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Radiation Oncology

Bergom Lab Areas of Research

Introduction

Breast cancer is the most common cancer in U.S. women, and over half of all breast cancer patients receive radiation therapy, but currently there are no tools that personalize radiation doses delivered or select the patients most likely to benefit from radiation. In our laboratory, we use unique animal models to identify novel gene(s) in the tumor microenvironment (TME) that improve the response of breast cancers to radiation therapy in breast cancer. The ultimate objective of these studies is to use these TME targets to better tailor breast cancer therapies in patients.

Genetic Changes in the Tumor Microenvironment Influencing Treatment Responses

We are interested in identifying genetic changes in the tumor microenvironment that alter responses to cancer treatment. This concept evolved from patient experiences where breast cancer patients with the same age, type of tumor (estrogen receptor, progesterone receptor, and HER2/neu status), and stage have different treatment outcomes for unknown reasons (Figure 1).

Breast Cancer Radiation Treatment Graphic

We hypothesized that host factors beyond those expressed on the tumor cells can alter tumor growth and treatment responses. To test our hypothesis, we are utilizing a newly developed Consomic Xenograft Model (CXM), the first experimental genetic tool to map the effect of germline variants in the TME on tumor therapy responsiveness (1). In the CXM, we manipulate the genetic backgrounds of immunodeficient rat strains while keeping the tumor cells the same, enabling us to assess the effect of host TME differences on radiation responses of human tumor xenografts. The immunodeficient genetically distinct rat strains used for these experiments are consomic rats from a consomic panel developed here at MCW. Consomic rats are rats in which a chromosome from one inbred rat strain is selectively substituted into another inbred rat strain (Figure 2).

Selective Breeding Graphic

This consomic rat panel derived from the inbred SS and BN rat strains has been extensively characterized for a number of phenotypes, including hypertension (2,3). Consomic rats within this panel also displayed a wide variation in the development of mutagen-induced breast cancers, with the SS.BN3 consomic demonstrating dramatically decreased incidence of the mutagen-induced breast tumors (4). Because this may be due to host TME factors, this hypothesis was tested with the CXM model, where the consomic rats were made immunocompromised by knockout of IL-2 receptor gamma (IL2Rg), and identical human breast cancer cells are injected into the mammary fat pad of the rats (Figure 3). Because the genetic background of the host is altered, but the tumor cells stay the same, any differences seen in tumor growth, metastasis, or treatment responses are due to host or TME factors.

The Consomic Xenograft Model

Using CXM, we discovered that BN strain-derived genetic variant(s) on rat chromosome 3 are important for tumor radiation sensitivity (Figure 4). In the xenografts from the parental SS strain and the SS.BN3 consomic strain, there are differences in vascular density (1). We have also performed perfusion and diffusion imaging of these tumors that has revealed functional vascular differences (data not shown).

Enhanced Radiation Sensitivity

To identify the element(s) on rat chromosome 3 that underlie differences in radiation sensitivity, we utilize a species-specific RNA-seq method developed with our collaborators in the Flister Laboratory that determines changes in gene expression between human tumor cells and the rat non-malignant TME with over 99% specificity (Figure 5). Using these techniques, we have a number of putative candidate genes that influence tumor radiosensitivity in the TME that are currently being tested. We also have profiled radiation-induced transcriptional changes that occur simultaneously in the tumor cells and non-malignant TME.
These studies may identify pathways to more specifically enhance the tumor radiation response.

Species-Specific RNA-Seq

Ongoing work related to this project includes characterizing radiation-induced transcriptional responses in the TME, non-malignant breast tissue, and tumor cells simultaneously using species-specific RNA Seq. These studies have the potential to identify pathways differentially stimulated in tumor but not normal tissue or vise versa, allowing more specific enhancement of tumor radiation responses while possibly decreasing normal tissue toxicity.

Our laboratory is very collaborative, and we utilize a number of multidisciplinary techniques to advance our research program. We have established collaborations with a number of investigators on our campus and at other institutions. Specific to this project identifying TME-specific mediators of radiation response, we work closely with the Flister Laboratory, as well as with the laboratories of Dr. Amit Joshi and Dr. Peter LaViolette.

Potential Impact  

Breast cancer radiation improves local control and can improve overall survival. Currently breast cancers are classified only by examining histologic markers on the tumor cells. Our results demonstrate that independent of tumor cells, host TME factors can alter breast cancer tumor growth and radiation responsiveness. By using innovative animal models, imaging, and gene expression techniques, the proposed studies will identify key TME genes and vascular changes that mediate tumor radiation sensitivity. We expect our findings to ultimately identify factors in the TME that predict better or worse responses to radiation therapy, allowing better tailored and more effective breast cancer treatment.

The Role of DiRas Family Tumor Suppressive Small GTPases in Cancer Progression

Members of the Rho and Ras small GTPase families regulate breast cancer development and progression. In addition, Ras and other small GTPases can alter the sensitivity of cancer cells to radiation and chemotherapy. Identifying new ways to suppress small GTPase activation in breast cancer may provide new treatment approaches. In breast cancer, the small GTPases RhoA and RhoC can promote breast cancer proliferation, migration, and metastasis, causing cancer progression and poorer clinical outcomes. Mutations in Ras pathways are also seen in up to 25% of breast cancer tumors and cell lines, suggesting that Ras promotes tumorigenesis in a subset of breast cancers. In addition, more than 10% of breast cancer patients have highly active NF-kB signaling in their tumors and these tumors are more likely to be resistant to chemotherapy. These lines of evidence provide strong evidence that RhoA, RhoC, and K-Ras are therapeutic targets in breast cancer. Identifying signals that can diminish Ras, Rho, and/or NF-kB pathways has the potential to dramatically antagonize the malignant properties of breast cancer and enhance breast cancer treatments.

To date it has proven exceedingly difficult to suppress small GTPase activation in cancers. Identifying new ways to suppress small GTPase activation in cancers may provide new treatment approaches. Interestingly, in non-cancerous tissues there are a number of small GTPases in the Ras family which have tumor suppressive features (Figure 6).Thus far, most of these family members are poorly characterized. However, our data and that of others suggest that some of these members of the Ras GTPase superfamily can antagonize oncogenic small GTPase signaling, although the exact mechanisms of many of these functions are poorly characterized. Our long-term goal is to understand how tumor suppressor small GTPase signaling can be manipulated to enhance the treatment of breast, and potentially other, cancers. Because these molecular mechanisms are utilized to prevent tumor development in normal tissues, they have the potential to be especially efficacious.

Dendrogram of Select Ras Family Members Highlighting a Subset of Proteins with Tumor Suppressive Actions

We are initially focusing on the unique DiRas (Distinct subgroup of the Ras family) family of small GTPases. DiRas1 is closely related to its family members DiRas2 (also known as Di-Ras2) and DiRas3 (also known as ARHI or Noey2). DiRas1 (also known as Di-Ras1 or Rig) has been reported to be a tumor suppressor in brain and esophageal cancers. DiRas2 appears to be predominantly expressed in the brain, while DiRas3 is a tumor suppressor in breast, ovarian cancers, and other cancers. Our studies also demonstrated that, like DiRas3, DiRas1 is also expressed in normal breast epithelial tissue, and this expression is lower in breast cancers (Figure 7) (5). The mechanisms by which DiRas1 inhibits tumor growth are not fully characterized. We hypothesized that DiRas1 may act as a tumor suppressor by binding and sequester­ing proteins that promote activation of the oncogenic small GTPases, such as guanine nucleotide exchange factors (GEFs), which activate small GTPases by ultimately promoting the binding of GTP to small GTPases. One such protein is SmgGDS (also known as Rap1GDS1), a protein that promotes the pro-oncogenic function of several Ras and Rho family small GTPases, both by acting as a GEF for RhoA and RhoC and also by promoting the addition of lipid groups - known as prenylation - to many small GTPases, which promotes small GTPase activation by localizing the proteins to the plasma membrane (6,7). SmgGDS also promotes NF-kB transcriptional activity in a number of cancer types, which is critical to cancer cell growth and proliferation.

DiRas1 Expressed in Normal Breast Epithelium and Exhibits Lower Expression in Many Breast Cancers

We recently identified DiRas1 as a direct binding partner for SmgGDS. In silico docking analysis predicted that DiRas1 can compete with other small GTPases, such as RhoA and K-Ras, for SmgGDS binding (Figure 8A-D). Consistent with this prediction, DiRas1 potently inhibited interactions of SmgGDS with a broad range of pro-oncogenic small GTPases, including RhoA, K-Ras, and Rap1 (Figure 8E-G). In addition, DiRas1 inhibited NF-kB activity in a number of cell lines, including brain cancer and breast cancer cells (5). Taken together, these findings identify a novel way in which the tumor suppressive small GTPase DiRas1 represses signals mediated by several pro-oncogenic Ras and Rho family GTPases (Figure 9). The actions of DiRas1 thus appear, at least in part, to be due to binding to SmgGDS and essentially sequestering it to prevent interactions of pro-oncogenic small GTPases with SmgGDS. This is similar to how dominant negative small GTPases inhibit GTPase-mediated signaling in cells.

DiRas1 Expression Potently Inhibits SmgGDS Association with Other Small GTPases

Proposed Model of DiRas1-Mediated Tumor Suppressive Actions

Additional lines of research on the DiRas family of tumor suppressors includes the creation of conditional knockout mice for DiRas proteins, utilizing mass spectrometry to identify new DiRas protein binding partners, and performing in vivo tumor xenograft studies using tetracycline-inducible tumor cell lines to determine whether loss of DiRas proteins promote tumor growth and progression. We are also examining potential signaling mediators of the tumor suppressive phenotypes mediated by DiRas1, 2, and 3. Learning more about the ways in which the DiRas-family GTPase exerts tumor suppressing signals may allow these pathways to be used to antagonize oncogenic signaling in tumors where expression of DiRas proteins is lost.

References

  1. Flister MJ, Endres BT, Rudemiller N, et al. CXM: A New Tool for Mapping Breast Cancer Risk in the Tumor Microenvironment. Cancer Res 2014;74:6419–6429.
  2. Mattson DL, Kunert MP, Kaldunski ML, et al. Influence of diet and genetics on hypertension and renal disease in Dahl salt-sensitive rats. Physiol Genomics 2004;16:194–203.
  3. Cowley AW, Roman RJ, Kaldunski ML, et al. Brown Norway chromosome 13 confers protection from high salt to consomic Dahl S rat. Hypertension 2001;37:456–461.
  4. Adamovic T, McAllister D, Wang T, et al. Identification of novel carcinogen-mediated mammary tumor susceptibility loci in the rat using the chromosome substitution technique. Genes Chromosomes Cancer 2010;49:1035–1045.
  5. Bergom C, Hauser AD, Rymaszewski A, et al. The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases. J Biol Chem 2016;291:6534–6545.
  6. Hauser AD, Bergom C, Schuld NJ, et al. The SmgGDS Splice Variant SmgGDS-558 Is a Key Promoter of Tumor Growth and RhoA Signaling in Breast Cancer. Mol Cancer Res MCR 2014;12:130–142.
  7. Berg TJ, Gastonguay AJ, Lorimer EL, et al. Splice variants of SmgGDS control small GTPase prenylation and membrane localization. J Biol Chem 2010;285:35255–35266.