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Tumor Biology |
Departments of Microbiology [W. X.], Pathology [P. U.], Clinical Immunology [L. M.], and Medicine [I. H. G.], Mount Sinai School of Medicine, New York, New York, 10029-6574, and Department of Urology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 [J. N.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6. Our data suggest a role for the loss of SSeCKS/Gravin in the metastatic progression of human prostate cancer. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular pathogenesis of MPA is a multistep process involving the activation of endogenous oncogenes as well as the loss of tumor suppressor/cancer susceptibility genes. No single oncogene has been associated with MPA, yet 40% of cases studied contain activating mutations or increased expression of oncogenes such as ras, myc, or fos (4
, 5)
. Even more significant is the loss of critical tumor suppressor functions such as p53, DCC, and pRb, or cytoskeletal/adhesion molecules such as E-catenin/
-cadherin (6
, 7)
. These losses correspond to allelic deletions in chromosomes 5q, 17p, 18q, and 13q, found in a small but significant population of MPAs, and deletions in 10q and 6q, found in >60% of MPAs.
To date, only a handful of metastasis-suppressing genes have been identified, usually by cDNA subtraction and microcell-mediated chromosomal transfer. Examples include nm23 (NME1), KAI1, KiSS1, BrMS1, MKK4/SEK1, CD44, and maspin (reviewed in Ref. 8 ). Typically, the expression of these gene products is inversely proportional to metastatic potential. The reexpression of these proteins, besides suppressing the production of spontaneous and experimental metastases in animal models, correlates with decreased colony-forming activity in soft agar, motility, and invasiveness into Matrigel and increased cell-cell adhesion and immunosensitivity.
We identified a potential tumor suppressor, SSeCKS, of which the expression is down-regulated in src- and ras-transformed fibroblasts (9)
. Reexpression of SSeCKS suppresses src-induced oncogenesis such as growth factor- and anchorage-independence, loss of contact inhibition, and metastatic potential, mainly by reorganizing actin-based cytoskeletal architecture (10)
. SSeCKS is likely the rodent orthologue of human Gravin/AKAP 12, a scaffolding protein for PKC and PKA (11)
. SSeCKS also plays a role in G1
S progression by modulating cyclin D expression and by sequestering G1-phase cyclins in the cytoplasm (12)
. Here, we show that gravin maps to a single chromosomal site, 6q24–25.2, a hotspot for deletion in advanced prostate cancer (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
. We show that SSeCKS/Gravin expression is abundant in untransformed human and rat prostate cell lines, in normal prostatic epithelial cells, and in undifferentiated human prostate cancers in vivo but down-regulated in prostate cancer cell lines and in high-grade cancers in vivo. Moreover, reexpression of SSeCKS in MLL cells suppresses cell rounding, anchorage-independent growth, and the generation of secondary lung metastases in nude mice and increases cell-cell adhesion. These data suggest a role for the loss of SSeCKS/Gravin expression in the onset of prostate cancer metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production of Tetracycline-regulated MLL Cell Lines.
MLL/tTAK cells, expressing a tet-regulated tTA transactivator (25)
, were produced by transfecting with CaPO4/DNA precipitates containing 3.5 µg of pTet-tTAK and 0.6 µg of pRSV/hygro followed by selection of stable transfectants in 400 µg/ml of hygromycin (Sigma Chemical Co.). Individual clones were tested for the ability to induce expression of tetO/luciferase (pUHD13–3) in the absence of tet (26)
. Clones 2 and 7 were chosen for secondary transfection with 3.8 µg of pUHD10–3/SSeCKS (27)
and 1.6 µg of pBABE/puro (28)
, and stable transfectants were isolated after selection in hygromycin and puromycin (8 µg/ml). All of the cells were selected in 5 µg/ml tet and then maintained on 0.7 µg/ml.
FISH Analysis.
FISH analysis was performed by See DNA Biotech, Inc. (Downsview, Ontario, Canada). A 6.2-kb Gravin cDNA fragment was labeled with Biotin-14-dATP using a Life Technologies, Inc. BioNick kit according to the manufacturer’s specifications. Slides were prepared with human lymphocytes grown in -minimal essential medium containing 10% FCS, phytohemagglutinin, and bromodeoxyuridine (180 µg/ml; Sigma Chemical Co.), then grown for 6 h in medium containing thymidine (2.5 µg/ml). FISH detection was performed as described previously (29
, 30)
. Among 100 mitotic figures that were checked, 81 showed signals on one pair of the chromosomes (i.e., 81% hybridization efficiency). DAPI banding patterns mapped the signals to the long arm of chromosome 6, and based on the summary from 10 independent photographs, Gravin was mapped by higher resolution to 6q24–25.2. No additional loci were identified by FISH detection, suggesting the absence of highly conserved gene family members.
Colony Assay in Soft Agar.
Cells (104) were plated into soft agar in 6-cm wells as described previously (10)
and then grown for 3 weeks at 37°C with feedings of fresh medium twice/week.
Northern and Western Blotting.
Total or poly(A)-selected RNAs were electrophoresed, blotted, and probed with 32P-rat SSeCKS cDNA as described (9)
. Radioimmunoprecipitation assay lysates containing 40–100 µg of total protein were prepared and immunoblotted using rabbit polyclonal anti-SSeCKS immunoglobulin as described (31)
, using either alkaline phosphatase- or horseradish peroxidase-labeled secondary antibodies followed by Western Blue substrate (Promega, Madison, WI) or ECL (New England Nuclear, Boston, MA) for visual or chemiluminescence detection, respectively. Images were scanned on an Agfa Duoscan T1200, digitized on a PowerMac G3 (Apple) computer using Adobe Photoshop version 4.01, and quantified using the UN-SCAN-IT Gel program version 4.3 (Silk Scientific, Orem, UT).
IFA and Immunohistochemistry Analyses.
Cells seeded onto 22-mm2 coverslips were fixed and stained with immunoaffinity-purified rabbit polyclonal anti-SSeCKS (31)
or rabbit anti-Gravin sera (32)
as described previously (27)
. Immunocytochemistry was performed as described (32)
. Slides were viewed on an Olympus IX-70 fluorescent microscope and digitized using a Sony Catseye camera connected to a PowerMac G3 computer. Image analysis was performed using Adobe Photoshop 4.01.
Tumor and Metastasis Formation in Nude Mice.
Six-week-old female nude mice (Taconic Farms, Germantown, NY) were injected s.c. with 105 MLL/vector or MLL/SSeCKS clones. The viability of the cells was >90% as determined by trypan blue exclusion. All of the mice were fed water containing 100 µg/ml tet plus 5% sucrose until the primary tumors were palpable (2–4 mm), at which point the tet-water was withdrawn. Mice were sacrificed 3 weeks after injection. The primary tumors were measured and weighed, and the lungs were stained for metastases by injecting India ink (30 ml of ink plus 4 drops of 1 M ammonium hydroxide, diluted into 200 ml of dH2O) into the trachea for 10 min at room temperature followed by several washes in PBS. Surface metastases, which exclude the dye, were then counted.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1000 amino acid (a.a.), <20% similarity over the next 500 a.a., and identity in two 15-a.a. stretches at the C-termini, one of which encodes a PKA anchoring site (11)
. Full-length SSeCKS cDNA recognizes Gravin mRNA under conditions of stringent hybridization (32)
. Using a Gravin cDNA probe, we mapped gravin by FISH to chromosome 6q24–25.2 (Fig. 1)
. These map coordinates are confirmed by microsatellite markers (Sanger Sequencing Center, Cambridge, United Kingdom). Secondary hybridization signals were not detected, which might reflect a second family member. FISH analysis using a full-length SSeCKS cDNA probe identified the same, singular region (data not shown). Moreover, mouse SSeCKS maps to the Tsga12 locus at the centromeric end of chromosome 10p, which is syntenic with human chromosome 6q24–274
, strongly suggesting that SSeCKS and Gravin/AKAP12 are orthologues. Deletions in this region are associated with advanced, nonorgan confined prostate cancer cases (13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
, suggesting a possible role for SSeCKS/Gravin in prostate oncogenesis.
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shows a progressive loss of SSeCKS RNA signal in MLL and AT-3.1 cancer cells (5- and >20-fold lower, respectively, as defined by densitometry) compared with the mildly transformed G cell line. AT-3.1 cells, which are androgen-independent, are slightly more metastatic in nude mice than MLL (3)
. SSeCKS RNA levels in R3327-H-, MatLu-, MLL-, or AT-3-induced tumors are reduced >5-fold compared with levels in the G cell line. Fig. 2B
shows a similar reduction in Gravin/AKAP12 RNA levels in various human prostate cancer cell lines compared with normal human prostate. In addition to the expected 6.5-kb gravin message, PC-3 and PPC-1 cells displayed an additional RNA transcript (8.5 kb) that hybridized to the rat SSeCKS probe. Growth of the androgen-responsive LNCaP cells (33)
in the absence of androgens for >9 months had no effect on Gravin/AKAP12 transcript levels, possibly because LNCaP cells contain a 6q24-ter deletion (34)
, a region encompassing gravin (Fig. 1)
. Thus, the loss of SSeCKS/Gravin message is typical in prostate cancer lines.
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The P69 series (24)
consists of human prostate epithelial lines with increasing oncogenic characteristics in nude mice: P69 are nontumorigenic cells immortalized with SV40 Tag; M2182 are nonmetastatic variants that form tumors at the primary injection site; and M12 are variants selected in nude mice that form lung metastases after i.p. or intraprostatic injection. Fig. 2C
shows a severe decrease (>10-fold) in the levels of the Mr 305,000/287,000 major Gravin isoforms in both M2182 and M12 cells compared with the parental P69 cells.
Our previous data indicated that the level of SSeCKS RNA and protein increases dramatically under contact-inhibited growth conditions (9
, 12
, 31
, 35)
. Fig. 2C
shows a >10-fold increase in SSeCKS protein levels in rat EP12 cultures in transition from subconfluence (L) to several days of confluent growth (H), as determined by densitometry. In contrast, SSeCKS levels in MLL cells, which are 4-fold lower than EP12 cells, are not affected by culture density. However, the abundance of the Mr 240,000 SSeCKS isoform as well as a novel Mr 80,000 isoform increases in MLL after growth at high density (Fig. 2D)
. The Mr 240,000 isoform (Mr 250,000 in human cells) is a proteolytic cleavage product lacking NH2-terminal sequences (32)
. Thus, SSeCKS/Gravin protein levels are induced in both epithelial cells and fibroblasts in response to increased culture density, and this induction mechanism is inactive in prostate cancer cells.
Tet-regulated SSeCKS Reexpression Suppresses MLL-induced Oncogenesis.
We demonstrated previously that the tet-regulated reexpression of SSeCKS led to the suppression of src-induced oncogenic growth fibroblasts (10)
. In that study, reexpression of SSeCKS to levels 2–25-fold over those in untransformed parental cells led to the selective loss of src-induced oncogenic growth parameters such as anchorage- and growth-factor independence and Matrigel invasiveness but did not inhibit cell proliferation, indicating that the SSeCKS effects were not simply attributable to toxicity. Because MLL cells encode at least one activated Ha-ras allele (4)
, we addressed whether SSeCKS could suppress ras-associated tumorigenic growth by producing MLL cells with tet-regulated SSeCKS expression. Fig. 3A
shows similar levels of SSeCKS protein in the MLL/tTAK cells compared with the parental MLL cells. The MLL/tTAK cells express a tet-regulated form of the tTA transactivator (25)
, which we found to be much less toxic than the constitutively expressed tTA. Expression of the Mr 290,000 SSeCKS isoform was induced 4–20-fold in several independently derived clones grown without tet (Fig. 3A)
. These clones also express background levels of SSeCKS in the presence of tet.
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. SSeCKS induced a fibroblast-like morphology (Fig. 3B)
rather than the epithelial morphology typified by EP12 cells (Fig. 4B, c)
. tTAK caused mild cell flattening (Fig. 3B)
, and although this was always significantly less than that induced by SSeCKS, it is impossible to exclude that SSeCKS and tTAK work cooperatively in this regard. In contrast, increased cell-cell adhesion was induced by SSeCKS reexpression and not by tTAK.
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shows enrichment of SSeCKS in the perinuclear regions of EP12 cells as well as a cortical cytoskeletal distribution. SSeCKS was enriched in actin-dense membrane ruffles (Fig. 4A, a and a'
; arrows), in focal complexes connected to actin stress fibers (Fig. 4A, b and b'
; 
), and in actin-dense bundles at the lagging edge (Fig. 4A, b, b'
; arrows). In MLL[tet/SSeCKS] cells grown without tet, reexpressed SSeCKS was enriched in membrane ruffles (Fig. 4B, a)
and in lamellipodia (Fig. 4B, b)
.
We then addressed whether SSeCKS expression affected parameters of in vitro oncogenic growth. Fig. 5A
shows that expression of tTAK alone (V-2 or V-7) was somewhat inhibitory to proliferation in medium with 10% serum, probably attributable to squelching of transcription factors by the VP16 moiety of tTAK (10
, 27
, 36) . Although SSeCKS expression in some clones decreased proliferation rates slightly more than vector controls, it is impossible to separate SSeCKS- and tTAK-induced effects. However, fluorescence-activated cell-sorted MLL cells transiently cotransfected with SSeCKS and Green Fluorescent Protein expression vectors showed no significant changes in proliferation rates compared with Green Fluorescent Protein controls (data not shown). Thus, we conclude that SSeCKS reexpression does not significantly alter the proliferation rate of MLL cells.
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shows that SSeCKS expression inhibited anchorage-independent growth 4–5-fold over that induced by tTAK alone. The 40% decrease in colony-forming activity in vector control cells probably relates to the inhibitory effects of tTAK. We sought to determine whether SSeCKS could inhibit the anchorage-independent growth of MLL in the absence of the tet system. Fig. 5C
shows that transient expression of SSeCKS decreased the colony-forming efficiency of MLL 3-fold compared with vector controls. Thus, the suppressive effects of SSeCKS on anchorage-independent growth are separable from those of tTAK.
The SSeCKS/Gravin Mr 80,000 Isoform: A Marker for Prostate Cancer?
We noted that the Mr 80,000 isoform was present in the MLL/tTAK cells and in all of the MLL[tet/SSeCKS] clones grown with tet, whereas tet removal correlated with a 2–10-fold decrease in the abundance of this isoform. This isoform was also detected in the human tumorigenic lines M2182 and M12 but not in untransformed parental P69 prostate epithelial cells (Fig. 2C
; arrow). We investigated whether the Mr 80,000 isoform was produced by a factor present in either prostate cancer or untransformed cells. NP-40 lysates from MLL and EP12 cells were mixed at a 1:1 ratio to determine whether factors from one lysate would decrease the presence of the Mr 80,000 isoform. Table 1
shows that mixing of the lysates at 30°C for 5 min caused a decrease in the abundance of the Mr 80,000 isoform (compared with incubation of the MLL lysate alone). Preboiling of the MLL lysate did not effect the EP12-induced decrease, whereas boiling of the EP12 lysate did. This indicates that EP12 encodes a heat-labile factor that is antagonistic to the production or stability of the Mr 80,000 isoform.
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. However, analysis of progressing primary-site MLL[tet/SSeCKS] tumors revealed a loss of tet-regulated SSeCKS expression (Fig. 6C)
, suggesting that the progressors were variants that had defeated the inducible expression of SSeCKS. Growth of cells in vitro from these primary tumors (in DME + hygromycin/puromycin) showed a lack of inducible SSeCKS in the absence of tet (data not shown). These data indicate that SSeCKS reexpression did not significantly decrease the formation or growth rate of primary-site tumors.
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; Fig. 6B
). The inhibition occurred with two independent clones with varying degrees of inducible SSeCKS expression (2–6 and 7–2). Because expression of SSeCKS had no effect on cell motility in a monolayer wounding assay (Fig. 6D)
, it is unlikely that these cells are defective for cell motility in vivo. These data indicate that even mild reexpression levels of SSeCKS, as in clone 7–2, are sufficient to suppress metastatic potential in MLL cells.
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. Abundant SSeCKS/Gravin staining was detected in benign prostatic hyperplastic lesions (Fig. 7b)
and in well-differentiated carcinomas (Fig. 7c)
. In contrast, SSeCKS/Gravin staining was absent in undifferentiated carcinomas (Fig. 7d)
. Noncancerous ducts in the same prostate contain epithelia with robust SSeCKS/Gravin staining. A larger survey of human samples showed a consistent loss of SSeCKS/Gravin expression in MPAs with Gleason sums >6 (Table 3
). MPAs were all positive for prostatic acid phosphatase as a control for stainability. These data correlate the loss of SSeCKS/Gravin with the onset of aggressive prostate oncogenesis in humans.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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6. These data strongly suggest that the loss of SSeCKS/Gravin contributes to the progression of prostate cancer and that SSeCKS/Gravin encode tumor suppressor activity. Relative to untransformed control epithelial cell lines or normal prostate tissue, SSeCKS/Gravin RNA and protein levels are markedly reduced in rat and human prostate cancer cell lines or rat prostate tumors grown in syngeneic animals. There was no consistent gradation of SSeCKS/Gravin down-regulation correlating with tumorigenic or metastatic phenotype between the cancer cell lines. For example, LNCaP cells had no detectable levels of Gravin RNA, yet these cells are less tumorigenic in nude mice than PC-3 cells, which had down-regulated, yet detectable levels of Gravin transcript. The P69 series showed a similar down-regulation of SSeCKS/Gravin in the tumorigenic M2182 and metastatic M12 variants. However, these clones were selected for spontaneous oncogenic growth in nude mice, and as such, do not reflect the biology of prostate cancer progression. For example, although SSeCKS/Gravin RNA levels are decreased in M2182 and M12, they retain chromosome 6q. Indeed, the chromosomal deletions and amplifications in M12 cells are more typical of lung metastases from nonprostate cancers than bony metastases from prostate cancer (24) . On the basis of the ability of SSeCKS to inhibit formation of MLL-induced lung metastases (below), we would have expected an additional decrease in relative SSeCKS/Gravin levels in M12 versus M2182 cells. However, it is most likely that the metastatic potency of our M12 cells has been decreased by repeated passage in culture, because s.c. injection of 106 M12 cells induced robust primary-site growth but no lung metastases (data not shown).
As in the case of SSeCKS reexpression in src-transformed NIH3T3 fibroblasts (10) , SSeCKS reexpression in MLL cells inhibited anchorage-independent growth. We also show that SSeCKS could inhibit MLL colony-forming activity in the absence of the tet-regulated system. SSeCKS expression itself was not significantly more inhibitory to proliferation than tTAK. However, SSeCKS reexpression in the absence of the tet system clearly indicates that SSeCKS does not inhibit MLL cell proliferation on its own. Thus, it is unlikely that the decreased colony-forming activity detected after SSeCKS reexpression in MLL cells is attributable to inhibition of proliferation by SSeCKS. Although SSeCKS induced more cell flattening than tTAK alone, we cannot rule out that these effects result from synergy between SSeCKS and tTAK. In contrast, SSeCKS but not tTAK, could induce cell-cell adhesion in the MLL system.
SSeCKS expression antagonizes MLL-induced oncogenesis in vivo, especially the generation of lung metastases. Preliminary data indicate that some MLL[tet/SSeCKS] cells metastasize to the lungs, based on the ability to select for rare (<25 cells/lung) puromycin/hygromycin-resistant cells in the lungs after collagenase treatment (data not shown). Taken together with our demonstration that SSeCKS reexpression does not decrease MLL cell motility, these data suggest that SSeCKS induces dormancy of metastatic cells in the lung.
The ease at which the MLL cells override the tet-regulated SSeCKS expression in vivo probably belies the plasticity of these tumor cells. Nonetheless, our results suggest that either the sustained expression of SSeCKS or its repeated dosage in prostate tumors would decrease primary tumor growth and metastatic potential.
The association of SSeCKS with cortical, actin-based cytoskeletal structures (27)
leads us to believe that the tumor-suppressive activity of SSeCKS is a direct result of its ability to reorganize cytoskeletal architecture and, subsequently, to reestablish feedback control of signaling pathways. Tumor cells often have normal steady-state levels of major cytoskeletal proteins such as actin and tubulin yet have increased turnover of their polymerized, filamentous forms compared with untransformed cells (37)
. In contrast, cancer cells have decreased levels of minor or intermediate cytoskeletal proteins such as vinculin, -actinin, tropomyosin, or cytokeratins (38)
. Many groups have shown that reexpression of any one of these latter proteins is sufficient to induce a cytoskeletal reorganization that stabilizes microtubule and microfilament structures. SSeCKS seems to function in a similar manner.
Taken together with the putative scaffolding role for PKA, PKC, and cyclin D (11
, 12)
in SSeCKS, as well as preliminary data suggesting a role for these scaffolding functions in the control of cyclin D1 expression and G1S progression (12)
, we suggest that SSeCKS controls tumorigenic and metastatic growth by acting as a gating function for signaling and cytoskeletal pathways. Indeed, a paradoxical effect of SSeCKS overexpression is a decrease in apoptosis and anoikis (27)
, suggesting that SSeCKS suppresses oncogenic pathways leading to caspase activation.
We also noted that SSeCKS expression induced increased cell-cell adhesion in most of the MLL[tet/SSeCKS] clones. E-cadherin expression is lost in MLL (39
, 40)
, possibly because of deletion of both alleles (41)
. We could not detect E-cadherin or -catenin in MLL[tet/SSeCKS] cells even after ectopic SSeCKS expression; ß-catenin, which is detectable in parental MLL cells, was not affected by SSeCKS (data not shown). A pan-cadherin polyclonal antibody (Sigma Chemical Co.; Ref. 42
) to E-, EP-, N-, P, L-, and V-cadherins failed to show any increase in cadherin levels after SSeCKS expression. Thus, cell-cell adhesion may be facilitated by atypical cadherins or by noncadherin moieties such as selectins or integrins. The latter notion is strengthened by data that MLL cells show increased levels of ß1 and 5 integrins as well as fibronectin (40)
, which might antagonize mechanisms of normal cell-cell adherence.
An Mr 80,000 SSeCKS/Gravin isoform was detected in human and rat prostate cancer cells but not their untransformed control lines or in SSeCKS reexpressors. The likelihood that this is an SSeCKS isoform rather than an induced cross-reactive protein is based on its being recognized by two different monoclonal antibodies raised to rat SSeCKS protein (data not shown). We postulate that untransformed prostate epithelia express a heat-labile factor which either degrades the Mr 80,000 isoform or inhibits a tumor-specific protease that generates the Mr 80,000 isoform. This protease is unlikely a caspase, because SSeCKS/Gravin lacks any of the three recognition motifs (43)
. It is interesting to speculate that the Mr 80,000 fragment either plays a positive role in tumorigenesis or acts as a dominant-interfering isoform against full-length SSeCKS/Gravin much in the same way FAK-related nonkinase (FRNK) antagonizes focal adhesion kinase (FAK) function (reviewed in Ref. 44
). If the former role is correct, then antagonists of the putative SSeCKS protease might inhibit parameters of prostate tumorigenesis. The latter role might explain why cells such as MLL have metastatic potential although they express limited levels of SSeCKS. Thus, SSeCKS/Gravin functions might be lost by either chromosomal deletion, transcriptional down-regulation, proteolytic cleavage, or hyperphosphorylation, as we showed in the case of ras-transformed Rat-6 cells (31)
.
In summary, we have demonstrated that the loss of SSeCKS/Gravin expression is typical in prostate cancer cell lines and in human MPA. We also show that SSeCKS/Gravin can suppress prostate oncogenesis in vitro and in vivo, suggesting that reexpression would antagonize progression of prostate cancer in patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by CaP CURE Awards (to J. N. and I. H. G.), Department of Defense Grant DAMD17-99-1-9006, and National Cancer Institute Grant CA65787 (to I. H. G.). 
2 To whom requests for reprints should be addressed, at Department of Medicine, Box 1090, One Gustave L. Levy Place, Mount Sinai School of Medicine, New York, NY 10029-6574. Phone: (212) 241-3749; Fax: (212) 828-4202; E-mail: irwin.gelman{at}mssm.edu
3 The abbreviations used are: MPA, malignant prostatic adenocarcinoma; MLL, MatLyLu; tet, tetracycline; IFA, immunofluorescence; FISH, fluorescence in situ hybridization; DAPI, 4',6-diamidino-2-phenylindole; PKA, protein kinase A; PKC, protein kinase C.
4 Internet address: http://www.informatics.jax.org.
Received 2/28/01. Accepted 5/ 9/01.
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REFERENCES |
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(PKC-) inhibits invasive and metastatic abilities of Dunning R-3327 MAT-LyLu rat prostate cancer cells. Cancer Res., 56: 4137-4141, 1996.This article has been cited by other articles:
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  H. Z. Li, Y. Gao, X. L. Zhao, Y. X. Liu, B. C. Sun, J. Yang, and Z. Yao Effects of Raf Kinase Inhibitor Protein Expression on Metastasis and Progression of Human Breast Cancer Mol. Cancer Res., June 1, 2009; 7(6): 832 - 840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Frontini, M. Vijayakumar, A. Garvin, and N. Clarke A ChIP-chip approach reveals a novel role for transcription factor IRF1 in the DNA damage response Nucleic Acids Res., March 1, 2009; 37(4): 1073 - 1085. [Abstract] [Full Text] [PDF]
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|
|
|
 |
|
 Z. Jin, J. P. Hamilton, J. Yang, Y. Mori, A. Olaru, F. Sato, T. Ito, T. Kan, Y. Cheng, B. Paun, et al. Hypermethylation of the AKAP12 Promoter is a Biomarker of Barrett's-Associated Esophageal Neoplastic Progression Cancer Epidemiol. Biomarkers Prev., January 1, 2008; 17(1): 111 - 117. [Abstract] [Full Text] [PDF]
|
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|
 |
|
 D. C. Weiser, U. J. Pyati, and D. Kimelman Gravin regulates mesodermal cell behavior changes required for axis elongation during zebrafish gastrulation Genes & Dev., June 15, 2007; 21(12): 1559 - 1571. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. K. Choi, J. H. Kim, W. J. Kim, H. Y. Lee, J. A. Park, S.-W. Lee, D.-K. Yoon, H. H. Kim, H. Chung, Y. S. Yu, et al. AKAP12 Regulates Human Blood-Retinal Barrier Formation by Downregulation of Hypoxia-Inducible Factor-1{alpha} J. Neurosci., April 18, 2007; 27(16): 4472 - 4481. [Abstract] [Full Text] [PDF]
|
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 |
|
 C. W. Rinker-Schaeffer, J. P. O'Keefe, D. R. Welch, and D. Theodorescu Metastasis Suppressor Proteins: Discovery, Molecular Mechanisms, and Clinical Application. Clin. Cancer Res., July 1, 2006; 12(13): 3882 - 3889. [Full Text] [PDF]
|
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|
 |
|
 N Nathoo, A Chahlavi, G H Barnett, and S A Toms Pathobiology of brain metastases J. Clin. Pathol., March 1, 2005; 58(3): 237 - 242. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Sato, N. Maehara, and M. Goggins Gene Expression Profiling of Tumor-Stromal Interactions between Pancreatic Cancer Cells and Stromal Fibroblasts Cancer Res., October 1, 2004; 64(19): 6950 - 6956. [Abstract] [Full Text] [PDF]
|
|
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 |
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 P. S. Steeg PERSPECTIVES ON CLASSIC ARTICLES: Metastasis Suppressor Genes J Natl Cancer Inst, March 17, 2004; 96(6): E4 - E4. [Full Text]
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V.-M. Wasenius, S. Hemmer, E. Kettunen, S. Knuutila, K. Franssila, and H. Joensuu Hepatocyte Growth Factor Receptor, Matrix Metalloproteinase-11, Tissue Inhibitor of Metalloproteinase-1, and Fibronectin Are Up-Regulated in Papillary Thyroid Carcinoma: A cDNA and Tissue Microarray Study Clin. Cancer Res., January 1, 2003; 9(1): 68 - 75. [Abstract] [Full Text] [PDF]
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 H. Kitamura, K. Okita, D. Fujikura, K. Mori, T. Iwanaga, and M. Saito Induction of Src-suppressed C Kinase Substrate (SSeCKS) in Vascular Endothelial Cells by Bacterial Lipopolysaccharide J. Histochem. Cytochem., February 1, 2002; 50(2): 245 - 256. [Abstract] [Full Text] [PDF]
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identifies an aberrant 
) compared with lung metastases (•). Results compare two independent experiments of MLL[tet/vector] (clones V-2 and V-7) in mice receiving no tet to "SSeCKS," MLL[tet/SSeCKS] clones 2-6 and 7-2, in mice receiving tet (+ tet) or regular water (- tet). C, eighteen-day tumors from mice in the experiment in A were excised and analyzed by Western blotting for SSeCKS. Note the loss of tet-regulated SSeCKS expression in MLL[tet/SSeCKS] clone 2-6 from mice initially fed tet and then fed water lacking tet (+/-), compared with 2-6 cells grown in culture. D, monolayer wounding assay showing no effect of SSeCKS expression on cell motility (clone 2-6) compared with vector (v-2) controls.