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

Tissue Samples

Forty-eight formalin-fixed, paraffin-embedded and nondecalcified blocks of tissue specimens obtained from lytic bone lesions of MM diagnosed between 1991 and 2001 were selected from the files of the Institute of Pathology, Technical University of Munich, Munich, Germany. Most of the material was obtained during surgery, and contained a high percentage of tumor cells with few bone trabeculae. Clinical information was obtained from the patients’ medical records. Hematoxylin and eosin-stained slides and immunoperoxidase studies were reviewed in all cases by three of the authors (LQ-M, MK, and FF). The cases were graded according to the histological grading criteria described by Bartl and colleagues 17 The clinical staging was done according to Durie and Salmon. 18 Some of the cases have been reported previously as part of another study. 19

Immunohistochemistry

Immunohistochemistry was performed on an automated immunostainer (Ventana Medical Systems, Inc., Tucson, AZ) according to the company’s protocols, with slight modifications. After deparaffinization and rehydration, the slides were placed in a microwave pressure cooker in 0.01 mol/L citrate buffer, pH 6.0, containing 0.1% Tween 20, and heated in a microwave oven at maximum power for 30 minutes. The antibody panel used included cyclin D1 (clone P2D11F11; Novocastra, Newcastle, UK), Stat3 (BD Transduction Laboratories; San Diego, CA), Stat3 phosphorylated (P) (New England Biolabs Inc., Beverly, MA), p21 (BD Transduction Laboratories), Bcl-2 (DAKO, Glostrup, Denmark), Bcl-xL (BD Transduction Laboratories), Mcl-1 (Chemicon, Temecula, CA), CD20 (clone L26, DAKO), and p27KIP1 (BD Transduction Laboratories). The proliferation rate was assessed with the monoclonal antibody against the Ki67 antigen (clone MiB-1, DAKO). Appropriate positive controls were used to confirm the adequacy of the staining. The Stat 3 antibody from Transduction Laboratories recognizes both the latent (cytoplasmic) and the activated (nuclear) form of Stat 3, whereas the Stat 3P antibody recognizes exclusively the nuclear or activated form of Stat 3. As control for the anti-Stat 3 antibodies, cell blocks from two MM cell lines were used. To assure the staining quality of cyclin D1, a cyclin D1-positive mantle cell lymphoma carrying the t(11;14) translocation was included in every run. Because p21 expression is found only in isolated lymphocytes, any staining >10% was considered positive. A grid ocular objective was used to count 300 cells over three high-power fields (×40) and the percentage of positive cells was reported as 0 to 100%.

Cell Lines

In addition to the primary cases, eight human myeloma cell lines (KMM1, OPM2, U266, KMS5, KMS11, KMS12, KMS18, KMS20) were used in this study. A mantle cell lymphoma cell line, Granta 519, was used as control. All cell lines were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum, 2 mmol/L glutamine, 10 U/ml penicillin, and streptomycin (Gibco, Life Technologies). Protein concentration was determined by the BCA protein assay reagent kit (Pierce, Rockford, IL).

Preparation of Cell Line Blocks

Tumor cell lines were grown in RPMI 1640 supplemented with 15% fetal calf serum. The cells were harvested during their exponential growth phase, centrifuged at 550 × g for 5 minutes, and the supernatant removed. The cell pellet was washed once and resuspended in three drops of human plasma to which three drops of thrombin were added. The resulting clot was fixed in 10% formalin and paraffin-embedded.

Western Blot Analysis

A total of 60 μg of protein extracts was separated by 7.5% and/or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to pure nitrocellulose immobilization membranes (Amersham Pharmacia Biotech, Freiburg, Germany). Membranes were blocked for 1 hour in 5% nonfat dry milk and incubated with the primary antibody for 1 hour. Subsequently, membranes were washed five times for 5 minutes each in a wash buffer (10 mmol/L Tris, pH 7.6, 100 mmol/L/L NaCl, and 0.1% Tween) and incubated with a biotinylated secondary antibody for 1 hour. Membranes were washed five times in the same wash buffer and detection was performed by chemiluminescence with the ECL detection system (Amersham Pharmacia Biotech) for 2 to 15 minutes, and then the membranes were exposed to a Kodak X-OMAT AR film. All assays were repeated several times and gave similar results.

IL-6 Enzyme-Linked Immunosorbent Assay

IL-6 was measured in the supernatant of the cell cultures, 5 days from splitting during their exponential growth phase using the Quantikine human IL-6 kit from R&D Systems (Minneapolis, MN). The enzyme-linked immunosorbent assay was performed according to the recommendations of the manufacturer without modifications. The optical density was measured with the microplate reader DigiScan from Asys Hithec, Germany. The used wavelength was 450 nm with a wavelength correction at 570 nm as recommended. The standard curve was created by reducing the data using a computer software (MiKrotek Labor Systems GmbH, Overath, Germany) capable to generate a four-parameter logistic (4-PL) curve fit as recommended.

Real-Time Quantitative Reverse-Transcriptase-Polymerase Chain Reaction (RT-PCR)

Tissue preparation, microdissection of pure tumor cell populations, and RNA extraction from formalin-fixed tissues for real-time quantitative RT-PCR were performed as described previously. 20 Real-time RT-PCR reactions were performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). Intron-spanning primers and probes for cyclin D1, Stat 3, and TATA box-binding protein (TBP) as housekeeping gene control were designed using Primer Express software (Applied Biosystems) (Table 1) ▶ .

Ten μl of RNA extracted from microdissected cells was transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) and 250 ng of random hexamers (Roche, Penzberg, Germany) following the manufacturer’s recommendations in a final volume of 20 μl. PCR reactions were performed in at least two replicates and were performed with the TaqMan Universal PCR Master Mix (Applied Biosystems) using 6 μl of diluted cDNA, 200 nmol/L of the labeled probe, and 300 nmol/L of primers (except cyclin D1-307 sense that was used at 900 nmol/L) in a 30-μl final reaction mixture. After initial incubation at 50°C for 2 minutes and 95°C for 10 minutes, samples were amplified for 50 cycles of 95°C for 15 seconds, followed by 60°C for 1 minute.

Amounts of cyclin D1, Stat 3, and TBP mRNAs were calculated using linear regression analysis from an external standard curve generated from Granta 519 RNA. To determine the threshold value for altered cyclin D1 and Stat 3 expression in MM, the mean values of cyclin D1/TBP and Stat 3/TBP ratios in reactive tissues from lymph nodes and bone marrow were evaluated. The mean cyclin D1/TBP ratio in reactive tissues was 0.84 (range, 0.31 to 1.38). Cyclin D1/TBP ratios greater than the value of 2.33 (mean + 5 SD) were arbitrarily considered to represent increased cyclin D1 expression. The Stat3/TBP ratio in reactive tissues from BM and LN was also determined (mean, 1.27; range, 1.15 to 1.38). Stat3/TBP ratios below 1.79 (mean + 5 SD) were arbitrarily considered to represent normal Stat3 levels.

Statistical Analysis

The significance of the association of different clinicopathological parameters with the expression of Stat 3 and cyclin D1 or with the lack of expression of both proteins was assessed with the chi-square test. The association between Bcl-2 and proliferation rate was assessed using the Mann-Whitney U-test and the Fisher’s exact test. The association between the expression of Stat 3P and cyclin D1 was assessed using the Fisher’s exact test.

Patient Characteristics and Histological Findings

Clinical data and histology are summarized in Table 2 ▶ . Of the 48 patients, 25 were male and 23 were female (M:F ratio, 1:1), with a median age of 64 years (range, 32 to 81 years). In 12 patients the clinical information was not available. Histologically, 39 cases (81%) were composed of well-differentiated plasma cells (Bartl grade I) and 9 cases (19%) were moderately differentiated (Bartl grade II). Ten cases revealed lymphoplasmacytoid differentiation. None of the cases showed plasmablastic morphology.

Western Blot Analysis for Stat 3, Stat 3 Phosphorylated, Cyclin D1, Bcl-xL, Mcl-1, and Bcl-2 in Human MM Cell Lines

To validate the specificity of the Stat 3 antibodies used in this study, Western blot analysis was performed in well-characterized human MM cell lines (Figure 1) ▶ . Western blot analysis with the N-terminal-specific anti-Stat 3 antibody that recognizes both Stat 3α- and Stat 3β-phosphorylated and -unphosphorylated proteins, demonstrated two specific protein bands of the expected size (92 and 83 kd, respectively) in all human MM cell lines. The mantle cell lymphoma cell line, Granta 519, used as control for cyclin D1 expression showed significantly weaker bands. In contrast, phosphorylated Stat 3 (Y705) (92 kd), which specifically recognizes Stat 3 phosphorylated at tyrosine 705, was strongly expressed only in U266 and KMS20 MM cell lines. As expected, cyclin D1 was expressed only in the two cell lines known to have a t(11;14); Granta 519 and the MM cell line KMS12. Bcl-xL and Mcl-1 were expressed in all cell lines and their levels of expression varied from one cell line to another (Figure 1) ▶ . Five MM cell lines expressed high levels of Bcl-xL (U266, KMS18, KMS5, KMS11, and KMM1), and three expressed moderate levels (KMS20, KMS12, and OPM2). The expression of Mcl-1 was more homogeneous and only U266 expressed higher levels when compared to the other cell lines. Bcl-2 was negative in three of the eight cell lines (KMS-20, KMS-5, and KMM1). Of note is that the two cell lines with t(11;14) and expression of cyclin D1 showed very high levels of Bcl-2.

IL-6 Expression in Human MM Cell Lines and Correlation with the Expression of Anti-Apoptotic Proteins

IL-6 was measured in the supernatant of all cell lines to identify those MM cell lines with autocrine production of IL-6. We found that KMS-20, U266, KMS18, KMS5, and KMM1 produce IL-6 in an autocrine manner, whereas the mantle cell lymphoma cell line Granta 519 and the MM cell lines KMS11, KMS12, and OPM2 are independent of IL-6 for their growth. Of note is that even though all cell lines expressed Stat 3 in steady-state, those cell lines with autocrine production of IL-6 had higher expression levels of Stat 3, with the exception of KMS18. However, the autocrine production of IL-6 in some cell lines did not universally result in the presence of Stat 3P, nor did it influence the expression of Bcl-xL, Mcl-1, and Bcl-2.

Immunohistochemical Findings in Primary MM Cases

The results of the immunohistochemical studies are summarized in Table 3 ▶ . All cases were previously immunophenotyped for CD20 and CD138. All cases were positive for CD138. Four of the 48 cases (8.3%) showed expression of CD20 in a proportion of the tumor cells. To confirm the staining pattern of the Stat 3 antibodies in paraffin tissues, cell blocks from two MM cell lines were used. The KMS18 cell line showed a purely cytoplasmic staining with the Stat 3 antibody demonstrating the presence of steady-state Stat 3 (Figure 2A) ▶ and was negative for the Stat 3P antibody (Figure 2A ▶ , inset). In contrast the U266 cell line revealed both a cytoplasmic and nuclear positivity with the anti-Stat 3 antibody (Figure 2B ▶ , inset). The activation of Stat 3 in U266 cell line was confirmed with the Stat 3P-specific antibody, rendering a crisp nuclear staining (Figure 2B) ▶ . For the purpose of this study, only cases with nuclear staining with both antibodies, indicating Stat 3 activation, were considered positive for Stat 3 (Figure 2, C and D) ▶ . Cases with only cytoplasmic staining for Stat 3 but negativity for Stat 3P were considered Stat 3-negative (Figure 2, E and F) ▶ . Following this definition, Stat 3 activation was observed in the majority of tumor cells in 23 of the 48 cases (48%). The staining varied from case to case; 13 cases showed strong (+++), 7 moderate (++), and 3 weak nuclear expression (+). Of the 25 cases considered negative for Stat 3P, 13 cases revealed cytoplasmic staining with the Stat 3 antibody, indicating the presence of steady-state Stat 3. Cyclin D1 expression was observed in 18 cases (37.5%). In 11 of these 18 cases, homogenous, strong nuclear staining of cyclin D1 was observed in >80% of the tumor cells (+++) (Figure 3, A and B) ▶ , in 4 cases in 20 to 50% of the tumor cells (++) (Figure 3, C and D) ▶ , and in the remaining 3 cases, in 10 to 20% of the neoplastic cells (+) (Figure 3E) ▶ . In contrast to cases with +++, the intensity of cyclin D1 staining in cases with ++ and + positivity was weak to moderate and varied from cell to cell (Figure 3D) ▶ . In three cases, unequivocal, rare plasma cells were positive for cyclin D1 (Figure 3F) ▶ . These cases were interpreted as negative for cyclin D1. Mcl-1 was expressed in the cytoplasm of tumor cells in all cases (100%) (Figure 4A) ▶ . However, in 7 of 18 cases with cyclin D1 expression, the intensity of the positivity was weaker (++) (Figure 4B) ▶ . Bcl-2 was strongly positive in 32 of 48 cases (67%) (Figure 4, C and D) ▶ . Bcl-xL was positive in 42 cases (88%) (Figure 4, E and F) ▶ , the intensity of the expression varied from case to case (++ to +++). Expression of p21 in >10% of the tumor cells was observed in 18 cases (38%). The percentage of p21+ cells ranged from 14 to 100% with a median of 39%. The proliferation rate varied from <5 to 80% with a median value of 15% as assessed by MiB1 staining.

Quantitation of Stat3 and Cyclin D1 mRNA in MM Cases

Stat 3 and cyclin D1 mRNA expression levels quantitated by real-time TaqMan RT-PCR analysis are summarized in Table 3 ▶ . Under the selected experimental conditions, reproducible quantitative RT-PCR results were obtained in almost all cases. The observed Stat 3/TBP mRNA ratios were generally low (median, 0.94; range, 0.15 to 2.8), when compared to the additionally investigated control lymph node, spleen, and bone marrow samples (mean, 1.27; range, 1.15 to 1.38). In contrast to Stat3, mRNA cyclin D1 transcript levels showed remarkable variation in the tumors. The mRNA levels clustered in three different groups: 1) high cyclin D1 transcript levels with cyclin D1/TBP ratios greater than 100 were observed in 11 of 48 (23%) cases, (range, 101 to 425; median, 163); 2) intermediate cyclin D1 transcript levels with cyclin D1/TBP ratios between 10 to 20 were observed in 6 of 48 (12.5%) cases (range, 10 to 18.72; median, 15.3); and 3) negative or low cyclin D1 transcript levels with cyclin D1/TBP ratios less than 7.0 were found in 31 of 48 (64.5%) cases. The correlation between immunohistochemistry and real-time RT-PCR was excellent (Table 3) ▶ . The 11 cases with strong (+++) nuclear cyclin D1 staining in the majority of the tumor cells, expressed high levels of cyclin D1 mRNA (cyclin D1/TBP ratio greater than 100). In contrast, cases with 20 to 50% cyclin D1-positive cells showed intermediate cyclin D1 mRNA expression (cyclin D1/TBP ratio between 10 to 20).

Correlation between Stat 3P and Cyclin D1 Expression

Three different groups determined by the expression of Stat 3P and cyclin D1 (protein and mRNA) were identified. In general, cases expressing cyclin D1 did not express Stat 3P and vice versa. This negative association between cyclin D1 and Stat 3P was statistically significant (P = 0.0066).

Group 1 contained 23 (48%) cases that revealed activated expression of Stat 3 in the majority of tumor cells; however, none of the cases showed increased expression of Stat 3 mRNA (Table 3) ▶ . This finding indicates that the increased levels of activated Stat 3 in MM cases is most probably a consequence of posttranscriptional changes. Three cases in this group showed intermediate to low cyclin D1 protein and/or mRNA expression. Group 2 included the 15 (31%) cases with cyclin D1 staining and absence of activated Stat 3. Group 3 included 10 (21%) cases with neither expression of cyclin D1 nor activated Stat 3.

Correlation between Activated Stat3 and Cyclin D1 Expression with Clinicopathological Characteristics and Outcome

The tumor morphology and the clinicopathological characteristics of the patients were analyzed in the three different groups described above (Tables 2 and 3) ▶ . Group 1 included 23 cases of MM with Stat 3 activation. Eleven patients were male and 12 were female with a median age of 64 years (range, 32 to 79 years). Histologically, 17 cases were composed of well-differentiated plasma cells and 6 cases of intermediately differentiated plasma cells. Three of the cases (13%) showed lymphoplasmocytoid differentiation, including case 18, which expressed cyclin D1. Twelve of 20 (60%) patients presented with stage IIIA-B disease. Twelve patients are alive 4 to 96 months later (mean, 26 months), two with no evidence of disease. Six patients died with disease and five cases were lost to follow-up. Group 2 included 15 cases with cyclin D1 expression and no activation of Stat 3 protein. However, in eight cases cytoplasmic expression of steady-state Stat 3 was observed. Ten patients were male and five were female with a median age of 64 years (range, 40 to 81 years). Histologically, all cases were composed of well-differentiated plasma cells; seven cases (47%) revealed lymphoplasmocytic differentiation. Eight of 13 (62%) patients presented with stage IIIA-B disease. Nine patients are alive with disease 1 to 91 months later (mean, 36 months). Four patients died with disease and two were lost to follow-up. Group 3 included 10 cases with no cyclin D1 expression or Stat 3 activation. Five of these cases showed cytoplasmic expression of Stat 3. Four patients were male and six female with a median of 66 years (range, 47 to 76 years). Histologically, seven cases were composed of well-differentiated plasma cells and three cases of intermediately differentiated plasma cells. Eight of nine (89%) patients presented with stage IIIA-B disease. Four patients are alive with disease 27 to 54 months later (mean, 37.5 years). Two patients are dead and four patients were lost to follow-up.

No significant differences in the frequency of the main clinical parameters, ie, stage III (60% versus 62% versus 89%) and overall survival (67% versus 69% versus 67%) were observed between groups 1, 2, and 3. Nevertheless, it is of clinical interest that MM cases with cyclin D1 expression showed a male predominance (male:female, 2:1). In addition, cyclin D1+ cases were always well differentiated, and were more likely to show lymphoplasmacytoid differentiation (chi-square = 9.55).

Correlation between Activated Stat3 and Cyclin D1 Expression and Proliferation Rate and Expression of Anti-Apoptotic Proteins

The proliferation rate and the expression of anti-apoptotic proteins were analyzed separately in the three different groups described above (Table 4) ▶ . There were no significant differences among the three groups concerning proliferation rate, which showed a wide range in all groups, and expression of anti-apoptotic proteins. However, Bcl-2-positive cases showed a lower proliferation rate (mean, 10%) than cases that were Bcl-2-negative (mean, 30%), and this difference was statistically significant (P = 0.0003) (Figure 5) ▶ . The best discriminatory cutoff for proliferation rate was 20%. Cases with proliferation rate greater than 20% usually lacked expression of Bcl-2 and this was significant (P = 0.0001). Although there were no significant differences between the three groups and expression of anti-apoptotic proteins, cases expressing cyclin D1, tended to have a lower proliferation rate, to express more often Bcl-2 and p21, and less frequently Bcl-xL.

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