Expression of Smad Proteins as a Potential Prognostic Factor in Chronic Lymphocytic Leukemia

Background: The Smad family proteins are crucial element of one of the most versatile cytokine signaling pathways- the transforming growth factor beta (TGF- ß). TGF-ß is a multipotent cytokine that participates in a wide range of cellular processes in the human body both in physiology and pathology. TGF-ß is involved in angiogenesis, regulates apoptosis, stimulates differentiation and divisions of several target cells and exhibits effects on the cell cycle by G₁ phase arrest. This strongly suggests that disruption of Smad signaling pathway could be involved in cancerogenesis. The Smad proteins are unique group of particles, that after receiving a signal from activated TGF-β, act on transcription factors in the nucleus, leading directly to the expression of the corresponding genes. Based on the differences in their function, the Smad family is divided into three classes: receptor-associated Smads including Smad 1, Smad 2, Smad 3, Smad 5 and Smad 8, co-operating Smads (Smad 4), and inhibitory Smads (Smad 6, Smad 7). Smad 1 and Smad 5 are prefentially involved in the BMP (bone morphogenetic protein) dependent pathway, while Smad 2 , Smad 3 are associated with activin (Act) pathway. According to current knowledge, disturbances in the functioning of Smad proteins are present in a numerous solid tumors. Low expression of Smad 4 protein correlates with progressive outcome and promote metastasis of many solid tumors including pancreatic cancer, colorectal and breast cancer. However, overexpression of Smad 1/8 and Smad 2/3 protein was observed in both patients with colorectal cancer and lung cancer So far, studies in hematological malignances are limited, with no data published in chronic lymphocytic leukemia (CLL) patients. The aim: This is the first study assessing the expression of Smad 1/8, Smad 2/3, Smad4 and Smad 7 proteins in CLL cells in an aspect of their clinical significance and potential prognostic value in this disease. Material and Methods: CLL cells isolated from peripheral blood of overall 214 previously untreated CLL patients were examined on the expression of Smad1/8, Smad 2/3, Smad4 and Smad 7 proteins. Results were compared with data obtained from 42 healthy volunteers. Moreover, expression of Smad proteins was correlated with stable/progression status of the disease, as well as with several prognostic factors known for CLL, including clinical stage according to Rai system, lymphocyte doubling time (LTD), cytogenetics, ZAP-70 and CD38 expression, lactate dehydrogenase (LDH) activity or beta-2-migroglobulin (β2-M) expression. Additionally, Smad protein expression was correlated with the level of spontaneous in vivo apoptosis of CLL cell and expression of three vascular endothelial growth factor (VEGF) receptors – R1, R2 or R3. All measurement were made using flow cytometry methods. Apoptotic index (AI) was calculated as a percentage of Annexin-V-positive cells. Results: Significantly lower expression of Smad 4 was showed in CLL cells than in healthy lymphocytes in controls (p<0.001). Overexpression of Smad 1/8 and low expression of Smad 4 was found in CLL patients with more progressive compared to stable course of the disease (p<0.005). Moreover, high level of Smad 1/8 and low expression of Smad 4 correlated with other well establish prognostic factors, such as clinical stage according to Rai (p<0.001), LDT (p<0.015), LDH (p<0.001), β2-M (p<0.010) and CD38 (p<0.003). In a multivariate analysis low expression of Smad 4 was independent negative prognosic factor. In contrast, there were no statistical differences observed according to ZAP-70 and cytogenetics. Moreover, we did not find differences in expression Smad 2/3 and inhibitory Smad 7 expression was not related to all investigated prognostic factors among CLL patients. Low expression of Smad 4 correlated with lower apoptotic index of CLL cells and higher expression of R1 and R2 VEGF receptors. Conclusions: Our study demonstrated significant correlation between expression of Smad 1/8 and Smad 4 proteins and progressive outcome of disease and poor prognosis. The data presented provides supporting evidence that expression of Smad family proteins may be a valuable prognostic factor for CLL patients.