Blocking Thyroid Hormones Induced MAPK Activation -Novel Target for Therapy In Myeloma
Authors: Ellis Martin , Cohen Keren , Fabian Ina , Hercbergs Aleck , Ashur-Fabian Osnat 
Published: October 2019

Abstract
Abstract 2964

Background:
Basic/epidemiological/clinical data has established that thyroid hormones (T3/T4) may modulate neoplastic cells and T3/T4 deprivation removes this stimulus. Recently, growing body of epidemiological and clinical evidence suggests improved survival in individuals with primary/secondary hypothyroidism in a variety of tumor types, e.g., breast, lung, renal, prostate, head and neck, glioblastoma and more. On the other hand, hyperthyroidism was associated with an increased risk of lung and prostate cancers in a large-based population study as well as in pancreatic and ovarian cancers. Finally, enhanced response rates to chemo/radiation therapy in hypothyroidism patients have also been reported in preclinical/clinical studies. Taken together, these observations suggest a role for T3 and T4 in tumor growth and a protective effect of hypothyroidism in various cancer types.
T3/T4 affects cell division/angiogenesis by activating MAPK/PI3K pathways through binding integrin αvβ3, an integrin over-expressed on many cancer cells. Thyroid binding site upon the integrin is at close proximity to its well known RGD recognition site and is blocked by tetrac, a natural analog of T4. Interestingly, in multiple myeloma (MM), a highly resistant hematological malignancy, the tumor cells interact with αvβ3 for their invasion/proliferation and thyroid diseases were associated with increased MM risk. As most MM patients still resist treatment, drugs with new mechanisms of action are urgently needed.
We have recently demonstrated that, similar to results obtained from other cancer types, thyroid hormones induce myeloma cell viability and proliferation through MAPK activation. In the current study, we further studied the ability of thyroid hormones to induce MAPK activation in myeloma cells. We show that this MAPK induction is quick, specific and is initiated through thyroid hormones binding to the integrin αvβ3 receptor site and propose ways to block this potent action.

Methods:
Cell lines: MM cell lines, RPMI 8226 and CAG are cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS/antibiotics. Reagents and chemicals: T3, T4, tetrac, RGD and RGE peptides, αvβ3 monoclonal antibodies (LM609), phosphorylated and total MAPK ERK1/2 antibodies. Cell proliferation assay: WST-1 (10% final concentration) is incubated at 37°C for 2 h and read using microELISA reader at 440nm. Flow cytometry: Cell cycle: Cells are harvested, fixed and stained with DNA propidium iodide (PI) (50 μ g/ml)/RNAse A (10mg/ml) and analyzed for DNA content by FACS. Analysis of apoptosis/necrosis: Cells (105) are incubated with 10 μ l Annexin V (FITC conjugated)/5 μ l PI and analyzed by FACS. Western blotting: Whole cell lysates were separated on 8–12% polyacrylamid gels and analyzed by western blot using above indicated antibodies. Results were repeated 2–4 times, in triplicates and were analyzed using unpaired students t test.

Results and Discussion:
Results demonstrate that physiological T3 and T4 concentrations (1nM and 100nM respectively), induce within minutes MAPK activation in myeloma cells, lasting up to 24 hours. This induction is completely blocked by a selective MAPK inhibitor, U0126. We further show that this thyroid-induced MAPK activation is initiated through αvβ3 integrin site, by using specific functional antibody to the integrin (LM609), as well as blocking the binding site using RGD but not RGE peptides. In addition, by using a T4 analog, tetrac, that selectively blocks thyroid hormones binding to the integrin site, we demonstrate a novel and specific way to block the induction of MAPK by T3 and T4. We present here indications that thyroid hormones induce a quick and potent MAPK activation in myeloma cells, similar to reports from other cancer types. This MAPK activation can be blocked using several inhibitors, including tetrac, previously demonstrated by us as a sensitizer of bortezomib action in myeloma.

Conclusions:
In order to surpass myeloma cancer treatment resistance, new mechanisms of action are urgently needed. Our unique approach, by inhibiting thyroid-induced MAPK pathway initiated at the αvβ3 integrin site, may potentially become a new promising chemosensitizing treatment in this disease.

Disclosures:
No relevant conflicts of interest to declare.