Mechnisms of a unique tumor inhibitor that downregulates Ras and upregulates the tumor suppressor p53

Research project P 13970 Mechanisms of a unique tumor inhibitor that downregulates Ras and upregulates the tumor Burkhard JANSEN 11.10.1999 Ras proteins are essential components of signaling pathways that regulate cell growth, differentiation and survival. To act as signal regulators they must be tethered specifically with the inner leaflet of the plasma membrane. This anchorage is promoted inter alia by the farnesyl isoprenoid moiety of Ras. The mechanism of the isoprenoid - dependent Ras membrane anchorage is, however, not known. This is a major unresolved enigma. In our previous studies we demonstrated that, depending on their structure, isoprenoid analogs release Ras from its interactions with membrane components, evident by the isoprenoid- induced increase in the lateral mobility of Ras that precedes its dislodgment from the membrane to the cytosol. Evidently, this leads to decreased levels of oncogenic Ras in rodent and human tumor cells, inhibition of Ras signaling, and upregulation of the p53 tumor suppressor and the cell cycle regulator waf 1. These results are consistent with the possibility that protein-protein interactions are involved in Ras membrane association necessary for the development and progression of human malignancies. Indeed, we showed that isoprenoid analogues carry the potential to inhibit human tumor growth. Moreover, preliminary experiments using selective chemical cross-linking procedures led us to the identification and partial purification ofa membrane Ras-interacting protein. Furthermore, the cross -linking of Ras with this protein could be competitively prevented by an isoprenoid analog that blocks Ras functions and Ras dependent tumor growth. Our goals are to purify the protein to homogeneity, to determine whether it acts as anisoprenoid-dependent Ras anchorage (IDRA) protein and to examine its effects on Ras membrane anchorage and signaling. To achieve these goals we will obtain partial amino acid sequence of the protein and subsequently clone its cDNA from both a rat and a human cDNA library. A full-length clone will be used to express the encoded protein in attempts to evaluate its ability to serve as a specific IDRA protein. Namely: 1.Test the dependence of its association with Ras on the Ras isoprenoid moiety. 2. Test the ability of isoprenoid analogs to interfere with this interaction. 3. Examine the dependence of the putative IDRA-Ras interactions on the guanine nucleotide binding state of Ras. 4. Examine the specificity of the putative IDRA towards various Ras isoforms and towards other GTPases. 5. The biological relevance of IDRA will then be evaluated by examining the effects of its over-expression or down-regulation (by the anti-sense approach) on Ras membrane anchorage and on Ras signaling and transformation. 6. We shall furthermore investigate the influence of isoprenoid analogues on wt or mutant p53 and p53 associated or regulated factors such as MDM2, waf 1, c-fos, c-jun and JNK The relation between these effects and Ras and IDRA interactions will be examined. As regulators of fundamental physiological processes Ras proteins appear to play a key role in homeostasis. Indeed, Ras malfunctions have a major impact in the development, progression and treatment resistance of numerous human malignancies. Therefore, understanding of the normal and abnormal functioning of Ras is of utmost importance and may lead to the development of new strategies of the treatment of human cancer.

Summary: During this project we investigated the mechanisms of action and the biological activity of the novel Ras inhibitor, Farnesyl thiosalicylic acid (FTS). Our studies showed that FTS is not only able to reduce oncogenic Ras but it also activated the tumor suppressor protein p53 in colon cancer cells, leading to a profound cell growth reduction. Furthermore, FTS chemosensitized both colon and pancreatic cancer cells as well as human melanoma xenotransplants when given in combination with standard chemotherapeutic agents. Background: Ras proteins are essential components of signaling pathways that regulate cell growth, differentiation and survival. They are anchored into the inner plasma membrane via a farnesyl isoprenoid moiety. This membrane targeting of Ras is a strict requirement for the biological activity. In our previous studies we could demonstrate that isoprenoid analoga, such as Farnesyl thiosalicylic acid (FTS) can dislodge Ras proteins from their specific membrane anchorage sites into the cytosol where they are rapidly degraded. This leads to a reduction of total cellular Ras proteins in tumor cells (rat, human) causing an inhibition of the Ras signaling pathway. Treatment of various human tumor cells leads to a clear reduction of the cell growth triggered by this described mechanism. Results: We showed in a colon cancer model that FTS not only dislodges Ras from the membrane and therefore changes the signaling pattern through the Ras pathway, but also upregulates the tumor suppressor protein p53. p53 is a potent regulator of both the cell cycle and the programmed cell death (apoptosis). p53 activation caused an arrest of the cell cycle and therefore a stop of cell growth. The co-regulation of these two key elements of cell division resulted in a dramatic reduction of colon cancer cell growth in both cell culture experiments and mouse xenotransplantation studies. Moreover, oncogenic Ras is well known for its major role in establishing chemoresistence against classic chemotherapeutic agents in a series of human neoplasias. Therefore, we tested the hypotheses if the reduction of Ras proteins leads to a chemosensitization of the treated cells. We could proof that FTS has indeed a high potential to chemosensitize human colon and pancreas cancer cells in vitro. Cells treated with FTS showed a much higher sensitivity to chemotherapeutic agents in vitro when given in combination with the either doxorubicine or gemcitabine. Chemosensitization of pancreatic tumor xenografts by FTS was also shown in nude mice with a synergistic effect on the survival rate of the combined treatment of gemcitabine and FTS with an increase in the overall survival of 65%. We also tested a combination chemotherapy of FTS and the standard chemotherapeutic agent for human melanoma treatment, dacarbazine (DTIC), in a SCID mouse xenotransplantation study. We showed an enormous increase in the sensitivity of the xenografts against DTIC. The tumor growth inhibition of the combination was 56% compared to the control groups. Conclusion: The studies which were performed within the framework of this project clearly elucidated a new mechanism of action of FTS and proofed its therapeutic value for the treatment of human malignancies such as colon and pancreas carcinoma as well as malignant melanoma. Therefore, FTS might well proof to be an important partner in the fight against human neoplasias, where mutated Ras genes play an important role in the survival strategies of these cancer cells. FTS is also active in single agent treatment regimes but we conclude that the most promising application might be combination treatments of FTS with chemotherapeutic drugs.