It is known that bone marrow (BM) angiogenesis is increased in multiple myeloma (MM) patients in relationship with disease progression and supports MM cell growth. MM-induced angiogenesis is mainly due to an overproduction of pro-angiogenic molecules by MM cells and the BM microenvironment. However, the molecular mechanisms at the basis of the angiogenic process are currently under investigation. The deregulation of the homeobox genes has been previously associated to tumor progression and neoangiogenesis. Particularly, overexpression of the homeobox HOXB7 is involved in tumor-associated angiogenic switch in solid tumors. Interestingly, we have recently shown that HOXB7 is one of the overexpressed genes in BM micorenvironment cells in MM patients as compared to healthy subjects. In this study we have investigated the expression of HOXB7 by MM cells and its potential role in MM-induced angiogenesis in vitro and in vivo.
First, by microarray analysis we evaluated HOXB7 expression in MM cells finding that HOXB7 was overexpressed in 43% of 23 human myeloma cell lines (HMCLs) tested as compared to normal plasmacells. HOXB7 expression was further confirmed by real time PCR and western blot analysis. On the other hand a small fraction of patients (about 10% in a representative database of 133 MM patients) showed HOXB7 overexpression with a preferential distribution based on the characteristics of molecular MM subtypes being significantly overexpressed in those cases which did not show any of the primary IGH translocations, corresponding to the TC2 and TC3 groups in the translocations/cyclins (TC) classification.
In order to investigate the potential role of HOXB7 in MM cells either we enforced HOXB7 expression by lentivirus vectors in JJN3 HMCL (JJN3-HOXB7) or we silenced its expression by appropriate siRNA in the KMS20 HMCL that constitutively over-expressed HOXB7. Interestingly we found that cell proliferation was significant higher in JJN3-HOXB7 as compared to JJN3 transduced with the empty vector (JJN3-pWPI) as well as HOXB7 downregulation in KMS20 significantly reduced their cell proliferation and viability. Following we evaluated the whole transcriptional and specifically the pro-angiogenic profile of JJN3-HOXB7 as compared to JJN3-pWPI using Genechips® HG-U133Plus 2.0 and an angiogenesis oligonucleotide arrays. We found that the pro-angiogenic cytokines as VEGFA, FGF2, MMP2 and PDGFA were significantly upregulated whereas we identified the specific downregulation of the angiogenic inhibitor TSP2 in JJN3-HOXB7. Data were then validated at protein level by ELISA assay in cell-conditioned media (CM). Consistently, we found that CM of JJN3-HOXB7 significantly stimulated vessel formation as compared to JJN3-pWPI using both in vitro angiogenic model (Angiokit) and chorioallantoic membrane assay. In line with these observations, we found that HOXB7 silencing by siRNA in KMS20 reduced the expression of pro-angiogenic molecules, including VEGF and FGF2, and growth factors. Finally the potential role of HOXB7 in MM-induced angiogenesis was tested in vivo in NOD/SCID mice. We found that mice injected with JJN3-HOXB7 cells developed a significantly bigger tumor mass than mice inoculated with the JJN3-pWPI (p=0.0039) with higher number of vessels stained by CD31 antigen. Moreover, the transcriptional and angiogenic profiles of the tumor mass indicated the upregulation of VEGFA, FGF2, MMP2, PDGFA and WNT5a as well as the downregulation of TSP2, at both mRNA and protein level in mice colonized by JJN3-HOXB7 as compared to those inoculated with JJN3-pWPI, confirming in vitro observations.
In conclusion our data demonstrate the critical role of HOXB7 in the production of pro-angiogenic molecules by MM cells and in the regulation of MM-induced angiogenic switch suggesting that HOXB7 could be a potential therapeutic target in MM.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.