In: Anatomy and Physiology
You have a patient with an aggressive tumor that needs treatment right away, but to determine the correct treatment you need to discover the cause of the cancer. You have two hypotheses to consider. (1) The tumor is caused by a viral oncogene consisting of an active viral gene fused with the coding region of the DVL1 gene (the human homolog of the Drosophila Dsh gene). (2) The tumor cells have two loss of function point mutations that alter the structure of the BRCA1 protein product and render the protein nonfunctional. Each type of tumor responds well to only one specific treatment.
a. What test or tests would you do to determine which is the correct cause of this tumor?
b. What results would you expect from your test or tests for each?
c. Explain to the patient (who is not a scientist) how these results have helped you decide which treatment to use.
a. DVL1 is a gene that encodes a phosphoprotein that induces cell proliferation. When the promoter of active viral gene is fused to the coding region of DVL1 gene, the DVL1 protein is expressed in these cells as an oncogene. Thus, it will induce cell proliferation resulting in formation of tumors. Oncogenes are known to induce excessive cell proliferation in cells due to their effect on DNA replication in S phase.
In case of second scenario, the tumor may be caused by effect of tumor suppressor gene inactivation. Tumor suppressor genes such as BRCA1 inhibit cell division and cause apoptosis. They are mostly DNA repair genes that will repair mutations caused in DNA. Loss of function mutation in BRCA1 would cause production of nonfunctional protein. This would cause increased cell proliferation.
In order to analyze whether the tumor is caused by DVL1 or BRCA1 mutation, the cells from the patient can be isolated and the expression of DVL1 and BRACA1 can be analyzed. Another assay that can be performed is to treat the tumor cells individually with both drugs and assessing whether any of the treatment works and reduces cell proliferation in those cells.
The expression of DVL1 can be assessed by specific antibodies against phospho-DVL1 (active form) and BRCA1 protein by Western blot (DVL1).The mRNA expression for DVL1 can also be assessed using quantitative RT-PCR. The expression of protein and mRNA can be compared with cells from normal person that does not have the tumor. The normal cells act as control. BRCA1 expression can be assessed by immunofluorescence using specific antibodies. For these, the cells will have to be synchronized to undergo mitosis and BRCA1 expression should be assessed during mitosis.
Similarly, Apoptosis assays can also be performed by TUNEL staining whether there is loss of apoptosis in tumor cells. DVL1 is involved in canonical and non-canonical Wnt signaling. If DVL1 is involved, it would cause increased nuclear translocation of beta catenin. Hence, beta catenin localization can be tested in tumor cells by immunofluorescence. Cell proliferation can be detected by analyzing Ki67 expression (immunofluorescence) in nucleus of the cells.
b. If the tumor is caused by expression of viral oncogenes, then there will be increase expression of DVL1 mRNA and phospho-DVL1 protein in the tumor cells as compared to the control cells. BRCA1 protein expression will not be altered in control and tumor cells during mitosis. BRC1 should be expressed on centromere during mitosis. There will be increased expression of beta catenin (non-phosphorylated) in nucleus of the cells. When the tumor is treated with the drug specific for viral oncogene, there will be regression of the tumor as observed by decreased Ki67 staining. The other tumor treatment will not cause reduced cell division. Further, this treatment should decrease DVL1 expression in tumor cells and inhibit beta catenin translocation.
In case the loss of function BRCA1 mutations causes the tumor, there will no expression of BRCA1 protein expression in tumor cells during mitosis in centromere as compared to control cells. BRCA1 expression should be lost in centromere during mitosis. Phospho-DVL1 expression will be same in both types of cells. There will be no translocation of beta catenin to the nucleus. The DNA fragmentation of tumor cells will be absent as detected by TUNEL staining, indicating decreased apoptosis. Further, reduced cell proliferation will only be seen with other specific treatment for BRCA1 mutations. There will be increased apoptosis seen in these cells. Beta catenin expression and DVL1 remains unaltered and there in no nuclear beta catenin expression seen in treated tumor cells.
c. The DVLI protein regulates one cell signaling pathway called Wnt beta catenin pathway. If DVL1 is expressed, beta catenin is not phosphorylated (obtain phosphate groups on its amino acids) and will move to nucleus, only in tumor cells. Here it will bind to DNA and cause increased transcription/expression of genes that it regulates. There will also be increased phosphorylation of DVL1 seen in tumor cells.
If BRCA1 is causing the tumor, there will be no effect on Wnt beta catenin signaling in tumor cells. In this case, beta catenin will be phosphorylated and hence, will not move to nucleus. Similarly, BRCA1 is known to be expressed in centromere during mitosis. However, if BRCA1 is non-functional, it is not associated with centromere during mitosis. Mitosis is the stage of cell cycle where the chromosomes (which is coiled DNA) separate into daughter cells. Further, BRCA1 loss will cause reduced apoptosis. Thus, in the tumor cells, the DNA is intact and not seen as fragments.
Confirmation will be decided by treatment of tumor cells with the specific drug. If the drug shows effect in vitro, only then will it have an effect in vivo. These treated cells should show either decrease Phospho-DVL1 and beta catenin translocation or should show increased apoptosis by TUNEL.