Activation of the mTOR Signaling Pathway in Multiple Myeloma Patients and Its Prognostic Significance.

INTRODUCTION The mammalian target of rapamycin (mTOR) is a serine/threonine-specific protein kinase, downstream of the phoshatidylinositol 3-kinase (P13K)/AKT pathway. It controls many aspects of cellular physiology, including transcription, translation, cell size, cytosckeletal organization, autophagy and progression from the G1 to S phase of the cell cycle. Constitutive activation of the mTOR related downstream effectors including P13K, AKT, p70S6K and 4EPB1 was found in numerous malignancies. Rapamycin and its analogues are mTOR inhibitor currently being tested in solid and hematological tumours. Previous studies demonstrated that rapamycin have preclinical potential as therapy for multiple myeloma, especially when associated with other drugs. We first determined the frequency of the activation status of AKT/mTOR/p70S6K pathway in multiple myeloma patients and correlated its activation with FISH analysis and clinical data.

METHODS Immunohistochemical analysis with phospo-AKT (Ser 473), phospho-mTOR (Ser2448) and phosphor-p70S6K (Thr389) (Cell Signaling Technology) was performed on a series of 44 multiple myeloma bone marrow sections. Slides were scored by two independent observers and staining was defined as weakly positive (>10–30, +), moderately positive (>30–70, ++) and strongly positive (>70, +++). FISH analysis was performed on CD138 purified plasma cells of 31 myeloma patients with specific probes for the detection of the t (4;14)(p16.3;q32) and del 13q14 (Vysis). Clinical data of all patients including age, presence of bone lesion, isotype, serum beta2-microglobulin, C-reactive protein, haemoglobin level and albumin were evaluated and Fisher exact test was used to compare the expression of p-mTOR with p-AKT and p-p70S6K signaling protein and clinical data.

RESULTS p-mTOR and p-AKT were expressed respectively in 26 of 44 (59.9%) and 23 of 44 (52.3%) patients, with a predominance of cytoplasmic staining pattern. p-mTOR immunoreactivity was strongly, moderately and weakly positive in 36%, 36% and 28% of the samples, respectively. p-p70S6S was detected in 26 patients (59.9%) with a predominantly nuclear staining pattern. Of the 26 myelomas stained positive for p-mTOR, 22 expressed p-AKT and all of them demonstrated p-p70S6S positivity. p-mTOR expression significantly correlated with both p-AKT (p<0.05) and p-p70S6K (p<0.001) expression consistent with the hypothesis that the AKT/mTOR/p70S6K pathway is activated in multiple myeloma. t(4;14) and del 13 were detected respectively in 5 and 10 out 31 of 44 patients and all of them demonstrated p-mTOR positivity. Fisher exact test significantly correlated p-mTOR expression with high beta2-microglobulin (P<0.01).

CONCLUSION mTOR signaling protein are activated in a fraction of multiple myeloma cases and may contribute to tumour cell proliferation and survival of myeloma cells. Our results also suggest that the AKT/mTOR/p70S6K pathway may have prognostic implications and may provide novel therapeutical targets in this subset of myeloma patients.