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Analysis of signal transducer and activator of transcription 3 (Stat 3) pathway in multiple myeloma: Stat 3 activation and cyclin D1 dysregulation are mutually exclusive events - PubMed

Analysis of signal transducer and activator of transcription 3 (Stat 3) pathway in multiple myeloma: Stat 3 activation and cyclin D1 dysregulation are mutually exclusive events

Leticia Quintanilla-Martinez et al. Am J Pathol. 2003 May.

Abstract

The signal transducer and activator of transcription molecules (Stats) play key roles in cytokine-induced signal transduction. Recently, it was proposed that constitutively activated Stat 3 (Stat 3 phosphorylated) contributes to the pathogenesis of multiple myeloma (MM) by preventing apoptosis and inducing proliferation. The study aim was to investigate Stat 3 activation in a series of multiple myeloma (MM) cases and its effect on downstream targets such as the anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2, and the cell-cycle protein cyclin D1. Forty-eight cases of MM were analyzed. Immunohistochemistry was performed on paraffin sections using antibodies against cyclin D1, Bcl-2, Bcl-xL, Mcl-1, p21, Stat 3, and Stat 3 phosphorylated (P). Their specificity was corroborated by Western blot analysis using eight human MM cell lines as control. The proliferation rate was assessed with the antibody MiB1. In addition, the mRNA levels of cyclin D1 and Stat 3 were determined by quantitative real-time reverse transcriptase-polymerase chain reaction of paraffin-embedded microdissected tissue. Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified: group 1, Stat 3-activated (23 cases, 48%). All cases revealed nuclear expression of Stat 3P. No elevation of Stat 3 mRNA was identified in any of the cases. Three cases in this group showed intermediate to low cyclin D1 protein and mRNA expression. Group 2 included 15 (31%) cases with cyclin D1 staining and lack of Stat 3P. All cases showed intermediate to high levels of cyclin D1 mRNA expression. Group 3 included 10 (21%) cases with no expression of either cyclin D1 or Stat 3P. High levels of anti-apoptotic proteins Bcl-xL and Mcl-1 were identified in 89% and 100% of all cases, respectively. In contrast to Bcl-xL and Mcl-1, the expression of Bcl-2 showed an inverse correlation with proliferation rate (P: 0.0003). No significant differences were found between the three groups in terms of proliferation rate or expression of anti-apoptotic proteins. However, cyclin D1+ cases were always well differentiated and were more likely to show a lymphoplasmocytoid differentiation (chi-square = 9.55). Overall, constitutive activation of Stat 3 was found in almost half (48%) of the investigated MM cases. However, this does not seem to have a major impact on the expression of anti-apoptotic proteins and proliferation. We showed that cyclin D1 overexpression and Stat 3 activation are, mutually exclusive events in MM (P = 0.0066). The universal expression of Mcl-1, independent of activated Stat 3, suggests that its expression is constitutive and that it might play an important role in the pathogenesis of MM.

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Figures

Figure 1.
Figure 1.

Western blot analysis for Stat 3, Stat 3P, cyclin D1, and anti-apoptotic proteins Bcl-xL, Mcl-1, and Bcl-2 in human MM cell lines. Each lane contains 60 μg of protein extract from the following cell lines: lane 1, Granta 519; lane 2, KMS-20; lane 3, U266; lane 4, KMS-18; lane 5, KMS-5; lane 6, KMS-11; lane 7, KMM1; lane 8, KMS-12; lane 9, OPM2. Lane 1 (Granta 519, mantle cell lymphoma cell line) and lane 8 (KMS12) represent the t(11;14) translocated cell lines. Expression of cyclin D1 is limited to these two cell lines. Lane 3 (U266) represents the cell line with known constitutive activation of Stat 3. Western blot analysis with the N-terminal anti-Stat 3 antibody demonstrates both Stat 3α and Stat 3β (92 and 83 kd, respectively) in all cell lines. In contrast only KMS20 and U266 show strong positivity for Stat 3 phosphorylated. Bcl-xL and Mcl-1 expression is present in all cell lines with no apparent correlation between these proteins and the presence of phosphorylated Stat 3. Bcl-2 is negative in three of the cell lines (KMS20, KMS5, and KMM1). Note that the two cell lines with expression of cyclin D1 show high levels of Bcl-2.

Figure 2.
Figure 2.

Immunohistochemical analysis of Stat 3 and Stat 3P. A: KMS18 cell line block used as control. The tumor cells show cytoplasmic positivity for Stat 3 without nuclear staining. Inset: Note that the cells are completely negative when stained with the Stat 3P antibody. B: U266 cell line block used as control. The tumor cells show a strong, crisp nuclear positivity for Stat 3P without cytoplasmic staining. Inset: Note that by immunostaining with the Stat 3 antibody, the cells show strong nuclear and cytoplasmic staining. C–F: Primary MM cases stained for Stat 3 and Stat 3P. C: Case 2, group 1. The tumor cells show cytoplasmic and nuclear positivity for Stat 3. D: The same case immunostained with the Stat 3P antibody reveals a strong nuclear expression confirming the presence of activated Stat 3. E: Case 36, group 2. The vast majority of tumor cells show cytoplasmic expression of Stat 3 indicating the presence of steady-state Stat 3. Note the lack of nuclear staining. F: The same case is negative for anti-Stat 3P. Note the positive nuclear staining of the endothelial cells used as internal control. Immunoperoxidase staining; original magnifications, ×400.

Figure 3.
Figure 3.

Immunohistochemical analysis of cyclin D1 in primary MM cases. A: The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++) (case 29, group 2). B: MM with lymphoplasmacytoid features (case 32, group 2). The majority of tumor cells show strong nuclear positivity for cyclin D1 (+++). C: MM case with nuclear positivity in 20 to 50% of tumor cells (++) and co-expression of Stat 3P (case 20, group 1). D: MM with nuclear positivity in 20 to 50% of tumor cells (++) (case 35, group 2). Note that the intensity of the staining varies from cell to cell. E: MM with nuclear positivity in 10 to 20% of tumor cells (+) and expression of Stat 3P (case 19, group 1). F: MM negative for cyclin D1. Note the presence of rare, positive plasma cells (arrows) (case 46, group 3). Immunoperoxidase; original magnifications: ×200 (B); ×400 (A, C–F).

Figure 4.
Figure 4.

Immunohistochemical analysis of anti-apoptotic proteins in MM cases. A: Mcl-1 expression in a MM case with activated Stat 3 (case 17, group 1). Note the strong cytoplasmic staining. B: Mcl-1 expression in a MM case with cyclin D1 overexpression. Note the weaker cytoplasmic positivity in comparison with the previous case and with the small reactive lymphocytes (arrows) (case 34, group 2). C and D: Bcl-2 expression in MM cases. C: Bcl-2 is strongly expressed in the cytoplasm in the vast majority of tumor cells. D: An example of a MM case negative for Bcl-2. Note the Bcl-2 positivity of a reactive lymphocyte used as internal control. E and F: Bcl-xL expression in MM cases. E: Bcl-xL is positive in the cytoplasm of the tumor cells. F: MM with lack of expression of Bcl-xL. Note the positivity in the cytoplasm of reactive histiocytes used as internal control. Immunoperoxidase; original magnifications, ×400.

Figure 5.
Figure 5.

Comparison of proliferation rate in Bcl-2-positive and Bcl-2-negative cases. Statistical analysis was done using the Mann-Whitney U-test. Cases that are Bcl-2-positive have a mean proliferation rate of 10%, whereas cases with Bcl-2-negative have a mean proliferation rate of 30% (P = 0.0003).

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