UOK257 is the only renal cancer cell line available to date that has been established from a BHD patient's tumor tissue. This cell line is particularly valuable for study of the biological role of FLCN
inactivation in tumorigenesis because it harbors a FLCN
mutation predicted to produce only truncated mutant protein and induces the growth of tumors in vivo
with histology resembling the BHD-associated renal tumor from which it was derived [23
]. In this study, we have established and characterized UOK257 cell lines in which wild-type or mutant FLCN
was stably expressed. Although anchorage dependent cell growth in vitro
was not affected by wild-type FLCN
expression, cell growth in vivo
and anchorage-independent growth in soft agar were severely diminished by the expression of wild-type FLCN
. We have searched for downstream target genes regulated by FLCN
through gene expression microarray analysis and identified a number of genes that were differentially expressed in wild-type FLCN
(UOK257-2, -4, and -6) compared with mutant FLCN
-null (UOK257-H255R and -P) cells. We found three prominent groups of genes involved in cadherin signaling, TGF-β signaling, and angiogenesis. Notably, several key genes involved in TGF-β signaling, such as TGFB2
, were down-regulated in FLCN
-null and mutant FLCN
cells as well as in the BHD-associated renal tumors. Consistently, GREM1
, the antagonist of BMP that signals through SMADs was highly up-regulated in mutant FLCN
-null UOK257 cells although its expression was low in BHD-associated renal tumors.
We observed that the expression level of FLCN is important for tumor suppression, since the UOK257 cell lines (UOK257-4, -2 and -6) expressing high levels of FLCN did not develop tumors whereas the UOK257-3 cell line expressing a very low level of FLCN, did develop tumors with a low incidence (2 out of 10). It is likely that the FLCN expression level in UOK257-3 cells is marginal for tumor suppression, allowing tumor growth in some animals but suppressing tumor growth in others. In support of this idea, the expression levels of the downstream target genes in UOK257-3 cells were either similar to FLCN-null
mutant cells (UOK257-P and UOK257-H255R), or midway between the FLCN-null
mutant group and the FLCN
-restored group, which expressed high levels of FLCN (UOK257-2 and UOK257-6) (See additional file 1
: Fig. S3).
UOK257-H255R cells expressed a low level of FLCN protein resulting in loss of tumor suppressor function and deregulation of TGF-β signaling, even though they expressed slightly more FLCN mRNA than UOK257-4 cells (Fig. and ). These data suggest that FLCN-H255R missense mutant protein found in the canine model of BHD syndrome is less stable than wild-type FLCN. Thus decreased stability of mutant FLCN is likely to contribute to the loss of FLCN tumor suppressor function.
It has been suggested that Drosophila BHD
) regulates germline stem cell (GSC) maintenance downstream or in parallel with Jak/Stat and dpp (BMP ortholog in Drosophila
) signaling [24
knockdown by siRNA suppressed overproliferation of GSC induced by hyperactivation of Jak/Stat or dpp signaling. Interestingly, Jak1
, encoding a kinase that transmits signals by phosphorylating Stats in cells, was identified by microarray analysis as a downregulated gene in the mutant FLCN
-null cells (Fig. ). We also identified several key genes in TGF-β/BMP signaling such as TGFB2
(a regulatory SMAD) that were down-regulated in the mutant FLCN
cells. On the other hand, GREM1
, which encodes a protein that binds and inactivates BMP activity, was upregulated in the mutant and FLCN-null
cells. Thus the genetic interactions between dBHD
, and Jak/Stat and dpp (BMP) signaling may be partially explained by FLCN
deregulation of genes involved in these pathways.
The human TGF-β superfamily consists of 42 members including TGF-βs, activins, bone morphogenic proteins (BMPs), and growth and differentiating factors (GDFs) [25
]. TGF-βs are multi-functional cytokines that modulate cell proliferation, apoptosis, differentiation, adhesion and migration. TGF-β shows a biphasic effect on tumor cell growth [27
]. It inhibits tumor cell growth in the early phase of tumorigenesis but promotes cell growth when cells escape the anti-proliferative effect of TGF-β in the late phase of tumorigenesis. Interestingly, TGF-β2 induced anchorage independent growth of UOK257 cells (Fig. ), suggesting that UOK257 cells are refractory to the growth suppressive effect of TGF-β. The possibility exists that reduced expression of TGF-β2 in FLCN
-null cells contributed to cell growth in the early phase of tumorigenesis.
Disruption of TGF-β signaling has been reported in many cancers. TGF-β type II receptor is often mutated in gastro-intestinal cancers [28
]. Mutations in SMAD2 or SMAD4 occur frequently in pancreatic and colorectal carcinomas [31
]. Although mutations in SMAD3 have not been reported, 3 out of 8 (37.5%) gastric tumors in one report showed low to undetectable levels of SMAD3 expression and restoration of SMAD3 suppressed tumorigenicity of gastric cancer cells [34
]. Low levels of SMAD3 expression in the BHD tumors may contribute to the ability of these renal tumor cells to escape the growth suppressive effect of TGF-β.
Activins are homo- or heterodimeric proteins consisting of two β subunits (βA and βB), while inhibins are heterodimers of α and β subunits (inhibin-A [αβA] and inhibin-B [αβB]) [35
]. INHBA is one of the β subunits (βA) that comprise activin A (βAβA), activin AB (βAβB) and inhibin A (αβA). Activin A regulates kidney organogenesis, tubular regeneration and renal fibrosis [reviewed in [36
]]. Activins also induce apoptosis, and inhibit cell proliferation and tumor growth in numerous types of cells. In contrast to TGF-β2, activin A inhibited growth of UOK257 cells in soft-agar (Fig. ), suggesting that activin signaling is intact in UOK257 cells. Thus reduced expression of INHBA
, β subunit of activin A, in UOK257 cells and BHD tumors, may be permissive for tumor cell growth. It would be interesting to examine whether activin A treatment can suppress BHD tumor growth in vivo
Thrombospondin-1 (THBS1) is one of the five members of a family of thrombospondins that mediate the interaction of normal and cancer cells with the extracellular matrix and surrounding tissue. THBS1 suppresses tumor growth by activating TGF-β and by inhibiting angiogenesis. THBS1 exerts direct effects on endothelial cell migration and survival through interaction with CD36. It also reduces availability of VEGF by inhibiting MMP9, therefore releasing VEGF from the extracellular matrix. There are several reports suggesting that reduced expression of THBS1 or hypermethylation of THBS1
is associated with poor prognosis of cancer patients and higher tumor grade [37
]. Accordingly THBS1 regulation may be an important part of the tumor suppressor function of FLCN
We examined whether TGF-β signaling is dysregulated by the inactivation of the FLCN gene. TGF-β or BMP4 induced SMAD3 or SMAD1/5/8 phosphorylation was not affected by FLCN inactivation suggesting receptor mediated SMAD phosphorylation is not altered by FLCN. However, several genes whose expressions are regulated by TGF-β were dysregulated by the inactivation of FLCN. The basal and maximal induced levels of the downstream target genes (TGFB2, INHBA and SMAD7) regulated by TGF-β were reduced in cells with FLCN inactivation. These data suggest that FLCN may regulate TGF-β signaling through a non-SMAD mediated mechanism. As a result of such regulation, the level of TGF-β ligands, such as TGF-β2 and activin A, could be highly induced in cells expressing FLCN by a positive feedback control.
A possible function of FLCN in energy sensing and metabolism has been suggested by its interaction with AMPK through FNIP1/2 and by the observation that FLCN phosphorylation is affected by mTOR signaling (20-21). Here we demonstrated that an AMPK activator, AICAR, and an AMPK inhibitor, Compound C, as well as an mTOR inhibitor, rapamycin, affected the expression of the same key molecules involved in TGF-β signaling, which appear to be regulated by FLCN. Thus FLCN could be a key molecule connecting energy-sensing signals to growth suppressive TGF-β signaling.