Amanda Hummon

Amanda Hummon

Proteomics of Cancer Cells


Adjunct Associate Professor
Associate Professor, University of Notre Dame
Charles L. Huisking Associate Professor, University of Notre Dame
Charles L. Huisking Assistant Professor, University of Notre Dame
Walther Cancer Assistant Professor, University of Notre Dame
Sallie Rosen Kaplen Postdoctoral Fellow, National Cancer Institute
Postdoctoral Research Associate, University of Illinois at Urbana-Champaign
Ph.D. Chemistry, University of Illinois at Urbana-Champaign
A.B. in Chemistry, Cornell University

Selected Awards

American Chemical Society Rising Star Award
Joyce Award for Excellence in Undergraduate Teaching
NSF Early CAREER Award
Young Investigator Award, Spectroscopy Society of Pittsburgh
WICR Brigid G. Leventhal Scholar In Cancer Research Award

Research Interests

Cancer arises from insults to the genome. With genomic damage, the expression levels of genes are altered from their normal state. Changes in the genome, transcriptome and proteome have been found to be highly conserved among samples from adenomas to carcinomas to metastases. Because genetic changes are commonly repeated among cancer patients, a better understanding of which genes, transcripts, and proteins are affected could have broad health implications. Therefore, the best way to understand the molecular underpinnings of cancer is to dissect the deregulated pathways that are contributing to the cancer phenotype, identify the aberrantly expressed genes and their products, and decipher their effect on downstream targets. The Hummon Research Group develops high-throughput methods to evaluate the proteome in cancer cells. On-going projects include:
• Interrogating the distribution of analytes in three-dimensional cell cultures using imaging mass spectrometry
Three dimensional cell cultures are popular model systems in health-related biological research. They combine the flexibility of cell culture with structural information not possible with standard two-dimensional cultures. Our research group is developing mass spectrometric tools to enable the characterization of three-dimensional cell culture systems.
We are the first research group to examine three dimensional cell culture systems via imaging mass spectrometry. We are developing approaches to manipulate 3D cultures and prepare them for imaging. We are working with colon adenocarcinoma cell lines, which form 3D structures called spheroids. In our mass spectrometric images, we detect changes in the spatial distribution of proteins throughout the spheroid structures. We are expanding our studies to genetically manipulated 3D cultures to explore molecular changes associated with the beginning of metastasis and the epithelial to mesenchymal transition. To examine the changes in protein expression and distribution that accompany this transition, we are growing cultures containing inducible shRNAs that silence a critical regulatory gene, E-Cadherin. We will map the alterations in protein expression that accompany silencing of E-Cadherin on the protein level. Finally, we are applying our approach to examine drug concentrations and penetration depth into 3D cultures.
• Investigating the impact that individual and clusters of miRNAs have on the cancer proteome and transcriptome.
We investigated the effect that the miR-143/145 cluster has on the transcriptome and proteome of colorectal cancer. Expression of the miR-143/-145 cluster is reduced in colon cancer. As the deregulation of the miR-143/145 cluster is implicated in tumorigenesis, we combined SILAC and microarray analyses to systematically interrogate the impact of miR-143/145 on the colon cancer proteome and transcriptome. Our results indicate that the summed effects of individually introduced microRNAs produce distinct molecular changes from the consequences of the assembled cluster. This finding has broad implications to additional clusters in a number of disease states and cellular processes. We conclude that there is a need to investigate both the individual and combined functional implications of a microRNA cluster. We are expanding our studies to the miR-23a~27a~24-2 cluster.
• Examining changes to the phosphoproteome that accompany cancer progression.
We are investigating alterations in the phosphoproteome that occur at two critical points in cancer progression. First, we are examining the changes in the phosphoproteome that occur with exposure to very low doses of ionizing radiation, which can lead to tumorigenesis. We are also comparing changes in the abundance of phosphoproteins in primary versus metastatic colon cancer cells. To conduct this research, we have developed a phosphoproteomic enrichment strategy that greatly advances the number of phosphoproteins identified in a complex biological sample.
• Exploring changes to the proteome following RNA interference-based reduction of the expression of pivotal regulatory genes
We are using loss-of-function analysis via RNA interference to investigate the role of specific genes in colorectal cancer. For example, we are examining the changes to the transcriptome and proteome following the reduction of the gene FLASH. Knockdown of FLASH has been shown to drastically reduce the viability of colon cancer cells. We have also investigated the distinct gene expression patterns on the right and left side of the colon and their relationship to relapse. In particular, expression of the genes CDX2 and NOX4 are strong predictors of survival. We are further exploring the functional role of NOX4 with in vitro loss-of-function studies.
•Developing novel sample preparation strategies for proteomic analysis
As a complement to our mechanistic studies, we are also developing proteomic sample preparation protocols. We have devised an improved method for phosphopeptide enrichment. We are also developing a novel method to selectively enrich and isolate peptides from archived tissue samples embedded in optimal cutting temperature (OCT) compound. Many primary biobanked tissue samples are embedded in OCT, a substance that interferes with mass spectrometric analysis. Successful analysis of these bio-banked tissue samples would be an important resource in understanding cancer.

Recent Papers

  • Chen, D., Ludwig, K. R., Krokhin, O. V., Spicer, V., Yang, Z., Shen, X., Hummon, A. B., Sun, L. "Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Large-Scale Phosphoproteomics with the Production of over 11,000 Phosphopeptides from the Colon Carcinoma HCT116 Cell Line" 2019 Analytical Chemistry, 91 (3), pp. 2201-2208. DOI:10.1021/acs.analchem.8b04770.
  • Wangsa, D., Braun, R., Stuelten, C. H., Brown, M., Bauer, K. M., Emons, G., Weston, L. A., Hu, Y., Yang, H. H., Vila-Casadesús, M., Lee, M. P., Brauer, P., Warner, L., Upender, M., Hummon, A. B., Camps, J., Ried, T. "Induced Chromosomal Aneuploidy Results in Global and Consistent Deregulation of the Transcriptome of Cancer Cells" 2019 Neoplasia (United States), 21 (7), pp. 721-729. DOI:10.1016/j.neo.2019.04.009.
  • Liu, X., Lukowski, J. K., Flinders, C., Kim, S., Georgiadis, R. A., Mumenthaler, S. M., Hummon, A. B. "MALDI-MSI of Immunotherapy: Mapping the EGFR-Targeting Antibody Cetuximab in 3D Colon-Cancer Cell Cultures" 2018 Analytical Chemistry, 90 (24), pp. 14156-14164. DOI:10.1021/acs.analchem.8b02151.
  • Ludwig, K. R., Schroll, M. M., Hummon, A. B. "Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up Proteomic Studies" 2018 Journal of Proteome Research, 17 (7), pp. 2480-2490. DOI:10.1021/acs.jproteome.8b00235.
  • Schroll, M. M., Hummon, A. B. "Employing proteomics to understand the effects of nutritional intervention in cancer treatment" 2018 Analytical and Bioanalytical Chemistry, 410 (25), pp. 6371-6386. DOI:10.1007/s00216-018-1219-z.
  • Shields, P. G., Song, M., Freudenheim, J. L., Brasky, T. M., Mathe, E., McElroy, J. P., Hummon, A. B., Wewers, M. D. "Electronic cigarettes and the lung proteome" 2018 American Journal of Respiratory and Critical Care Medicine, 198 (10), pp. 1350-1351. DOI:10.1164/rccm.201806-1151LE.

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