Profiling of Tyrosine Phosphatases in Breast Cancer

Institution: University of California, Davis
Investigator(s): Clifford Tepper, Ph.D. -
Award Cycle: 2000 (Cycle VI) Grant #: 6KB-0042 Award: $133,392
Award Type: New Investigator Awards
Research Priorities
Detection, Prognosis and Treatment>Imaging, Biomarkers, and Molecular Pathology: improving detection and diagnosis
Etiology and Prevention>Etiology: the role of environment and lifestyle

Initial Award Abstract (2000)
Breast cancer is characterized by the overgrowth of cells and movement of cells out of the confines of the breast. Some breast cancers are also characterized by their resistance to drug treatments. Many of these processes are controlled by the presence or absence phosphate molecules on cellular proteins (phosphorylation of proteins). The enzymes that are responsible for phosphorylation are called tyrosine kinases (enzymes that add a phosphate molecule to a protein) and tyrosine phosphatases (enzymes that remove a phosphate molecule from a protein). It is the balance between the actions of these two sets of enzymes that control the fate of the cell. The importance of these enzymes is reflected by the fact that the causes of many cancers can be traced back to mutations in the genes that code for them and that the aberrant production and/or activity of these enzymes are often found in cancers. These genes are therefore valuable markers for cancer and can also be used as possible targets for therapy. One good example of a tyrosine kinase is HER2. Elevated levels of HER2 correlates with poor prognosis in some forms of breast cancer and HER2 is used as a marker. Herceptin, an antibody that interferes with HER2 function, has recently been introduced as an additional therapy for metastatic breast cancer. Having said that, it is important to point out that identifying a good tyrosine kinase marker is not trivial and thus far only very few have been identified. One good reason is that previously developed approaches have looked at one gene at a time. Because tyrosine phosphatases often interact with one another, the alteration of one kinase or phosphatase might affect the others; therefore, one needs to examine all kinases and phosphatases at once to gain a full picture of the aberrant levels.

In this project, we will to design and implement a method that allows us to "see" all the tyrosine phosphatases at once. This means that within one or two days, we can describe the tyrosine phosphatase profiles of a give breast cancer specimen or cell line. This information will give us a fingerprint of a tumor and allow us to rapid identify phosphatases that are present at aberrant levels in certain types of breast cancer. The approach, when developed, would provide early detection markers, as well as the response profiles of breast cancer to anti-estrogen and chemotherapeutic treatments.

Final Report (2003)
Breast cancer is caused by the uncontrolled growth of cells and movement of cells out of confinement. Some forms are also characterized by their acquired resistance to drug treatments, such as tamoxifen. any of these cellular processes are controlled by reversible phosphorylation of proteins. The enzymes that catalyze these reactions are tyrosine kinases and phosphatases. Tyrosine kinases add a phosphate to a protein and tyrosine phosphatases remove it. It is the balance between the actions of these two sets of enzymes that control the fate of the cell. The importance of these enzymes is reflected by the fact that the causes of many cancers are traced back to the mutations of these genes and that the aberrant production and/or activity of these enzymes are often found in cancers. Here we have utilized a novel method that allows us to rapidly see all of the protein tyrosine phosphatases in a single gel through a single reaction. This approach has been combined with microarray gene expression analysis, which permits us to simultaneously examine genome-wide gene expression. As a result, this information gives us a detailed fingerprint to the tumor and allows us to rapidly detect phosphatases that are aberrantly expressed in certain types of breast cancers or in response to therapies. These can then be used as biomarkers for detection and prognosis, as well as potential therapeutic targets. In order to know exactly what to look for, we have compiled a comprehensive protein tyrosine phosphatase (PTP) and dual-specificity phosphatase (DSP) database.

In summary, there are 59 PTPs: 20 receptor-type, 17 non-receptor type, 3 prenylated, non-receptor type, 1 low molecular weight, and 18 dual-specificity phosphatases. We have ascertained the basal expression of a total of 28 tyrosine and dual-specificity phosphatases in most breast cancer cells, such as MCF-7. We also wanted to determine if tyrosine phosphatases regulated the response of breast cancers to estrogen withdrawal and/or tamoxifen therapy. Our studies discovered five estrogen-regulated tyrosine phosphatases: PTPN3, PTPN21, DUSPI, DUSP8, and PTPRH. Our prime candidate was the dual-specificity phosphatase DUSPI, since it has a protective effect and promotes survival by inhibiting programmed cell death. Further, tamoxifen therapy induces cell death by inducing oxidative stress and activating c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) that DUSPI dephosphorylates and inactivates. That said, DUSPI up-regulation might potentiate the development of tamoxifen resistance. We therefore pursued validation studies to determine the value of our candidate phosphatases as biomarkers and/or novel therapeutic targets. To that end, we used quantitative PCR to validate that both hormone-withdrawal and tamoxifen treatment induced the expression of DUSP1 3.5- and 6.8-fold, respectively. This was indeed of clinical relevance, since we could demonstrate that DUSPI expression was elevated in 26.7% (4/15) of breast cancer specimens, ranging from 2.2- to 4.9-fold compared to the mean expression level found in non-cancerous tissue. The ability for DUSP1 function to modulate the sensitivity of breast cancer cells to therapy was investigated next. For this, we generated stable, DUSPI-overexpressing MCF-7 sublines, MCF7-DUSPI. This cell line exhibited marked resistance to tamoxifen-induced apoptosis with only 7.5% of the cells undergoing apoptosis in comparison to parental MCF-7 cells (43%). This was evidently the result of DUSPI-mediated suppression of the JNK/SAPK pathway, as activated JNK (phosphoČJNK) could not be detected in MCF7-DUSPI. Consistent with the ability for tamoxifen to induce oxidative stress, hydrogen peroxide treatment led to heightened levels of JNK activation and apoptosis, both of which were attenuated in MCF7-DUSPI cells. We next wanted to determine if antagonism of DUSP 1 function could increase sensitivity of cells to apoptosis. As a genetic approach, we used RNA interference to silence DUSP 1 gene expression. Treatment of MCF-7 cells with small interfering RNA duplexes (siRNAs) suppressed tamoxifen-induced DUSP 1 mRNA and protein expression, led to heightened levels of phospho-JNK, and augmented apoptosis. Additionally, DUSPI gene silencing rendered MCF-7 cells more susceptible to Fas death receptor-mediated apoptosis. This was especially important, as death receptor ligands are an emerging therapeutic modality. Finally, although specific small molecule inhibitors of DUSP1 are not presently available, this possibility was further pursued by evaluating the influence of the phosphatase inhibitor orthovanadate upon signaling and apoptosis. In combination with orthovanadate, the kinetics and magnitude of JNK phosphorylation were significantly enhanced compared to tamoxifen treatment alone. This was apparently due to DUSP 1 inhibition, as these treatments did not alter expression levels of DUSPI, JNK, or SEKl/MKK4. Accordingly, apoptosis was increased by 23% with the combination treatment. Similarly, MCF-7 cells could be sensitized to apoptosis induced by anti-Fas stimulation alone. Together, this data demonstrates that antagonism of DUSP 1 expression or function with nucleic acids (siRNAs, antisense oligodeoxynucleotides) or small molecule inhibitors, respectively, might be a beneficial therapeutic adjuvant to breast cancer therapies.