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Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

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PAX5− cells overexpress specific pathways involved in cell survival and exhibit increased resistance to bortezomib by facilitating plasmacytic differentiation(A) Immunoblot analyses of pmTOR, p-p70, p-AKT, pERK, and pMAPK in control and PAX5− MCL cells; β-actin served as the loading control.(B) ELISA of the IL-6 levels using conditioned media from SP53 PAX5− MCL cells or control cells. The IL-6 levels were analyzed at steady state (Left) or during serum withdrawal (Right). HS5-CM, which are enriched for IL-6, were used as a positive control. (C) The levels of IL-6 signaling pathway components were quantified via qRT-PCR. Cells were cultured under low serum conditions for 3 days prior to mRNA harvesting, and the relative expression levels were normalized to GAPDH. (D) Immunoblot analyses of pSTAT3 expression in PAX5− and control MCL cells upon the addition of HS5-CM during culturing. HS5-CM were added to the culture for up to 3 days prior to protein harvesting. Total STAT3 and β-actin served as loading controls. (E) PAX5− and control MCL cells exhibited increased pSTAT3 signaling upon HS5-CM addition (30 mins), and this alteration was mediated by IL-6. An IL-6 neutralizing antibody (1 µg) was used to neutralize IL-6 activity in 1 mL of HS5-CM.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 4: PAX5− cells overexpress specific pathways involved in cell survival and exhibit increased resistance to bortezomib by facilitating plasmacytic differentiation(A) Immunoblot analyses of pmTOR, p-p70, p-AKT, pERK, and pMAPK in control and PAX5− MCL cells; β-actin served as the loading control.(B) ELISA of the IL-6 levels using conditioned media from SP53 PAX5− MCL cells or control cells. The IL-6 levels were analyzed at steady state (Left) or during serum withdrawal (Right). HS5-CM, which are enriched for IL-6, were used as a positive control. (C) The levels of IL-6 signaling pathway components were quantified via qRT-PCR. Cells were cultured under low serum conditions for 3 days prior to mRNA harvesting, and the relative expression levels were normalized to GAPDH. (D) Immunoblot analyses of pSTAT3 expression in PAX5− and control MCL cells upon the addition of HS5-CM during culturing. HS5-CM were added to the culture for up to 3 days prior to protein harvesting. Total STAT3 and β-actin served as loading controls. (E) PAX5− and control MCL cells exhibited increased pSTAT3 signaling upon HS5-CM addition (30 mins), and this alteration was mediated by IL-6. An IL-6 neutralizing antibody (1 µg) was used to neutralize IL-6 activity in 1 mL of HS5-CM.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).

Mentions: We next examined the pathways expressed in various hematological malignancies such as the PI3K/AKT/mTOR and MAPK/ERK signaling pathways.27–31 Immunoblots showed strong activation of AKT/mTOR pathway signaling factors, including pmTOR, p-p70 and pAKT, and this activation could account for the increased proliferation of PAX5−MCL cells (Figure 4a). pERK and pMAPK have been implicated in cell survival, adhesion to the microenvironment and motility;32 the levels of these factors were also increased in PAX5− MCL cells (Figure 4a). Interestingly, CCND3, which encodes for cyclin D3, an important cyclin for early B cell growth,33 was also increased (Supplementary Figure 4a).


Differential PAX5 levels promote malignant B-cell infiltration, progression and drug resistance, and predict a poor prognosis in MCL patients independent of CCND1.

Teo AE, Chen Z, Miranda RN, McDonnell T, Medeiros LJ, McCarty N - Leukemia (2015)

PAX5− cells overexpress specific pathways involved in cell survival and exhibit increased resistance to bortezomib by facilitating plasmacytic differentiation(A) Immunoblot analyses of pmTOR, p-p70, p-AKT, pERK, and pMAPK in control and PAX5− MCL cells; β-actin served as the loading control.(B) ELISA of the IL-6 levels using conditioned media from SP53 PAX5− MCL cells or control cells. The IL-6 levels were analyzed at steady state (Left) or during serum withdrawal (Right). HS5-CM, which are enriched for IL-6, were used as a positive control. (C) The levels of IL-6 signaling pathway components were quantified via qRT-PCR. Cells were cultured under low serum conditions for 3 days prior to mRNA harvesting, and the relative expression levels were normalized to GAPDH. (D) Immunoblot analyses of pSTAT3 expression in PAX5− and control MCL cells upon the addition of HS5-CM during culturing. HS5-CM were added to the culture for up to 3 days prior to protein harvesting. Total STAT3 and β-actin served as loading controls. (E) PAX5− and control MCL cells exhibited increased pSTAT3 signaling upon HS5-CM addition (30 mins), and this alteration was mediated by IL-6. An IL-6 neutralizing antibody (1 µg) was used to neutralize IL-6 activity in 1 mL of HS5-CM.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
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Figure 4: PAX5− cells overexpress specific pathways involved in cell survival and exhibit increased resistance to bortezomib by facilitating plasmacytic differentiation(A) Immunoblot analyses of pmTOR, p-p70, p-AKT, pERK, and pMAPK in control and PAX5− MCL cells; β-actin served as the loading control.(B) ELISA of the IL-6 levels using conditioned media from SP53 PAX5− MCL cells or control cells. The IL-6 levels were analyzed at steady state (Left) or during serum withdrawal (Right). HS5-CM, which are enriched for IL-6, were used as a positive control. (C) The levels of IL-6 signaling pathway components were quantified via qRT-PCR. Cells were cultured under low serum conditions for 3 days prior to mRNA harvesting, and the relative expression levels were normalized to GAPDH. (D) Immunoblot analyses of pSTAT3 expression in PAX5− and control MCL cells upon the addition of HS5-CM during culturing. HS5-CM were added to the culture for up to 3 days prior to protein harvesting. Total STAT3 and β-actin served as loading controls. (E) PAX5− and control MCL cells exhibited increased pSTAT3 signaling upon HS5-CM addition (30 mins), and this alteration was mediated by IL-6. An IL-6 neutralizing antibody (1 µg) was used to neutralize IL-6 activity in 1 mL of HS5-CM.*p < 0.05 (vs. PAX5control; Student’s t-test); **p < 0.005 (vs. PAX5control; Student’s t-test).
Mentions: We next examined the pathways expressed in various hematological malignancies such as the PI3K/AKT/mTOR and MAPK/ERK signaling pathways.27–31 Immunoblots showed strong activation of AKT/mTOR pathway signaling factors, including pmTOR, p-p70 and pAKT, and this activation could account for the increased proliferation of PAX5−MCL cells (Figure 4a). pERK and pMAPK have been implicated in cell survival, adhesion to the microenvironment and motility;32 the levels of these factors were also increased in PAX5− MCL cells (Figure 4a). Interestingly, CCND3, which encodes for cyclin D3, an important cyclin for early B cell growth,33 was also increased (Supplementary Figure 4a).

Bottom Line: On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6.Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells.Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

View Article: PubMed Central - PubMed

Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation, Institute of Molecular Medicine for the Prevention of Human Diseases (IMM), University of Texas-Health Science Center at Houston, Houston, TX, USA.

ABSTRACT
Reduced Paired box 5 (PAX5) levels have important roles in the pathogenesis of human B-cell acute lymphoblastic leukemia. However, the role of PAX5 in human lymphoma remains unclear. We generated PAX5-silenced cells using mantle cell lymphoma (MCL) as a model system. These PAX5(-) MCL cells exhibited unexpected phenotypes, including increased proliferation in vitro, enhanced tumor infiltration in vivo, robust adhesion to the bone marrow stromal cells and increased retention of quiescent stem-like cells. These phenotypes were attributed to alterations in the expression of genes including p53 and Rb, and to phosphoinositide 3-kinase/mammalian target of rapamycin and phosphorylated signal transducer and activator of transcription 3 pathway hyperactivation. On PAX5 silencing, the MCL cells displayed upregulated interleukin (IL)-6 expression and increased responses to paracrine IL-6. Moreover, decreased PAX5 levels in CD19+ MCL cells correlated with their increased infiltration and progression; thus, PAX5 levels can be used as a prognostic marker independent of cyclin D1 in advanced MCL patients. Importantly, high-throughput screening of 3800 chemical compounds revealed that PAX5(-) MCL cells are highly drug-resistant compared with PAX5 wild-type MCL cells. Collectively, the results of our study support a paradigm shift regarding the functions of PAX5 in human B-cell cancer and encourage future efforts to design effective therapies against MCL.

Show MeSH
Related in: MedlinePlus