inframe with CPD sequence YARKARRQARR sequence in a CPD vector (Fig. 1B). Both MAGE-A3 and CPD-MAGE-A3 were excised (Fig. 1c) for further cloning into expression vectors.
2. Induction of recombinant proteins: Both MAGE-A3 and CPD-MAGE-A3 were digested from pDRIVE T and ligated into the pQE-70 protein expression vector in-frame with downstream hexahistidine (Fig. 2A i and ii). Identity of the vectors was confirmed by restriction digestion analysis and DNA sequencing (data not shown). The pQE-70 expression vectors are designed to express a target protein under the regulation of promoter T5/lac in E. coli BL21 Star (DE3-pLysS) bacteria that is specifically designed for efficient lysis and induction of recombinant protein. Three clones of each recombinant protein were selected and protein induction was attempted at various time periods and temperatures in LB medium. The temperature shift from 37C to 33C after a 5h IPTG induction improved recombinant protein expression, with the highest expression observed in clone 3 of MAGE-A3 (Fig. 2B i) and clone 1 of CPD-MAGE-A3 (Fig. 2B ii).
3. Efficient DC penetration of CPD-MAGE-A3: We compared the ability of CPD-MAGE-A3 with MAGE-A3 to access the cytoplasm of DCs by pulsing the proteins on day six of culture with 3mmol/L of both MAGE-A3 control and CPD-MAGE-A3. We observed very little fluorescent staining in MAGE-A3-pulsed DCs after 2 h (Fig. 3A). In contrast, CPD-MAGE-A3 penetrated the DCs within 5 min after pulsing. This clearly demonstrated efficient DC penetration and a rapid way to introduce CPD-MAGE-A3 into the cytoplasm. Studies using deconvolution and confocal microscopy confirmed that CPD-MAGE-A3 was localized to the DC cytosol (Fig. 3B and 3C).