Holmen Lab
Sheri L. Holmen, Ph.D.
Associate Member, Drug Development
sholmen@nvcancer.org
Dr. Sheri Holmen earned her B.S. and M.S. in Biomedical Science with cum laude honors from Western Michigan University in 1994 and 1995, respectively and a Ph.D. with an emphasis in tumor biology from the Mayo Clinic College of Medicine in 2000. She was a Pfizer postdoctoral fellow of the Life Sciences Research Foundation at the Van Andel Research Institute (VARI) from 2000 to 2003 and established her own lab at VARI in 2003. Dr. Holmen joined the Nevada Cancer Institute's Division of Drug Development in 2007.
Laboratory Members
Staff
Adam Guilbeault, B.S.
James Robinson, Ph.D.
Eric Johnson, research intern
Stephen McKinney, research intern
Ryan Rimer, research intern
Christina Schaerer, research intern
Research Interests
Our laboratory is focused on defining critical targets in the signaling pathways of cancer cells that can become the focus for therapeutic intervention. Because of the high cost of developing new therapies, it is essential that we first identify which genetic alterations can be productively targeted. We are concentrating our initial efforts on tumors that demonstrate constitutive activation of Ras and Akt signaling. We plan to further validate the role of these pathways using pharmacological inhibitors of clinical importance.
The RCAS system
We use a series of replication-competent retroviral vectors based on Rous sarcoma virus (RSV), a member of the avian leukosis virus (ALV) family, to study the roles of different genes in tumor initiation and progression. RSV is the only known naturally occurring, replication-competent retrovirus that carries an oncogene, src. In the RCAS vectors, the region encoding src (which is dispensable for viral replication) has been replaced by a synthetic DNA linker. Foreign genes inserted into this linker are expressed from the viral LTR promoter via a subgenomic splice site (just as src is in RSV). RCAN vectors differ from RCAS vectors in that they lack the src splice acceptor; the gene of interest is inserted along with an internal promoter. Higher-titer viruses subsequently have been generated by replacing the RSV pol gene with the pol gene of the Bryan strain of RSV.
These vectors are termed RCASBP or RCANBP. The ability of these vectors to infect non-avian cells relies on expression of the corresponding receptor on the cell surface.
The viral receptor is typically introduced into mammalian cells (or mice) via an inducible and/or tissue-specific transgene. Therefore, this system allows for tissue- and cell-specific targeted infection of mammalian cells through ectopic expression of the viral receptor. Alternatively, when targeted infection of mammalian cells is not required (e.g., in cell culture), infection can be achieved through the use of non-avian envelopes, such as the amphotropic envelope from murine leukemia virus. The receptor for this envelope is endogenously expressed on almost all mammalian cells. We have used the RCASBP/ RCANBP family of retroviral vectors extensively in both cultured cells and in vivo systems for studies of viral replication and for cancer modeling. Most of these studies have analyzed gain-of-function phenotypes by delivering and overexpressing a particular gene of interest.
Recently, we engineered the RCASBP vector to reduce the expression of specific genes through the delivery of short hairpin RNA sequences in the context of an endogenous microRNA (miRNA). We also engineered this vector to control the expression of the inserted sequences using the tetracycline (tet)-regulated system. Sequences inserted into this region are transcribed from a tet-responsive element and not the viral LTR. This virus allows inserted sequences to be turned on and off, in order to determine if their expression is required for tumor initiation, maintenance, and progression. The ability to turn off expression will help determine if that gene or miRNA is a good target for therapy.
Melanoma
Activated NRAS oncogenes, which turn on mitogen-activated protein kinase (MAPK) signaling, are detected in approximately 20% of human melanomas. Recently, activating mutations in the BRAF gene, which also activate MAPK signaling, have been found in more than 65% of malignant melanomas. With mutually exclusive mutations in RAS and BRAF, the MAPK signaling pathway is constitutively activated in over 85% of cases of malignant melanoma, indicating its importance. Interestingly, it was recently observed that tumors harboring BRAF mutations are much more sensitive to pharmacological inhibition of downstream MAPK signaling than those with NRAS mutations. This suggests that single-agent therapeutic strategies may be ineffective in tumors containing Ras mutations and that rational combination therapeutic strategies will be required.
The RAS subfamily consists of H (Harvey)-RAS, N (neuroblastoma)-RAS, and two splice variants of K (Kirsten)-RAS (K-RAS4a and K-RAS4B). In many tumors, oncogenic mutations have been identified at positions 12, 13, or 61, which cause RAS to remain constitutively active. With the exception of thyroid cancers, most tumors are associated with mutation of only one isoform of RAS and this association cannot be explained solely on differential regulation of RAS gene expression in different tissues. HRAS mutations are more commonly associated with bladder and kidney cancers; KRAS mutations are found in lung, colorectal, ovarian, and pancreatic cancers; and NRAS mutations are most commonly associated with melanoma and hematologic malignancies.
We have been characterizing the transforming capabilities of the different Ras isoforms in a non-transformed, immortal Ink4a/Arf-deficient mouse melanocyte cell line. We have observed that activated NRas is able to transform these cells much more efficiently than either activated HRas, or KRas; whereas expression of NRas increases the proliferation of the melanocytes, expression of KRas does not. Interestingly, co-expression of c-myc with KRas in these cells mimics the proliferation and transformation capabilities of NRas alone, whereas coexpression of Akt with KRas in these cells mimics the proliferation and transformation capabilities of HRas alone. These data suggest that the different Ras isoforms have distinct non-redundant functions in melanocytes and may explain why most melanomas are associated with mutation of only one isoform of Ras.
Glioblastoma
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. It is also the most fatal: mean survival is less than one year from the time of diagnosis with less than 10% survival after two years. Despite major improvements in imaging, radiation, and surgery, the prognosis for patients with this disease has not changed in the last 20 years. Recently, genes that are differentially expressed in tumor tissue relative to normal brain tissue have been found. However, those that can be productively targeted for therapeutic intervention in patients remain to be identified.
A model of human GBM based on the avian RCAS/TVA system has been developed by Eric Holland. In this model, the retroviral receptor TVA is expressed under the control of the Nestin promoter, which is active in neural and glial progenitors. Malignant gliomas can be induced in vivo through the combined expression of activated forms of both KRas and Akt in glial progenitor cells. To determine the reliance of these tumors on continued KRas signaling in vivo, we generated a viral vector that allows the expression of KRas to be controlled post-delivery. Tumor-free survival rates were compared between cohorts with continued KRas expression and cohorts in which KRas expression was suppressed. KRas signaling was found to be required for the maintenance of these tumors in vivo; inhibition of KRas expression resulted in apoptotic tumor regression and increased survival. Subsequent reexpression of KRas reinitiated tumor growth, indicating that a percentage of the progenitor cells survived and retained tumorigenic properties.
This model shows the critical importance of the Ras pathway in glioblastoma maintenance and indicates that continuous suppression of Ras signaling is necessary and sufficient to suppress the tumorigenic potential of the glial progenitor cells. In addition, this regulated expression system will allow the evaluation of the role of other genes and pathways in this context. This has important clinical implications for pharmacologic agents targeting these pathways in GBM patients.
External Collaborators
John Brigande, Oregon Health & Science University, Portland, Oregon
Jerry Dodgson, Michigan State University, East Lansing, Michigan
Henry Hunt and Huanmin Zhang, Avian Disease and Oncology Laboratory, East Lansing, Michigan
Nita Maihle, Yale University School of Medicine, New Haven, Connecticut
Phillipe Monnier, University of Toronto, Toronto, Canada
Helmut Schaider, Medical University of Graz, Graz, Austria
Wei Zhang, MD Anderson Cancer Center, Houston, Texas
Recent Publications
Chen, Mo, Adam J. Granger , William S. Payne, Henry Hunt, Huanmin Zhang, Jerry B. Dodgson, and Sheri L. Holmen. 2007. Inhibition of avian leukosis virus replication by vector-based RNA interference. Virology. 365: 464-472.
Whitwam, Todd, Marleah E. Russo, Peter T. Haak, Devrim Bilgili, James H. Resau, Han-Mo Koo, and Sheri L. Holmen. 2007. Differential oncogenic potential of activated RAS isoforms in melanocytes. Oncogene. 26:4563-4570.
Park, Ki-Sook., Soung Hoo Jeon, Sung-Eun Kim, Young-Yil Bahk, Sheri L. Holmen, Bart O. Williams, K.wang-Chul Chung, Young-Joon Surh, and Kang-Yell Choi. 2006. APC inhibits ERK pathway activation and cellular proliferation induced by Ras. Journal of Cell Science. 119:819-827.
Wang, PengFei., Dong Kong, Jun Peng, Chun Zhang, Stephanie J. Potter, Xiang Gao, Bin T. Teh, Nian Zhang, Bart O. Williams, and Sheri L. Holmen. 2006. Simplified method for the construction of gene targeting vectors for conditional gene inactivation in mice. Transgenics. 4:215-228.
Holmen, Sheri L., and Bart O. Williams. 2005. Essential role for Ras signaling in glioblastoma maintenance. Cancer Research 65(18):8250-5.
Ai, Minrong, Sheri L. Holmen, Wim van Hul, Bart O. Williams, and Matthew W. Warman. 2005.Reduced affinity to and inhibition by DKK1 form a common mechanism by which high bone mass–associated missense mutations in LRP5 affect canonical Wnt signaling. Molecular and Cellular Biology 25(12): 4946-4955.
Holmen, Sheri L., Scott A. Robertson, Cassandra R. Zylstra, and Bart O. Williams. 2005.Wnt-independent activation of ß-catenin mediated by a Dkk-1-Frizzled 5 fusion protein. Biochemical and Biophysical Research Communications 328(2): 533-539.
Holmen, Sheri L., Cassandra R. Zylstra, Aditi Mukherjee, Robert Sigler, Marie-Claude Faugere, Mary Bouxsein, Lianfu Deng, Thomas Clemens, and Bart O. Williams. 2005. Essential role of ß-catenin in postnatal bone acquisition. Journal of Biological Chemistry 280(22): 21162-21168.
Sanchez-Perez, Luis, Timothy Kottke, Rosa Maria Diaz, Atique Ahmed, Jill Thompson, Heung Chong, Alan Melcher, Sheri Holmen, Gregory Daniels, and Richard G. Vile. 2005. Potent selection of antigen loss variants of B16 melanoma following inflammatory killing of melanocytes in vivo. Cancer Research 65(5): 2009-2017.
