The Speicher laboratory is pursuing seven major projects as well as additional smaller scope collaborative projects. Most projects utilize proteomics and mass spectrometry as major tools. These projects are briefly summarized below, and in greater detail in the following sections.
The first project uses proteomics to both identify and characterize cancer biomarkers associated with ovarian cancer and to explore proteome changes associated with ovarian cancer progression. This includes use of both xenograft mouse models and in vitro, short-term organ cultures of ovarian cancer tissue removed at time of surgery, as well as validation of biomarkers in patient sera.
The second project takes a proteomics-based systems biology approach to explore melanoma tumor progression and resistance to therapy, as well as the role of autophagy in these processes.
The third project takes similar approaches to those used in ovarian cancer to identify and validate colorectal cancer biomarkers.
The fourth project uses state-of-the-art proteomics and computational methods to identify plasma biomarkers of cardiotoxicity that is induced by cancer therapies in breast cancer patients.
The fifth project uses state-of-the-art proteomics approaches similar to Project 4 to identify plasma biomarkers that distinguish ectopic pregnancy from normal intrauterine pregnancy and non-viable intrauterine pregnancy.
The sixth project involves proteomics-based systems biology analysis of red cell changes induced by pathological conditions as well as a structural mass spectrometry analysis of macromolecular complexes in the red cell membrane skeleton that control membrane integrity in normal cells and in pathogical disorders.
A seventh project involves a proteomics-based systems biology analysis of NK cells to characterize their role in innate immunity and resistance to HIV infection.
One goal of this project is to discover and validate novel protein biomarkers of human ovarian cancer that can improve clinical management of this disease, and a second goal is to better understand tumor development and resistance in order to identify new therapeutic targets for this disease. Ovarian cancer is the leading cause of death from gynecological cancers in the United States. More than 24,000 women are diagnosed with ovarian cancer every year, and approximately 15,000 die of this disease. Ovarian cancers are biologically very heterogeneous with common epithelial tumors consisting of serous, endometrioid, mucinous, and clear cell sub-types. Serous sub-type tumors account for about half of the epithelial tumors and generally occur in women between 40 and 60 years of age. They are usually highly aggressive and account for the greatest number of ovarian cancer-related deaths.
The five-year survival for invasive epithelial ovarian cancer is about 90% when the disease is confined to the ovaries, but is only about 30% when the cancer has spread to other organs. Even high-grade serous tumors do well if diagnosis occurs when the tumor is confined to the ovary. However, about 75% of ovarian cancers are not diagnosed until after the cancer has spread, primarily because early-stage tumors are generally asymptomatic.
There is no effective molecular test for ovarian cancer at present. So far, the best plasma biomarker that detects ovarian cancer prior to symptoms is CA125, but CA125 suffers from a large number of false positives and false negatives, which prevents it from being used as a routine screen for ovarian cancer. Hence, one goal is to identify new, better, molecular biomarkers for ovarian cancer that would form the basis for a minimally invasive blood test for either early diagnosis or clinical management of the disease after initial diagnosis.
In pursuit of our first goal, several hundred ovarian cancer biomarkers were identified using a xenograft model coupled with in-depth proteome analysis of the resulting chimeric (human/mouse) serum to identify human proteins shed by the tumors into the blood. Multiplexed quantitative mass spectrometry assays using multiple reaction monitoring (MRM) were used for initial laboratory scale validation of approximately 40 high priority biomarkers. Approximately half of these biomarkers were significantly higher in ovarian cancer patients compared with normal donors or those with benign disease. Ongoing efforts include further testing of the best proteins in biomarker panels as well as evaluation of additional candidate biomarkers from the discovery experiments.
A second evolving goal of this project involves a systems biology approach to elucidate the role of protein acetylation in tumor progression and therapy resistance. These efforts also are focused on elucidation of the roles of several proteins identified in the above biomarker studies in ovarian cancer progression and response to therapy.
Malignant melanoma is one of the most aggressive forms of cancer, with a median survival of 6-10 months upon onset of metastasis, and it is one of the few cancers where the incidence in the general population continues to increase. Dramatic recent improvements in patient care have occurred using targeted therapies and immunotherapy. But for most patients, benefits are transient and hence long term patient survival rates remain low and additional therapeutic options are needed.
Multiple related aspects of melanoma progression and therapy resistance are being pursued. Systems biology analyses of protein profile changes associated with tumor progression and acquisition of therapy resistance are being used to gain novel insights into key mechanisms associated with metastatic and therapy resistant phenotypes. In addition, recent efforts have focused on autophagy, which is a common mechanism of therapy resistance that also correlates with melanoma aggressiveness. Clinical trials are currently underway involving combinations of anticancer therapies and hydroxychloroquine, an autophagy inhibitor. However, these efforts are limited by the lack of predictive biomarkers to select patients most likely to respond to autophagy targeting therapies and the lack of markers to monitor effects on autophagy levels during treatment using minimally invasive methods.
Current goals include identification of predictive and pharmacodynamic plasma biomarkers to assist in clinical management of patients receiving autophagy inhibitors and identification of new therapeutic targets. Proteome analyses of supernatants from 3D cultures of melanoma cells with high and low autophagy have led to the identification of a number of promising candidate biomarkers. Several of these biomarkers have been validated in additional cell lines and in a small group of patients where the biomarkers appear to correlate with tumor autophagy levels. Future efforts will expand the evaluation of candidate biomarkers in patients as well as pursue an unbiased proteomics approach to identify global changes in posttranslational modifications and protein levels related to autophagy and autophagy inhibitor therapies.
Colorectal cancer is a major international health problem that results in more than 100,000 deaths worldwide every year. In the United States, it is the second highest cause of cancer deaths, with approximately 140,000 new cases reported annually, and mortality approaches about 60,000 cases each year. About 90% of these colorectal cancers arise from adenomatous polyps, and surgical resection is an effective treatment for these precancerous lesions as well as localized disease.
An effective screening method for colorectal cancer, which can identify and remove pre-cancerous polyps as well as localized small tumors with good prognosis, is colonoscopy. Widespread use of this screening method could greatly reduce colon cancer mortality. However, although this procedure is effective, it is expensive, uncomfortable, has moderate risk of complications and is not readily available to all patients. As a result, only about 30% of the at-risk population has ever been screened using colonoscopy, and the percentage of patients who are screened on a regular basis consistent with existing clinical guidelines is substantially lower.
The primary goal of this project is to develop a first-tier screen using novel plasma protein biomarkers of colon cancer to pre-screen the general at-risk population to identify those patients who would benefit from colonoscopy. That is, patients who are very likely to have pre-cancerous or cancerous lesions. By focusing expensive colonoscopy resources on a subpopulation of patients with a high probability of precancerous or early stage cancer lesions, we could greatly reduce the financial burden associated with colonoscopy and increase compliance with this screening technique.
The approaches used to identify and validate novel biomarkers are similar to those described in Project 1 for ovarian cancer biomarkers and we have had similar levels of success here. Specifically, several hundred candidate biomarkers have been identified using a xenograft mouse model and approximately 40 high priority candidates were tested in plasma from human patients and controls. Several out-perform existing biomarkers such as CEA and CA-19-9. Based on the initial validation screen, at least 10 of these biomarkers warrant further testing in additional patient cohorts. In addition, a substantial number of high priority candidate biomarkers remain to be tested.
Millions of breast cancer survivors are at risk of developing cardiotoxicity resulting from therapeutic treatments, and biomarkers for predicting cardiotoxicity are urgently needed for these patients. Trastuzumab (Herceptin®) is used widely to treat HER2+ breast cancer, which has resulted in important survival gains, but treatment involves significant risk of cardiovascular morbidity and mortality. When it is used in combination with doxorubicin, it results in left ventricular (LV) dysfunction in 18% of treated individuals and severe, symptomatic heart failure (HF) in 2-4% of patients. Hence, there is a critical need for minimally invasive biomarkers for early identification of subclinical cardiac dysfunction that would: 1) enable earlier patient treatment with cardioprotective strategies; 2) prevent interruption of necessary cancer therapy; and 3) reduce morbidity and mortality. Use of either drug alone, also has substantial risk of inducing cardiotoxicity.
Proteomics methods are being used to identify novel blood biomarkers that can identify breast cancer patients at increased risk for therapy-induced cardiotoxicity prior to appearance of clinical symptoms. A substantial number of good candidate biomarkers have been identified and further discovery efforts are continuing. In addition, the most promising candidate biomarkers are being validated in longitudinal plasma samples collected from patients receiving various therapy treatment regimens to identify the biomarkers that detect cardiotoxicity at the earliest possible time. A working hypothesis is that a multi-protein panel of circulating biomarkers can identify patients with cancer therapy-induced cardiotoxicity earlier than conventional clinical methods. When available, ELISA assays are used for biomarker quantitation. When ELISA assays are not available, multiplexed MRM-MS assays are developed and used.
Ectopic Pregnancy (EP) occurs in about 1-2% of pregnant women and may compromise a woman’s health and future fertility. It is a leading cause of maternal mortality and morbidity accounting for 6% of pregnancy deaths due to a rupture of the fallopian tube with resulting intraperitoneal bleeding. Most patients present before tube rupture with nonspecific symptoms of abdominal pain and/or vaginal bleeding but these symptoms are neither sufficiently sensitive nor specific, and some women remain asymptomatic. If diagnosed early, EP can be effectively treated with little risk to the patient but current diagnostic methods, transvaginal ultrasound and serial quantitative serum human chorionic gonadotropin concentrations are inconclusive in up to 40% of patients. Also, the diagnosis is currently cumbersome requiring multiple office visits, serial blood tests for up to 6 weeks, multiple ultrasound examinations, and surgical procedures such as uterine curettage and laparoscopy.
The goal of this project is to develop more effective, minimally invasive blood tests that can reliably diagnose EP at an early stage. The proteomic strategies are similar to those described in project 4. In-depth quantitative comparisons of serum from patients with EP and normal intrauterine pregnancy have identified several novel biomarkers, several more specific isoforms of known biomarkers, and confirmed several previously known biomarkers. Followup validation in larger patient cohorts has verified the clinical utility of one biomarker in a large patient cohort. Future efforts are directed to further testing of recently discovered biomarkers as well as new, more-in-depth discovery efforts that should identify additional and potentially better biomarkers. As with other biomarker projects, the expectation is that an optimal clinical diagnostic assay will consist of a panel of multiple biomarkers rather than a single protein.
Mammalian red cells have highly developed cell membranes that exhibit unusual elasticity and integrity properties that are essential for cell survival in the circulation. Surprisingly, these membranes are more complex than initially appreciated, as they contain more than a thousand proteins. Although these membranes have been extensively studied, critical, fundamental properties remain poorly defined, including: 1) how the membrane skeleton imparts the biconcave shape to red cells; 2) how the membrane skeleton dynamically rearranges in response to shear stress; and 3) the mechanisms by which diverse red cell diseases destabilize red cell membranes and cause anemias. Because the mechanisms by which most pathological conditions disrupt red cell membranes are not defined, there are not effective therapeutic treatments for most red cell pathologies, including well studied diseases such as malaria, sickle cell disease, and anemias.
Recent technological advances make it now feasible to take a global, systematic approach to study red cell membranes in order to develop comprehensive, accurate models of protein organization, structure, and dynamics of the entire membrane. One goal of this project is to develop a detailed, experimentally-validated, medium-resolution structure (~5Å) of the entire red cell membrane skeleton as the basis for future development of therapeutic treatments for diverse membrane defects. A related goal is to model large conformational changes that have physiological functions and to understand how pathogenic mutations disrupt these molecular dynamics.
Key experimental approaches include quantitative proteomics and mass spectrometry analysis of chemical crosslinks to test, refine and validate molecular models of protein complexes in the red cell as well as probe and model large physiologically important conformation changes. One conformation change that is being investigated is the profound molecular shape changes that spectrin undergoes in response to tensile stress. The physiological form of spectrin in resting red cells is a compact super-coiled flexible, rope-like structure that can be stretched to three times its resting length. In addition, further extensions can occur by reversibly unfolding tandem three-helix bundles that comprise most of the molecule. Recent analyses also indicate that the membrane skeleton is not a uniform array of interacting protein complexes. Instead, it probably contains regions with specialized macromolecular complexes that include important proteins missing from current schematic models of the membrane. One specific goal is to determine the structure and molecular dynamics of full length red cell spectrin, a one million Dalton protein complex that is the major component of the membrane skeleton, which is either mutated or present at abnormally low levels in many hereditary anemias. A related goal is to determine the composition and structure of red cell junctional complexes, which are multi-million Dalton complexes linked together by spectrin tetramers to form the membrane skeleton that imparts flexibility and integrity to the red cell membrane. In related studies, global comparisons of changes in red cell membrane proteome compositions associated with inherited or acquired pathogenic mutations are being used to elucidate the molecular bases for these diseases as a foundation for developing future therapeutic interventions.
NK cells are an important component of the innate immune system that provides a level of immediate protection against pathogens including viruses. Interestingly, some individuals can be repeatedly exposed to HIV-1 and remain seronegative, such as multiply Exposed Uninfected IV opioid drug Users (EU-IVU). In this project, global protein profile differences in NK cells from exposed, uninfected individuals vs. unexposed, uninfected individuals are being compared to identify cellular mechanisms and regulatory receptors exhibited by circulating (NK) cells that may explain their sustained absence of infection.
Data show that HIV-infected cells can provide a potent activation signal for NK cells by TLR-mediated activation of Plasmacytoid Dendritic cells (PDC), and importantly, the EU-IVU exhibit evidence of an increased NK activation phenotype, compared to controls. These results support the hypothesis that a better understanding of this correlation could identify host factors associated with resistance to viral infection. Proteomics is being used to globally compare NK cell proteins with and without activation by PDC, and to compare NK cell proteomes of EU-IVU subjects and controls. Network analysis is then used to interpret the global changes observed and to identify upstream regulators.