The molecular mechanisms underlying the anti-proliferative effects of 1,25D are poorly understood, despite the well known role of 1,25D in the regulation of calcium homeostasis. In this study we show that the anti-proliferative effects of 1,25D may be functional in MCF7 breast tumor cell lines, without a role for the central player in calcium homeostasis, VDR. These results were unanticipated since several known regulators of cell growth, such as p21
, contain functional VDREs in their promoter region and are induced by the 1,25D-VDR complex in cancer cells [35
]. Furthermore, cell lines derived from VDR knockout (KO) mice are resistant to 1,25D mediated growth arrest [37
], and re-introduction of VDR into these cells restores sensitivity to 1,25D mediated growth inhibition (Keith and Welsh, unpublished). While additional studies are needed to determine whether residual VDR activity in the MCF7 cells expressing VDR-siRNA is sufficient to mediate growth inhibitory signals upon binding 1,25D, our data demonstrates that marked reductions (~90% at the transcript level and ~85% at the protein level) in VDR do not necessarily induce 1,25D resistance. Seemingly contradictive to the role of VDR, this finding is consistent with previous data from MCF7 sub-lines selected in vitro
for resistance to 1,25D, in which VDR function is retained [7
], indicating dissociation between VDR expression and 1,25D mediated growth inhibition.
In the light of these results, we hypothesized that driver/causative genes other than VDR, or alternative pathways unrelated to vitamin D calcium signaling might impact 1,25D sensitivity or replace its function. Therefore we used genome wide screens to identify genetic alterations that cause resistance in our model. Indeed, in all four 1,25D resistant cell lines one common chromosomal region had reduced copy number at 11q13.4-14.1 (Figure ). Eighty genes are present in this 13.5 Mb chromosomal region, none of which could readily be assigned to 1,25D mediated VDR signaling. In focally aberrant regions (<3 Mb) identification of driver genes is often straight forward, but it is our experience [38
], acknowledged by others [39
] that identification of candidate genes in larger chromosomal regions is problematic. One effort made was integration with our expression array data which did not proof helpful; hence pathway analysis of genes in this region would be arbitrary. Other authors performed similar array experiments in the presence of 1,25D, yet again none of the 80 genes in this region was identified [9
]. Our expression array analysis confirmed that 1,25D resistant cells did not exhibit significant reductions in VDR gene expression. Still, as described previously the VDR-transcriptome may be significantly altered [12
]. Indeed, these authors showed that CYP24A1
was the only induced gene that was common to the genetic profiles of the parental and the 1,25D resistant cell lines when treated with 1,25D treated [12
]. In contrast, our expression array analysis identified CYP24A1
as one of the top five down-regulated genes in the 1,25D resistant MCF7 cell lines. Since CYP24A1
is strongly regulated by 1,25D in a negative feed-back, but no 1,25D was present in our test, any discussion on this apparent down-regulation remains speculative.
Pathway analysis of the expression arrays did not identify any other known vitamin D pathway genes either. Instead, over-representation of multiple membrane receptor signaling pathways known to be involved in the control of proliferation and apoptosis were associated with the 1,25D resistant phenotype. The five membrane-bound receptor signaling pathways identified in our analysis share the Grb2 and SOS genes and drive cell proliferation. Interestingly, previous analysis of one set of these sub-lines indicated that, in the absence of 1,25D, the doubling time of the MCF7 D3res cells was shorter than that of the MCF7 sensitive cells from which they were derived [8
]. Furthermore, using proteomic approaches we previously identified several proteins in the MAPK-ERK-RAS mitogenic pathway that were differentially expressed in the MCF7 D3res cells relative to their sensitive MCF7 counterparts under basal conditions [9
]. Although mechanisms have yet to be clarified, chronic up-regulation of these mitogenic pathways may selectively disable VDR signaling, leading to 1,25D resistance. In support of this idea, RAS activation has been shown to induce 1,25D resistance in keratinocytes via direct phosphorylation of VDR [40
In a previous study we used the same genome wide tools to identify the mechanisms of Gemcitabine resistance [32
]. Results were simple and straightforward to interpret; Gemcitabine had selected for tumor cells with high level amplifications, leading to high expression levels of genes within that amplicon which resulted in drug resistance [32
]. Here a similar simple mechanism for 1,25D resistance was not identified. A possible explanation may be that most therapeutic drugs (e.g. Gemcitabine) are synthetic and 1,25D is generated endogenously. Consequently, malignant cells would need to develop resistance to the anti-proliferative effects of 1,25D in a very early stage of their development to allow tumor expansion. Accordingly, we hypothesized that many solid tumors would also contain alterations at 11q13.4-14.1. Indeed, screening of four datasets covering 358 individual array CGH profiles showed that the region in chromosome 11q13.4-14.1 is highly unstable strengthening this hypothesis.