Cells developing resistance to metronomic CPA therapy undergo a complex molecular evolution. Changes in many different factors and pathways contribute to the formation of a resistant phenotype. As shown earlier, most of the classic resistance mechanisms can be ruled out in this study, as neither increased multi-drug resistance (MDR) transporter activity nor neoangiogenesis, vascular mimicry, or impaired CPA activation was observed [6
]. A pharmacodynamic study of metronomically administered CPA suggests that it is unlikely that resistance to this kind of regimen is caused by impaired CPA activation [12
One of the four different categories of resistance to anti-angiogenic therapy is “reduced vascular dependence.” As shown previously, this resistance mechanism applies here, as the resistant tumor grows under conditions of hypoxia and restricted nutrients without the formation of additional tumor vessels [6
]. To investigate the molecular basis of this in vivo
resistance mechanism to anti-angiogenic CPA therapy, a genome-wide microarray was performed. This study clearly shows that it is important to use appropriate in vivo
controls, as already the comparison of the gene expression profile of in vivo
passaged and standard PC3 tumors (PC3-wt) displayed a dramatic difference. The gene expression profile of the standard PC3-wt samples was clearly distinct from all other in vivo
passaged tumor sublines ().
Moreover, gene expression of resistant tumors PC3 D3 and PC3 D4 clearly differed from passaged non-resistant PC3 A3 tumors. An acute CPA treatment of the respective tumors for 24 hours only influenced gene expression marginally (). All PC3 D3 samples from four mice clustered in one group, distinct from the PC3 D4 group. Approximately 50% of the differentially expressed genes in PC3 D3 and PC3 D4 versus
PC3 A3 were shared by both resistant sublines (). To rule out clonal variation instead of chemoresistance as cause of the observed differences, we performed a control experiment. Here, we compared the initial experiment of PC3 D3 and PC3 D4 versus
PC3 A3 to the control experiments PC3 A3 and PC3 D3 versus
PC3 D4 as well as PC3 A3 and PC3 D4 versus
PC3 D3 (see Figure W4
). We could identify less than 2.7% of the genes to be commonly regulated. Therefore, we regard the signature of resistant genes as considerable.
Taken together, we assume that not a single main mechanism is present in the resistant cell lines but more likely a complex molecular evolution, leading to the ability to survive the restrictive conditions of anti-angiogenic therapy.
In our model, expression of resistance-related genes in vivo
differs from gene expression in vitro
, indicating an involvement of micro-environmental factors leading to the observed in vivo
resistance. The following three pathways that potentially contribute to the resistant phenotype were identified: 1) axon guidance, 2) steroid biosynthesis, and 3) complement and coagulation cascades (). As prostate cancer cells are able to gain neuronal properties [13,14
], neuroendocrine-like differentiation might take place as a side effect of resistance formation. Steroid synthesis might be altered leading to hormone-promoted increased proliferation.
The most interesting functional group is “complement and coagulation cascades.” This group consists of six differentially expressed genes that are associated with the coagulation cascade. During antiangiogenic treatment, blood vessels are destroyed and coagulation takes place. In mice, the administration of flavone acetic acid, an anti-angiogenic drug, strongly reduces the coagulation time [15
]. We speculate that resistant tumor cells hamper the coagulation process to overcome the reduced oxygen and nutrient supply: 1) PLAT (reviewed in [16
]) and 2) ANXA3 [17
] showed increased expression in resistant tumors, whereas expression of pro-coagulation gene PROS1
(reviewed in [18
]) and three members of the serine protease inhibitor family, 1) SERPINA1
, 2) SERPINB7
, and 3) SERPIND1
, were decreased in resistant tumor tissue. Increased diffusion of oxygen and nutrients is facilitated by impaired thrombosis and fibrosis. Additionally, metronomic dosing of an inhibitor of systemic vasculogenesis was reported to cause increased blood flow in luciferase-tagged LM2-4 tumor xenografts [19
In contrast to our finding that the expression of anti-coagulation genes is increased and that of pro-coagulation genes is decreased, F3, the main mediator of the extrinsic pathway of blood coagulation [20,21
], was highly expressed in PC3 D4 tumor tissue. Interestingly, F3
shows an altered exon expression profile in PC3 D4 tumor samples. Comparing expression of PC3 D4 to PC3 A3, exons 3 to 6 are highly expressed in resistant PC3 D4 tumor tissue, whereas no differential expression of exons 1 and 2 can be seen. A version of F3
, missing exons 1 and 2, has not yet been described, whereas an alternatively spliced variant of F3, missing exon 5, has been reported [22
]. Exons 1 and 2 of F3
are coding for the first 70 amino acids of the protein. In this area, according to UniProt database [23
], a signal peptide and parts of a topological domain are located. Thus, we speculate that the expression of an altered F3 protein might result in an impaired activation of blood coagulation by resistant tumor tissue or even in facilitating blood flow. Such a hypothesis, however, remains to be verified by future studies.
In conclusion, resistance formation to metronomic CPA therapy can be seen as a molecular evolutionary process. Anti-coagulation properties of the cells (increased PLAT and ANXA3, increased exon deletion variant of F3, and decreased PROS1, SERPIND1, SERPINA1, and SERPINB7) could be part of a complex resistance mechanism. The functional relevance remains to be confirmed by subsequent studies.