To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 TSP1, we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium human no
Trang 1thrombospondin-1 secretion and angiogenesis
in renal cell carcinoma
Dorina Veliceasa1, Marina Ivanovic2, Frank Thilo-Schulze Hoepfner1, Praveen Thumbikat1,
Olga V Volpert1and Norm D Smith1
1 Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
2 Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
Keywords
angiogenesis; calcium metabolism; renal
cancer; thrombospondin
Correspondence
O V Volpert, Department of Urology,
Northwestern University, 303 East Chicago
Ave., Chicago, IL 60611, USA
Fax: +1 (312) 908 7275
Tel: +1 (312) 503 5934
E-mail: olgavolp@northwestern.edu
(Received 4 June 2007, revised 14
Septem-ber 2007, accepted 18 OctoSeptem-ber 2007)
doi:10.1111/j.1742-4658.2007.06159.x
Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel–Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor In HNK cells, 1,2-bis(o-aminophen-oxy)ethane-N,N,N¢,N¢-tetraacetic acid acetoxymethyl ester, a calcium chela-tor, simulated TSP1 retention, mimicking the RCC phenotype Further analysis revealed a profound decrease in transient receptor potential canon-ical ion channel 4 (TRPC4) Ca2+channel expression in RCC cells TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion Dou-ble labeling of the secretory system components revealed TSP1 colocaliza-tion with coatomer protein II (COPII) anterograde vesicles in HNK cells
In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfold-ing Our study indicates that TRPC4 loss in RCC leads to impaired Ca2+ intake, misfolding, retrograde transport and diminished secretion of anti-angiogenic TSP1, thus enabling anti-angiogenic switch during RCC progression
Abbreviations
BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid acetoxymethyl ester; bFGF, basic fibroblast growth factor; CEP, circulating endothelial precursor; CM, conditioned media; COP, coatomer protein; CXCR2, CXC chemokine receptor 2; EGF, epidermal growth factor; ER, endoplasmic reticulum; ERGIC, ER–Golgi intermediate compartment; FITC, fluorescein isothiocyanate; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; HIF, hypoxia inducible factor; HMVEC, human microvascular endothelial cell; HNK, human normal kidney, normal renal epithelial strain; HRP, horseradish peroxidase; HSP, heat shock protein; IL, interleukin; PDGFR, platelet-derived growth factor receptor; PEDF, pigment epithelial-derived factor; PTEN, phosphatase and tensin analog; RCC, renal cell carcinoma; TIMP, tissue inhibitor of metalloproteinase; TRPC4, transient receptor potential canonical ion channel 4; TSP1, thrombospondin-1; VEGF, vascular endothelial growth factor; VHL, von Hippel–Lindau.
Trang 2The prevailing treatments for kidney cancer are
sur-gery and immunotherapy Until 2005, only high-dose
interleukin-2 (IL-2) had been approved by the US
Food and Drug Administration (FDA) [1] As
immu-notherapy has unfavorable side-effects, new targeted
therapies to counter the molecular triggers of renal cell
carcinoma (RCC) are in high demand
Clear cell RCC is largely caused by inactivation of
the von Hippel–Lindau (VHL) tumor suppressor [2]
The main target of the VHL tumor suppressor is
hypoxia inducible factor-1a (HIF1a), an
oxygen-sens-ing transcription factor, which undergoes regulatory
hydroxylation at normal Po2[3] The VHL tumor
suppressor binds hydroxylated HIF1a, targets it for
proteasome degradation and thus suppresses HIF
pro-angiogenic targets, vascular endothelial growth factor
(VEGF) and erythropoietin, and pro-survival targets,
enabling stress-induced apoptosis [4] Novel RCC
therapies target VEGF (Avastin) [5] or its receptor
(sunitinib, sorafenib) [6] The latter also target
VEGF-producing tumor stroma by inactivating another
tyro-sine kinase, platelet-derived growth factor receptor-b
(PDGFRb) [1] However, VEGF induction by HIF1a
alone is insufficient to promote the growth of RCC
xenografts [7]
The exclusive role of VEGF in RCC
progres-sion⁄ angiogenesis has been challenged by the studies
of other angiogenic stimuli, including ELR+ CXC
chemokines, such as IL-8, and CXC chemokine
recep-tor 2 (CXCR2) ligands [8,9] or IL-2 via CXCR3 [10]
or basic fibroblast growth factor (bFGF) and
epider-mal growth factor (EGF) [11–14]
In contrast, antiangiogenic proteins in RCC
progres-sion and angiogenesis have been largely ignored A few
studies have implicated pigment epithelial-derived
fac-tor (PEDF) in Wilms’ tumor [15]; however, there are
no data that link PEDF and RCC Other studies have
demonstrated that the more aggressive Wilms’ tumors
are characterized by low levels of antiangiogenic
thrombospondin-1 (TSP1) [16] TSP1 is also secreted
by glomerular mesangial cells [17] In another study,
small, mildly angiogenic tumors were found to produce
more TSP1 than more aggressive counterparts [18]
We therefore hypothesize that TSP1 supports normal
kidney angiostasis, and that its loss contributes to the
RCC angiogenic phenotype
TSP1 is a multifunctional extracellular matrix
pro-tein, and a potent and versatile angiogenesis inhibitor
that is critical for the maintenance of the
antiangiogen-ic mantiangiogen-icroenvironment in multiple organ sites, including
breast, brain, colon and skin [19] Conversely,
re-intro-duction of TSP1 or its active peptides blocks
angiogen-esis in a variety of experimental tumors and metastases
[20] The tumor suppressor genes p53, phosphatase and tensin analog (PTEN) and SMAD4 maintain nor-mal, high levels of TSP1 expression (reviewed in [21]) Conversely, the oncogenes Id-1, Jun, Myc, Ras and Src repress TSP1 production and thus flip the angio-genic switch on and enable tumor growth [21] TSP1 inhibits multiple endothelial cell functions, such as migration, proliferation and lumen formation [20] In addition, TSP1 causes endothelial cell apoptosis and thus compromises the integrity of the tumor vascula-ture [22] Finally, TSP1 regulates the numbers of circu-lating endothelial precursor (CEP) cells, and thereby impinges on VEGF-mediated CEP cell recruitment to the sites of neovascularization [23] A knowledge of the molecular mechanisms that cause TSP1 loss in the tumor microenvironment is instrumental to determine
a subset of tumors that would benefit from TSP1-based therapies and to aid in the development of novel targeted therapies to control them
In this article, we show that disrupted TSP1 secre-tion renders RCC cells pro-angiogenic Seeking under-lying mechanisms, we found that RCC cells fail to mount calcium uptake in response to growth factors, probably as a result of the low expression levels of the two calcium exchange proteins, calbindin and transient receptor potential canonical ion channel 4 (TRPC4) Calcium deficiency is critical for the correct folding and secretion of TSP1: the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N¢,N¢-tetraacetic acid acet-oxymethyl ester (BAPTA-AM) caused retrograde transport and retention of TSP1 by otherwise normal renal epithelium (human normal kidney, normal renal epithelial strain, HNK) TSP1 misfolding caused by calcium deficiency led to its retrograde transport, intra-cellular retention and diminished secretion Thus, the loss of TSP secretion as a result of epigenetic changes may deplete antiangiogenic TSP1 in the tumor envi-ronment and cause conditions permissive for angio-genesis
Results
TSP1 suppresses angiogenesis in normal kidney epithelium
Seeking a role for TSP1 in the evolution of the angio-genic response in RCC, we stained 11 human RCC specimens and six specimens of adjacent normal tissue for TSP1 Based on the assumption that TSP1 main-tains angiostasis in the kidney, we expected TSP1 to
be lower in RCC tissues Surprisingly, RCC and adja-cent normal tissue showed similar staining intensities (Fig 1A; Table 1)
Trang 3D
E
F
Fig 1 Role of TSP1 in angiogenesis, and
localization in renal cells and tissue (A)
Sec-tions of RCC tumors and adjacent normal
tissue (HNK) were stained for TSP1 and
counterstained with hematoxylin (B, C) CM
from HNK and P769 RCC cells were tested
in the mouse corneal assay TSP1 in HNK
cells was silenced using siRNA; the
silenc-ing was verified by RT-PCR and western
blot of CM (B) VEGF was neutralized with
antibodies (C) Representative corneas are
shown There was a lack of angiogenic
response to HNK CM and a robust response
to RCC CM In addition, TSP1 neutralization
restored the angiogenic activity of HNK CM;
VEGF neutralizing antibodies reversed this
effect and abolished the angiogenic activity
of RCC CM (D) RNA isolated from the
indi-cated cell lines was subjected to
semiquan-titative RT-PCR with TSP1 primers HNK,
normal cell strain; P769, PRC9, SW839,
ARZ-1 and WT8, RCC cell lines (E) The
same cell lines were subjected to 24 h
serum deprivation, CM and cell lysates (CM
and L, respectively) were collected and
TSP1 was detected by western blotting.
Note the low TSP1 secretion and higher
intracellular levels in RCC cells (F) HNK and
P769 cells were cultured for 24 h in full
serum or serum-free medium, fixed and
stained for TSP1 Note the depletion of
TSP1 in the cytoplasm of normal cells.
Trang 4In contrast, HNK and RCC cell lines differed in
their ability to induce angiogenesis Conditioned media
(CM) from P769 and other RCC cell lines were
potently angiogenic in rat and mouse corneal assay for
angiogenesis CM from normal HNK cells was
non-angiogenic; however, it became angiogenic if TSP1
was either neutralized with antibodies or silenced using
siRNA (Fig 1B,C; Table 2) 786-O-WT8 cells were
weakly angiogenic in vivo and in vitro as a result of the
low levels of secreted VEGF; angiogenesis was only
marginally altered by TSP1 depletion (Table 2)
Both HNK and RCC cells produced VEGF
(mea-sured by ELISA); RCC cells produced three- to
four-fold more VEGF than HNK or 698-O-WT8 cells
(Table 3) In contrast, quantitative western blots
[ 2.6 lgÆ(100 lg total protein))1], whereas RCC cells secreted less than 0.12 lgÆ(100 lg protein))1, regardless
of VHL status (Table 3; Fig 1E) Using human micro-vascular endothelial cell (HMVEC) chemotaxis as an
in vitro measure of angiogenesis, we determined the specific activity (ED50) of each CM alone and with VEGF or TSP1 neutralized (Table 3) CM from 786-O-ARZ, PRC9, SW839 and p769 showed high specific activity, reflective of the VEGF levels In contrast, the HNK CM was nonangiogenic: TSP1 depletion with neutralizing antibodies revealed underlying angiogenic activity in HNK cells, which, in turn, was blocked by VEGF antibodies (Table 3)
RCC, but not normal kidney epithelium, retains TSP1
Despite the difference in secreted TSP1, TSP1 mRNA levels were similar in HNK and RCC cells (Fig 1D) RCC and HNK cells also produced roughly equal total TSP1 protein [43 ± 5.3 and 42 ± 7.1 ngÆ(10 lg protein))1, respectively, P¼ 0.48], as calculated using data from Fig 1E
However, RCC cells secreted noticeably less TSP1 than did HNK cells (Fig 1E) In contrast, lysates
of serum-starved HNK cells contained no detectable TSP1, whereas TSP1 was found at high levels in the cytoplasm of all RCC lines (Fig 1E) Immunocyto-chemistry of fixed cells showed robust cytoplasmic staining for TSP1 in both HNK and RCC cells cultured
Table 1 TSP1 and VEGF immunostaining of kidney cancer and
normal tissue Human tumor samples were stained for TSP1 and
VEGF, respectively The slides were scored by two independent
pathologists (double-blind study).
Tissue or
tumor type
Case (n)
Staining intensity Case (n)
Staining intensity
1
++
+
Table 2 Corneal angiogenesis by conditioned media (CM) Media conditioned by the indicated cell lines were tested in rat a or mouse b cor-neal neovascularization assay (see Experimental procedures) The results are expressed as positive corneas of the total implanted To evalu-ate the statistical significance of the changes in angiogenic activity as a result of inactivation of TSP1 and ⁄ or VEGF, the results were expressed as the percentage of positive responses, grouped and subjected to Student’s t-test TSP1 inactivation in the HNK CM (antibody
or siRNA silencing) significantly increased its angiogenic activity (P ¼ 0.023); further addition of VEGF inactivating antibodies returned the angiogenic activity to levels that were not significantly different from those of the initial HNK CM (P ¼ 0.085); angiogenesis by CM from all the tumor cell lines was significantly different from that of the HNK cells and WT8 revertant (P ¼ 0.0026) VEGF neutralizing antibody decreased angiogenesis by P769 to a value that was not significantly different from that of unaltered HNK CM (P ¼ 0.13) and was signifi-cantly lower than the activity of unaltered tumor CMs (P ¼ 0.0002).
Antibody
Positive responses per total implants for CM
5 ⁄ 6 b
(83.3%)
6 ⁄ 8 a (75%) 7 ⁄ 8 a (87.5%) 2 ⁄ 7 a (28.5%)
1 ⁄ 8 b
(12.5%)
2 ⁄ 8 a (25%) 1 ⁄ 8 a (12.5%) 0 ⁄ 5 a (0%) TSP1 Ab + VEGF Ab 2 ⁄ 9 b (2.2%)
Scrambled siRNA 1 ⁄ 9 b (11%)
(80%) TSP1 siRNA + VEGF Ab 4 ⁄ 10 b (40%)
a Tested in rat b Tested in mouse.
Trang 5in full serum After 12–24 h without serum, TSP1 was
depleted from the HNK cytoplasm as a result of
secre-tion, but retained by P769 RCC (Fig 1F) VHL tumor
suppressor had no effect on secreted TSP1: TSP1
secre-tion was comparable in 786-O-WT8, 786-O-ARZ and
other RCC lines (Fig 1C,D)
RCC cells show decreased calcium uptake
Improper folding may cause protein retention
Impor-tantly, calcium binding strongly affects TSP1 folding
[24] In the case of pseudoachondroplasia, TSP5
muta-tions in the calcium-binding cassette alter its ability to
transit endoplasmic reticulum (ER) and to undergo
secretion [25,26] We hypothesized that different TSP1
secretion may result from different calcium availability
in HNK and RCC cells We measured calcium uptake
by the cells stimulated by VEGF: 10 ngÆmL)1 VEGF
caused no measurable intake of Ca2+in RCC cells,
whereas HNK cells developed a robust response
(Fig 2A,B) Moreover, RCC cells responded poorly to
Ionomycin, a potent Ca2+ ionophore, relative to
HNK (Fig 2B) In addition, treatment of HNK with
BAPTA-AM, a cell-permeating calcium chelator,
caused a significant increase in cytoplasmic TSP1 and
a concomitant decrease in secreted TSP1 (measured by
western blot and immunostaining; Fig 2C,D) TSP1
appeared unique in this respect: 10 mm BAPTA-AM
had no effect on the intracellular content and secretion
of VEGF, but induced TSP1 retention and diminished
secretion (Fig 2E)
RCC expresses low TRPC4 and calbindin
Seeking reasons for the altered calcium metabolism,
we examined TRPCs, which mediate
agonist-stimu-lated Ca2+ influx [27] Semiquantitative and real-time
RT-PCR showed significant expression of TRPC1,
(Fig 3A,B) In RCC cells, TRPC4 expression was decreased four-fold (Fig 3A,B) TRPC4 expression and function are established in the vasculature, but not in the kidney Importantly, TRPC5, the TRPC4 analog, was not expressed in HNK or RCC cells (Fig 3A,B); thus, there was no functional redundancy HNK cells expressed high levels of the calcium-bind-ing protein, calbindin D28K [28] (Fig 3C) In normal kidney, calbindins transport calcium ions across the glomerular epithelium and serve as buffers, to prevent toxic concentrations of intracellular calcium [29] Con-sistent with published data, RCC cells expressed no calbindin D28K, probably because of their poorly differentiated state (Fig 3C)
Functional TRPC4 was indeed critical for TSP1 secretion: TRPC4 siRNA transfection of HNK cells caused an increase in cytoplasmic and a decrease in secreted TSP1 (Fig 3D)
RCC cells retain TSP1 in the ER One possible consequence of misfolding is protein
‘recall’ to the ER from the ER–Golgi intermediate compartment (ERGIC), a site for concentrating retro-grade cargo [30] Anteroretro-grade transport vesicles con-tain coatomer protein II (COPII), whereas retrograde vesicles contain COPI [31,32] In RCC cells and HNK cells treated with BAPTA-AM, TSP1 colocalized with
ER markers, but not with Golgi, suggesting retrograde transport (Fig 4A–E) When HNK and p769 cells were subjected to 4 h of serum deprivation to prompt secre-tion, fixed and stained for TSP1 and Sec23 (COPII component) or c2-Cop (COPI marker), TSP1 colocal-ized with Sec23⁄ COPII in HNK cells; colocalization with c2-Cop⁄ COPI was minimal in HNK cells,
Table 3 Angiogenic characteristics of the conditioned media (CM) CM from the indicated cell lines were collected and subjected to the fol-lowing analyses: (a) VEGF levels were measured by ELISA; (b) TSP1 levels were measured by densitometry analysis of western blots; (c)
ED50was measured in the endothelial cell chemotaxis assay; ED50of RCC CM was also measured in the presence of VEGF neutralizing antibody (1 lgÆmL)1) and TSP1 neutralizing antibody (2.5 lgÆmL)1) where shown; (d) corneal angiogenesis was tested in rat assay (see Experimental procedures) and scored Pellets contained 1.25 or 2.5 lg of total protein The antibodies were added at 2 and 5 lg per pellet where indicated N ⁄ A, not assessed.
CM
Secreted
VEGF (pgÆmg)1)
Secreted TSP (lgÆmg)1)
Trang 6D
Fig 3 Calcium channels and calbindin in HNK and P769 cells (A, B) mRNA levels for TRPC1–7 were evaluated in HNK and P769 cells by semiquantitative RT-PCR (A) or Q-PCR (B), using GAPDH message as control (C) Western blot for calbindin D28K (D) HNK cells were trans-fected with TRPC4 siRNA or scrambled control siRNA and cultured for 12 h in full medium After an additional 48 h in serum-free medium, RNA and CM were collected The silencing was ascertained by semiquantitative RT-PCR (approximately 45% decrease in the message level) Lysates (L) and CM were analyzed by western blotting for TSP1 content Note the cytoplasmic retention and decreased TSP1 secre-tion in HNK-siTRPC4.
C
D
E
Fig 2 Calcium uptake and mediators in HNK and RCC cells (A) Ca2+uptake in response to VEGF by HNK and RCC cells HNK and RCC cells were preloaded with fluo-4 acetoxymethyl ester and treated with 10 ngÆmL)1VEGF Ca 2+ uptake was measured at 10 s intervals by videofluorescence imaging (B) Representative images of fluo-4 acetoxymethyl ester-loaded cells prior to and after VEGF exposure (C, D) Changes in TSP1 secretion ⁄ retention in response to the calcium chelator BAPTA-AM HNK cells were cultured for 12 h in serum-free med-ium with the indicated BAPTA-AM concentrations (C) The TSP1 content per milligram of protein was calculated using comparison with serial TSP1 dilutions (standard curve) on western blot (D) Representative blots of cell lysates (L, top, 20 lg per lane) and CM (CM, bottom, 5 lg per lane) were collected in parallel experiments (E) HNK and P769 cells were cultured for 12 h with or without BAPTA-AM (1 n M ) CM and lysates were collected as above and analyzed by western blotting Note the retention of TSP1 in the cytoplasm and decreased secretion by the BAPTA-AM-treated HNK cells Also note the higher VEGF levels in the cytoplasm and CM of P769 cells, and the lack of response to BAPTA-AM C, purified TSP1 or VEGF, respectively.
Trang 7indicating anterograde transport and secretion (Fig 5).
By contrast, in p769 cells, TSP1 colocalized with
c2-Cop⁄ COPI, suggesting retrograde transport (Fig 5)
Discussion
Normal adult vasculature is quiescent as a result of the
balanced expression of pro- and antiangiogenic factors
[33,34] Multiple inducers of angiogenesis (VEGF,
bFGF, IL-8, stromal cell-derived factor-1, etc.), when
expressed at high levels, expand tumor vasculature
[35] Most strategies target angiogenic stimuli, their
receptors or receptor tyrosine kinase activity [36]
However, an expanding pool of natural molecules act
as brakes for angiogenesis [33] Similar to tumor
suppressors, inhibitors are frequently lost in tumors,
creating a permissive environment for expansion
Re-expression of such inhibitors in angiogenic tumors
impedes their progression: these include angiostatin,
endostatin, tumstatin, PEDF, SPARC (secreted
pro-tein, acidic and rich in cysteine), tissue inhibitor of
metalloproteinases (TIMPs) and TSP1 An emerging
concept is to view natural angiogenesis inhibitors as
endothelial-specific tumor suppressors [33]
TSP1 is one of the most studied angiogenesis
inhibi-tors [21], both in terms of regulation and mechanism
of action It is lost in multiple tumor types:
fibrosar-coma, glioblastoma and carcinomas of the breast,
bladder, colon, prostate and thyroid [19] TSP1
expres-sion is associated with dormancy of nonangiogenic
tumors, and predicts a favorable outcome in multiple
tumor types [37] It blocks angiogenesis via endothelial
cell apoptosis, which requires receptors CD36 and Fas,
and Fas ligand [38], and causes CD36-independent cell
cycle arrest [39] TSP1 suppresses recruitment of the
circulating endothelial progenitors [40] and signaling
by nitric oxide (NO) [41]
The causes of TSP1 loss vary They include genetic
alterations, e.g the loss of tumor suppressor genes
C
BAPTA
P769
P769 HNK
HNK
A
B
C
D
E
Fig 4 TSP1 localization in HNK and P769 cells (A) HNK cells were
serum-starved to prompt secretion and treated with BAPTA-AM
(1 l M ), where indicated After 12 h, HNK cells were fixed, stained
for TSP1 (green) and ER marker HSP-70 (red) Note the depletion
of TSP1 from the cytoplasm of untreated cells (C, top) and
accumu-lation in BAPTA-AM-treated cells (BAPTA-AM, bottom) (B–E) P769
and HNK cells were serum-starved for 24 h HNK cells were
trea-ted with BAPTA-AM to achieve TSP1 retention The cells were
then stained for TSP1 as in (A), and for either Golgi marker A58 (B,
C) or ER marker HSP-70 (D, E) Note the lack of TSP1 export in
BAPTA-AM-treated HNK cells and colocalization (shown in yellow)
with ER, but not with Golgi, in both RCC and BAPTA-AM-treated
HNK cells.
Trang 8(APC, p53, PTEN, SMAD-4 and THY-1) [42–46] or
the gain of activated oncogenes (Akt⁄ PI-3K, Id-1,
Jun, MCT-1, Mts1⁄ S100A4, Myc, Ras and Src) [47–
50] Some of these pathways interact: Ras can
acti-vate c-Myc [51], which acts via microRNA cluster
miR-17-92 [52] Epigenetic events may also
contrib-ute: TSP1 can be repressed by anoxia [53] or
hyper-glycemia [54] A knowledge of the pathways altering
TSP1 production may yield therapies to restore
angiogenic balance and reduce or arrest tumor
bur-den
Our study yielded two findings First, the loss of
secreted TSP1 contributed to angiogenesis by RCC
cells in cooperation with the increase in VEGF
Sec-ond, TSP1 secretion, which determines the state of the
angiogenic switch, was impaired in RCC because of
cytoplasmic retention, whilst healthy cells maintained
normal secretion Seeking molecular causes of failure
to secrete TSP1, we focused on misfolding caused by
limited calcium availability [55] This was indeed the
case: in RCC or BAPTA-AM-treated normal renal
cells, TSP1 resided in the ER, and not in the Golgi
apparatus In RCC cells, the analysis of transport
vesicles showed strong TSP1 association with COPI-positive vesicles responsible for retrograde transport, a mechanism by which the cells ‘recall’ misfolded pro-teins from the ERGIC [56] By contrast, in HNK cells, TSP1 was localized predominantly in COPII-positive anterograde vesicles, pointing to Golgi accumulation prior to secretion
Seeking reasons for impaired calcium metabolism,
we found that RCC cells expressed lower levels of TRPC4, which, together with TRPC1, forms hetero-meric channels [27] that mediate growth factor-stimu-lated calcium influx [27] Although TRPC4 expression
in the renal epithelium has been shown, its role in renal tissue is unknown Our data indicate that TRPC4
is a key regulator of calcium intake in this tissue Fur-ther analysis showed that, in agreement with published data [57], most RCC cell lines expressed no detectable calbindin, possibly because of their undifferentiated state In addition to transepithelial calcium transport, calbindin acts as a buffer, absorbing excess calcium [28,29] The lack of calbindin increases apoptosis in response to growth factor-initiated calcium intake [58] Thus, TRPC4 reduction may be an adaptation of RCC cells to the lack of calbindin protective function The protection from apoptosis despite the lack of cal-bindin could be explained by the decrease in TRPC4,
or by retention of the Ca2+-binding TSP1 However, TSP1 knockdown with siRNA had no effect on the viability of P769 cells (see supplementary Fig S1), sug-gesting that the loss of TRPC4 was sufficient to com-pensate for the lack of calbindin
Therefore, we have demonstrated diminished TSP1 secretion by RCC cells as a result of active retro-grade transport This active retroretro-grade transport was triggered by protein misfolding, which, in turn, was caused by changes in calcium metabolism Calcium intake in response to growth stimuli was reduced because of the decrease in TRPC4 and the lack of calbindin This is a novel pathway by which cancer cells down-regulate TSP1, an angiogenesis inhibitor, and flip their angiogenic switch Further analysis of calcium metabolism and its modifiers may yield novel strategies to suppress RCC angiogenesis and growth
Experimental procedures
Cells and reagents Human renal epithelial cells (HNK, P3-8; Clonetics, Walk-ersville, MD) were grown in keratinocyte growth medium (Gibco Invitrogen, Carlsbad, CA) with 10% fetal bovine serum RCC cells (PRC9, SW839, p769; American Tissue
A
B
Fig 5 TSP1 association with retro- and anterograde transport
vesi-cles The cells were starved for 6 h to initiate secretion, fixed and
stained for TSP1 (red) and for COPII component Sec23, a marker
of anterograde vesicles, or with COPI component c2-COP, a
mar-ker of retrograde vesicles (green) Note the predominant TSP1
colo-calization with Sec23 (anterograde vesicles) in normal HNK and
with c2-COP (retrograde vesicles) in P769 tumor cells.
Trang 9Type Culture Collection, Manassas, VA) and 786-O RCC
expressing wild-type (WT8) or inactive VHL tumor
sup-pressor (ARZ; a gift from R Kerbel, Sunnybrook and
Women’s Hospital, Toronto, Canada) were maintained in
keratinocyte growth medium with 10% fetal bovine serum
HMVECs (Clonetics) were maintained in MDCB131
(Sigma, St Louis, MO) with the endothelial cell bullet kit
(BioWhittaker, Walkersville, MD)
BAPTA-AM, fluo-4 acetoxymethyl ester and Pluronic
F127 were obtained from Molecular Probes (Invitrogen)
VEGF and EGF were purchased from R&D Systems
(Minneapolis, MN)
TSP1 antibodies (Ab-1, Ab-3, Ab-11) were obtained from
NeoMarkers (Fremont, CA) VEGF antibodies were
pur-chased from R&D Systems A-58 monoclonal antibody was
obtained from Sigma Antibodies for heat shock protein-70
(HSP-70), c2-Cop and Sec23 were obtained from Santa
Cruz (Santa Cruz, CA, USA) and Calbindin D-28K from
AbCam (Cambridge, MA) Fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse IgG were purchased
from Sigma Rhodamine (TRITC)-conjugated, horseradish
peroxidase (HRP)-conjugated and Alexa Fluor-conjugated
antibodies were obtained from Jackson Immunoresearch
(Westgrove, PA) TSP1 was purified from platelets as
described previously [59]
CM preparation
The cells were grown to 70–80% confluence, rinsed twice
and transferred to serum-free medium After 4 h, this
medium was removed and replaced by fresh medium After
24–48 h, CM were collected and concentrated in centifugal
filters (3 kDa cut-off; Millipore, Billerica, MA)
Transfection
TRPC4, TSP1, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and scrambled siRNA were obtained from
Dharmacon (Lafayette, CO) The cells were seeded in
six-well plates (4· 105
, 3· 105
and 2· 105
per well) in the growth medium siRNA in 200 lL of serum-free medium
(100 nm final concentration) and DharmaFECT reagent
(4 lL in 200 lL of serum-free medium) were incubated for
20 min at room temperature and added to the cells After
24, 48 and 72 h, CM were collected and the cells were
pro-cessed further (total RNA and⁄ or cell lysates)
Cell survival⁄ proliferation assay
The cells were seeded in a 96-well plate
3-(4,5-Di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide reagent
(Chemicon, Billerica, MA) was added at 24, 48 and 72 h
and incubated for 4 h at 37C The assay was performed
following the manufacturer’s instructions
Endothelial cell chemotaxis HMVECs starved overnight in MDCB131 with 0.1% BSA were plated at 1.5· 106
mL)1 on the lower side of porous membranes (8 mm, Nucleopore Corp., Kent, WA) in modi-fied Boyden chambers (Neuroprobe, Gaithersburg, MD); the samples were added to the top Cells migrating to the opposite side of the membrane were counted in · 10 400 fields (controls: 0.1% BSA, 10 ngÆmL)1bFGF)
Specific activity
CM were tested as above, at 0.01–40 lgÆmL)1, to generate dose–response curves The ED50values (concentrations pro-ducing 50% maximal response) were extrapolated from the best-fit curves (sigmaplot, Systat Software, San Jose, CA)
To evaluate VEGF and TSP1 contributions, the appro-priate neutralizing antibodies were added at 1.0 and 2.5 lgÆmL)1, respectively
Corneal angiogenesis
CM from HNK and RCC cell lines were analyzed in the rat or mouse corneal assay [60,61] Briefly, micropockets were aseptically created in the cornea of female Fisher 344 rats or C57Bl6 mice (Harlan), 1.5–2.0 mm and 0.5–1 mm from the limbus, respectively In rats, Hydron (HydroMed, Cranbury, NJ, USA) implants ( 5 lL, 2 lg CM protein) were placed in the micropockets and angiogenesis was scored on day 7 Animals were perfused with colloidal car-bon, and the corneas were fixed, flattened and photo-graphed Vascular growth from the limbus to the pellet was graded as positive or negative In mice, Hydron sucral-fate pellets (1 lL, 0.4 mg protein) were implanted and angiogenesis was scored on day 5 by slit-lamp microscopy All animals were handled following the National Institutes
of Health guidelines and protocols approved by the North-western University Animal Care and Use Committee
Statistical evaluation Quantitative results were evaluated using Student’s t-test
P< 0.05 was considered to be significant
Tissue acquisition and staining Deidentified specimens were obtained from the pathology department with Institutional Review Board approval for archived tissues Five micrometer sections were stained with hematoxylin–eosin to select the areas of carcinoma and noncancerous tissue Sections were deparaffinized, re-hydrated in graded ethanol solutions, treated for 5 min with 3% H2O2, rinsed and blocked for 30 min in 10%
Trang 10horse serum at room temperature The sections were
incu-bated with TSP1 antibodies in blocking solution (Ab-1,
1 : 250, 4C overnight), followed by rabbit anti-mouse IgG
(Vectastain ABC kit, Vector, Burlingham, CA, 1 : 125, 1 h
at room temperature), rinsed and incubated with avidin–
biotin complex (Vectastain; 1 h, room temperature) Slides
were developed with 2,4-diaminobutyric acid, counterstained
with hematoxylin, rehydrated and mounted
Western blotting
To detect TSP1, the cells were lysed for 1 h at 4C in 1%
Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS and
150 mm NaCl in 10 mm sodium phosphate pH 7.2, with
protease inhibitors The lysates were loaded at 30 lg per
lane; concentrated CM were loaded at 10 lg per lane The
blots were probed with TSP1 Ab-11 (1 : 400), and the
sig-nal was detected with a LumiGLO Kit (KPL, Gaithersburg,
MD) Calbindin antibodies (1 : 5000) were applied
over-night (4C)
Calcium imaging
Intracellular calcium was detected by videofluorescence
imaging Cells were grown on chamber slides, rinsed in
Hank’s balanced salt solution, 10 mm Hepes, 11 mm
glu-cose, 2.5 mm CaCl2and 1.2 mm MgCl2, loaded for 30 min
in 5 lm fluo-4 acetoxymethyl ester, Pluronic F127 (1 : 1,
Molecular Probes), treated and monitored (488 nm
excita-tion, 520 nm emission) with a fluorescent microscope
(Leica, Bannockburn, IL, · 20 objective) Images were
acquired with a Hamamatsu (Bridgewater, NJ) camera (10 s intervals, openlab software, Improvision, Waltham, MA) and analyzed with imagej software (minimum of 30 cells per treatment)
RT-PCR One microgram of total RNA extracted with an RNeasy kit (Qiagen, Valencia, CA) was used for reverse transcrip-tion with oligo(dT)15 primers (protocol and reagents from Promega, Madison, WI) Serial dilutions of cDNA were PCR-amplified in a 23-cycle reaction with b-actin primers (HotStartTaqTM, Qiagen) Dilutions yielding similar prod-uct amounts were chosen for analysis; prodprod-ucts were resolved on 1.5% agarose gels Primers⁄ conditions are given in Table 4
Immunofluorescence Cells grown on coverslips were fixed in ice-cold methanol– acetone (1 : 1) and blocked for 30 min (1% horse serum)
To detect TSP1, the cells were incubated for 1 h at room temperature with Ab-1 (1 : 50 in blocking solution), fol-lowed by the Alexa Fluor 488 goat anti-mouse IgM (5 lgÆmL)1 in blocking solution) A-58 Golgi protein anti-body (1 : 500) was followed by goat anti-mouse TRITC-IgG (1 : 100) ER marker antibody, HSP-70 (1 : 100), was followed by Alexa Fluor 546 goat anti-mouse IgG (5 lgÆmL)1) To analyze TSP1 localization to transport ves-icles, the slides were blocked for 30 min in 10% donkey serum and incubated with TSP1 Ab-3 (1 : 50) and Sec23 or c2-Cop antibodies (1 : 50) in 2% donkey serum for 1 h at room temperature The slides were rinsed three times and incubated for 1 h with FITC-conjugated donkey anti-mouse IgG and Texas Red conjugated donkey anti-goat IgG (1 : 100, 2% donkey serum) The slides were mounted in Fluoromount-G
Acknowledgements
This work was funded by National Institutes of Health (NIH) grant RO1 HL077471 (OV)
References
1 Brugarolas J (2007) Renal-cell carcinoma – molecular pathways and therapies N Engl J Med 356, 185–187
2 Kim WY & Kaelin WG (2004) Role of VHL gene mutation in human cancer J Clin Oncol 22, 4991–5004
3 Kaelin WG Jr (2003) The von Hippel–Lindau gene, kid-ney cancer, and oxygen sensing J Am Soc Nephrol 14, 2703–2711
4 Semenza GL (2003) Targeting HIF-1 for cancer therapy Nat Rev Cancer 3, 721–732
Table 4 Primers used in RT-PCR analysis.
Gene Primers (5¢- to 3¢)
Annealing
T (C)
Cycle number Actin TGTTGGCGTACAGGTCTTTGC 60 23
GCTACGAGCTGCCTGACGG
GAPDH TATCGTGGAAGGACTCATGACC 55 20
TACATGGCAACTGTGAGGGG
TSP1 CCGGCGTGAAGTGTACTAGCTA 65 25
TGCACTTGGCGTTCTTGT
TRPC1 GATTTTGGAAAATTTCTTGGGATGT 55 35
TTTGTCTTCATGATTTGCTATCA
TRPC2 CATCATCAT-GGTCATTGTGCTGC 55 35
GGTCTTGGTCAGCTCTGTGAGTC
TRPC3 GACATATTCAAGTTCATGGTCCTC 55 35
ACATCACTGTCATCCTCAATTTC
TRPC4 GCTTTGTTCGTGCAAATTTCC 55 35
CTGCAAATATCTCTGGGAAGA
TRPC5 CAGCATTGCGTTCTGTGAAAC 55 35
CAGAGCTATCGATGAGCCTAAC
TRPC6 GACATCTTCAAGTTCATGGTCATA 55 35
ATCAGCGTCATCCTCAATTTC
TRPC7 CAGAAGATCGAGGACATCAGC 55 35
GTGCCGGGCATTCACGTGGTA