Ph.D Thesis

Ph.D StudentShakya Jivan
SubjectRegulation of EMMPRIN in Cancer: a New Target for
DepartmentDepartment of Medicine
Supervisor ASSOCIATE PROF. Michal Rahat
Full Thesis textFull thesis text - English Version


Tumors overexpress the multifunctional protein EMMPRIN, which stimulates angiogenesis via induction of VEGF and MMPs. The level of its expression directly correlates with the grade and stage of cancer.  The tumor is a complex rogue organ composed of different types of cells in addition to tumor cells, including evidence of neuronal infiltration. Here, we show that EMMPRIN is regulated not only by the interaction between the tumor cells and the macrophages, but also by Isoproterenol (Isop), a norepinephrine analog. The interaction simulated by a co-culture between the LLC tumor cells and the PMJ2-R macrophage-like cells was sufficient to synergistically increase EMMPRIN secretion relative to each single culture and was further enhanced by stimulation with lower concentrations of Isop (0.5 mM). The increase in EMMPRIN expression was accompanied by increased levels of pro-angiogenic factors, VEGF and MMP-9. We also show that EMMPRIN is differently regulated in tumor cells and macrophages, as miR-146a post-transcriptionally increased EMMPRIN expression in the LLC tumor cells, while transcriptional regulation was demonstrated for the PMJ2-Rmacrophages. In an in vivo experiment where the brain's reward center of the Cre-transgenic C57BL/6 mice was activated, the subcutaneous LLC tumors were significantly reduced. This was accompanied by a significant decrease angiogenesis and in EMMPRIN expression, as evident from mean vessel density (MVD) and the intratumoral VEGF concentrations. This further strengthens the potent role of EMMPRIN in tumor angiogenesis.

Next, we show that active vaccination against EMMPRIN inhibited tumor growth. We synthesized a specific epitope, known to be responsible for the induction of MMP-9 and VEGF, as a multiple antigenic peptide (MAP). We therapeutically vaccinated 4T1 tumor-bearing mice with 161-MAP after tumor implantation, or prophylactically before implantation. The prophylactic, but not the therapeutic module of vaccination inhibited the 4T1 tumor growth. Similarly, in human derived A498 renal cell carcinoma established in nude mice, a single injection of splenocytes adoptively transferred from 161-MAP vaccinated BALB/c mice inhibited tumor growth. These results highlight the need to identify the therapeutic window and further characterize the potential of 161-MAP vaccination in human tumors.

Vaccination against a self-protein like EMMPRIN could potentially have adverse effects. To address this, we first assessed leukocyte function in a zymosan A induced peritonitis model.161-MAP vaccination did not alter the host’s ability to mount an effective immune response in comparison to the control SCR-MAP vaccinated mice. The peritoneal macrophages were equally competent in phagocytosing zymosan A particles and in secreting inflammatory cytokines between the control and the vaccinated groups. Similarly, the normal tissue morphology of kidney and colon, where EMMPRIN is expressed, was intact in 161-MAP vaccinated mice without affecting their level of EMMPRIN expression. In comparison, EMMPRIN expression was decreased only in the tumor tissues of 161-MAP vaccinated mice, suggesting that EMMPRIN vaccination induced an immune response that was specifically targeted to the tumors, but left the normal tissues unaffected. Moreover, upon repeated vaccination, the mice did not develop any autoimmune diseases. Collectively, this suggests EMMPRIN can be specifically targeted to inhibit tumor growth by vaccination without adversely affecting the host.