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Spot 14: A Marker of Aggressive Breast Cancer and a Potential Therapeutic Target

Departments of Medicine (W.B.K., J.L.Q., C.R.-J., J.T.M.) and Pathology (W.A.W.) and the Norris Cotton Cancer Center (W.B.K., J.L.Q., W.A.W., C.R.-J.), Dartmouth Medical School, Lebanon, New Hampshire 03756; and Department of Pathology (J.T.M.), Walter Reed Army Medical Center, Silver Spring, Maryland 20910

Address all correspondence and requests for reprints to: William B. Kinlaw, M.D., Dartmouth Medical School, Norris Cotton Cancer Center, 606 Rubin Building, 1 Medical Center Drive, Lebanon, New Hampshire 03756. E-mail: william.kinlaw{at}hitchcock.org.

	Abstract

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 
Spot 14 (S14) is a nuclear protein that communicates the status of dietary fuels and fuel-related hormones to genes required for long-chain fatty acid synthesis. In mammary gland, S14 is important for both epithelial proliferation and milk fat production. The S14 gene is amplified in some breast cancers and is strongly expressed in most. High expression of S14 in primary invasive breast cancer is conspicuously predictive of recurrence. S14 mediates the induction of lipogenesis by progestin in breast cancer cells and accelerates their growth. Conversely, S14 knockdown impairs de novo lipid synthesis and causes apoptosis. We found that breast cancer cells do not express lipoprotein lipase (LPL) and hypothesize that they do not have access to circulating lipids unless the local environment supplies it. This may explain why primary breast cancers with low S14 do not survive transit from the LPL-rich mammary fat pad to areas devoid of LPL, such as lymph nodes, and thus do not appear as distant metastases. Thus, S14 is a marker for aggressive breast cancer and a potential target as well. Future effort will center on validation of S14 as a therapeutic target and producing antagonists of its action.

	Introduction

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 
TUMOR CELLS EXHIBIT striking metabolic peculiarities (for review, see Ref. 1). Indeed, avid glucose uptake is such a predictable attribute of cancer that the accumulation of a labeled glucose analog is used clinically to localize tumors by positron emission tomography. Despite this, tumor metabolism has not received a great deal of investigative attention as a target for anticancer therapy. One key pathway for the disposition of glucose in tumors is long-chain fatty acid synthesis. This overview summarizes the data supporting the existence of a lipogenic tumor phenotype and focuses on the Spot 14 (S14, THRSP) gene as a key component of the lipogenic program in benign and malignant mammary epithelial cells.

	What Is S14?

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 
S14 was initially noticed as an in vitro-translated protein spot on two-dimensional electrophoresis that was rapidly induced by thyroid hormone in rat liver (2). Further study showed it to be abundant in tissues synthesizing fatty acids for use as a fuel (lactating mammary, adipose, liver) and that the gene is strongly activated by glucose metabolism. S14 is an acidic protein of approximately 16 kD with three domains that are conserved from the ancestral S14-related peptide (known as Strait11499, Mig12, or S14-related protein). The only recognizable motif is a C-terminal coiled-coil domain that facilitates the assembly of high-affinity multimers (3). Immunohistochemical analysis of rat liver, human mammary gland, and breast cancer localizes S14 primarily to the nucleus (4, 5, 6). Studies using antisense to knock down S14 expression in hepatocytes support the idea that S14 signals for expression of genes coding fatty acid-synthesizing enzymes when the dietary and hormonal milieux are propitious (7, 9).

	S14 in Mammary Physiology

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 
Using immunohistochemistry, we find the temporal and spatial pattern of S14 expression in mammary epithelial cells to be identical to that of fatty acid synthase (FAS) during pregnancy, lactation, and involution in the mouse (Fig. 1). Surprisingly, S14 and FAS are induced in early pregnancy (5 d), long before the onset of milk production (10).

View larger version (97K): [in this window] [in a new window]   FIG. 1. Immunohistochemistry of S14 and FAS during postnatal development of mouse mammary gland. Formaldehyde-fixed, paraffin-embedded mouse tissues were stained for S14 (as in Ref. 9 ) or FAS using an affinity-purified rabbit antihuman FAS IgG preparation (3 µg/ml; Immuno-Biological Laboratories, Gunma, Japan). Detection was by the biotin-streptavidin-amplified system. Slides were counterstained with hematoxylin (original magnification x20). S14 and FAS occur in adipocytes in the virgin adult mouse mammary fat pad, but ducts exhibit little expression. By pregnancy d 5, long before milk production, proliferating epithelial buds invade the fat pad, and intense expression of S14 and FAS is evident. >, Proliferating epithelial foci. At peak lactation (d 20), the fat pad is largely replaced with highly lipogenic lobuloalveolar units. Note that epithelial cells contain large lipid droplets, and the staining in adjacent adipocytes appears reduced for both S14 and FAS, consistent with the reported down-regulation of adipose lipogenesis at that stage (10 ). Two days after removal of suckling pups, apoptotic debris from involuting lobuloalveoli occupies the ducts and is immunoreactive for S14 and FAS. Substantial remodeling is apparent by the 5th day of involution, with reappearance of fat pad adipocytes that express both S14 and FAS.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Lipogenic T47D breast cancer and HC11 pregnant mouse mammary epithelial cells, but not nonlipogenic MCF10a cells, depend on fatty acid synthesis for growth. Cells were grown for 4 d in 10 µg/ml cerulenin or vehicle and assessed for viability in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (six wells/group; mean ± SEM; *, P < 0.05).

	Breast Cancers and Other Common Tumors Are Lipogenic

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

In addition to providing a marker for aggressive cancer, the fatty acid synthetic pathway and its regulatory apparatus present a novel array of potential therapeutic targets. This has been shown in experiments using pharmacological inhibitors of FAS enzyme activity, including cerulenin and its derivatives, as well as Orlistat, a drug approved by the Food and Drug Administration for the treatment of obesity. Inhibition of FAS with cerulenin causes apoptosis of lipogenic breast cancer cells (19) and inhibits growth of xenografts of human ovarian cancer cells in nude mice (17). Notably, cerulenin exerted a striking chemopreventive effect in a transgenic mouse model of Her2/neu-induced breast cancer, with delayed tumor appearance and actual prevention in some animals (20). Similarly, Orlistat shows activity against lipogenic prostate cancer xenografts in immunodeficient mice (21). In addition to inhibition of FAS activity, this compound also antagonizes lipoprotein lipase (LPL) (22), an enzyme that may be relevant to metastasis (discussed below).

The mechanism underlying apoptosis induced by FAS inhibition is not clear. On the one hand, Pizer et al. (23) concluded that accumulation of malonyl-coenzyme A, the precursor for FAS, is cytotoxic. Alternatively, lipogenic prostate cancer cells were rescued from Orlistat-induced apoptosis by provision of fatty acids, suggesting that deficient FAS product is important (21). Despite this mechanistic ambiguity, it is clear that interruption of fatty acid synthesis exerts a strong antitumor effect on lipogenic cancer cells.

	S14 in Human Breast Tumors

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 
Amplification of the S14 gene
The S14 gene resides on the cancer amplicon at 11q13 (24), a large region that is amplified in approximately 20% of breast cancers and also contains the cyclin D1 locus (25). Cyclin D1 is a mammary oncogene in humans and transgenic mice (26, 27). S14 gene amplification and concordant overexpression of lipogenic enzymes and S14 in breast cancers prompted our hypothesis that S14 acts to promote a virulent, lipogenic phenotype (5).

Breast tumors with high S14 content are aggressive
We generated a monoclonal antibody directed at human S14 (28) and used it to analyze 131 cases of breast cancer by immunohistochemistry (6). The frequency of S14 overexpression did not differ between cases of ductal carcinoma in situ and invasive disease, indicating that overexpression is not acquired during tumor progression. The level of S14 expression correlated with morphological indices of tumor virulence in both ductal carcinoma in situ and invasive cases. Most importantly, there was a striking association of high S14 content with reduced disease-free survival in invasive primary cancers (Fig. 3). Indeed, no tumor with a low S14 score recurred on prolonged follow-up, irrespective of lymph node status at initial surgery (6).

View larger version (12K): [in this window] [in a new window]   FIG. 3. Disease-free survival (Kaplan-Meier) in 88 patients with invasive breast cancer segregated by S14 score and nodal metastasis. No tumor with a low S14 score recurred, whether lymph nodes were involved (n = 10) or not (n = 11) (upper tracings, superimposed). One patient of 23 with high S14 and negative nodes recurred at approximately 2000 d. All other recurrences were in the high-S14, positive nodal metastasis group (14 of 44 recurred; lower tracing). Log rank, P < 0.0001. [Reproduced with permission from W. Wells, G. Schwartz, P. Morganelli, B. Cole, J. Chambers, and W. B. Kinlaw: Breast Cancer Res Treat 98:231–240, 2006 (6 ). © Springer Publishing.]

The conundrum of S14 and breast cancer cDNA microarrays
Recent publications recognize types of breast cancer by consistent molecular signatures on cDNA microarrays. Arrays used in initial studies did not contain S14 (30). Recently, however, Perou et al. studied 147 cases using a 24,000-gene array that contains S14 (S14 homolog rat). Dr. Perou has kindly made the data available at https://genome.unc.edu/pubsup/breastGEO/, allowing us to perform a cluster analysis of the unfiltered data using Java TreeView software. S14 expression clustered with a group of 29 genes, including several that are readily identifiable as adipocyte-specific, including perilipin, hormone-sensitive lipase, adipocyte fatty acid binding protein 4, adiponectin, and LPL. This strongly suggests that the probe used on the arrays contained mRNA from adipocytes of the mammary fat pad. The coregulation of some genes in this cluster may therefore reflect the variable admixture of adipocyte and tumor mRNA.

To examine this idea experimentally, we assessed a panel of cell lines for expression of LPL mRNA by RT-PCR (Fig. 4). Human preadipocyte and adipocyte mRNA served as (–) and (+) controls, respectively. We observed no expression of LPL mRNA in a variety of lipogenic breast cancer cell lines (ZR75.1, SKBR3, MCF7, T47D +/– progestin), or mammary epithelium (MCF10a). Hepatoma (HepG2) and embryonic kidney (HEK293) likewise do not express it. Importantly, a cervical carcinoma line (HeLa) that expresses negligible levels of FAS (31) does express LPL mRNA. This suggests that expression of LPL may confer an advantage to tumor cells that have a low capacity for de novo lipogenesis. Overall, these observations confirm the suspicion that the LPL mRNA detected on breast cancer microarrays is of adipose origin and support the conclusion that microarray data for genes that are expressed in both breast tumors and adipocytes, such as S14, FAS, and PPAR-, are not interpretable unless measures such as laser-assisted microdissection are used to acquire tumor samples for probe preparation. Moreover, this explains why immunohistochemistry was required to reveal the association of S14 expression and breast cancer virulence.

View larger version (36K): [in this window] [in a new window]   FIG. 4. RT-PCR analysis of LPL mRNA expression in cell lines and lymphoid tissue. A, LPL signal is only in adipocyte and HeLa cells, a line with negligible de novo lipogenesis. R1881 indicates exposure to the progestin (10 nM) x 48 h. B, Samples rerun to verify that LPL products from HeLa cell and adipocyte are of the expected size. C, mRNA from normal spleen yields no LPL signal; cyclo, cyclophilin signal amplified from the same samples.

As detailed above, lipogenic breast cancer cells depend upon a supply of fatty acids for growth and survival. We propose a working model (Fig. 5) in which only primary breast tumors with brisk de novo lipogenesis are equipped to survive metastatic transit through sites, such as local lymph nodes, that are not replete with LPL. Our observations that only breast cancers with high S14 expression recur on follow-up (Fig. 3) and that lymphoid tissue does not express LPL (Fig. 4C) are consistent with this formulation. The model also predicts that metastasis to distant sites replete with LPL, such as lung (33), fatty bone marrow, or liver, may be favored, although other factors are obviously involved. Moreover, interplay between tumor cell metabolism and the microenvironment could provide insight into interactions of diet and metastasis. Chlebowski et al. (34) reported that strict reduction of dietary fat yielded a substantial reduction of breast cancer recurrence, whereas a subsequent study found no influence of dietary fat content on breast cancer incidence (35). These clinical data therefore also support the concept that fatty acids are not carcinogenic per se but do act to fuel metastases.

View larger version (41K): [in this window] [in a new window]   FIG. 5. Hypothesized interaction of breast tumor lipogenesis and the availability of LPL in the breast cancer cell metabolic microenvironment. Box upper right, Hydrolysis of TG by LPL provides soluble fatty acids and glycerol. Top, Primary breast cancer may use fatty acids provided either by hydrolysis of circulating TG by adipose-derived LPL or from de novo synthesis from glucose in the tumor cell. Middle, After metastasis to local lymph node, which provides no LPL, only tumor cells with brisk de novo lipogenesis survive because access to exogenous fatty acids is shut off. Only lipogenic tumors survive to form distant metastases (lower panel). Distant sites that are replete with LPL may be metabolically hospitable to metastases, and abundant circulating substrate supplied by a high-fat diet may also provide fuel for metastases in sites supplied with LPL.

	S14 Is a Key Component of the Lipogenic Phenotype in Breast Cancer Cells

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

View larger version (21K): [in this window] [in a new window]   FIG. 6. Coordinate stimulation of FAS and S14 mRNAs by HER2/neu signaling. Total RNA harvested from mammary epithelial cells stably transfected with control or a HER2 expression construct was used as template for real time RT-PCR. Primers for FAS (left), S14 (right), and cyclophilin (internal control) were used (for details, see Ref. 28 ). Standards spanning the linear range of amplification were used for quantitation. Data (six wells/treatment) are mean ± SE; *, P < 0.05 by Student’s t test. Cells kindly supplied by Stephen Ethier (University of Michigan, Ann Arbor, MI).

View larger version (25K): [in this window] [in a new window]   FIG. 7. Synergistic effects of SREBP-1c and progestin on S14 mRNA and protein expression. A, S14 mRNA-T47D cells were exposed to adenoviruses expressing: 1) ß-galactosidase control (Ad-ß-gal), 2) dominant-negative SREBP-1c (SREBP-1c-DN), or 3) constitutively active SREBP-1c (SREBP-1c). R1881 or vehicle were added to media postinfection, and RNA was harvested 40 h later. Data are real-time RT-PCR signals (six per group, mean ± SEM) corrected for cyclophilin mRNA. *, Difference between hormone (–) and (+) treatments (P < 0.05). B, S14 protein-Western analysis of T47D cells treated with R1881, adenovirus harboring a constitutively active SREBP-1c gene mutant, or both. Cells were grown in stripped serum x 48 h, infected with Ad-SREBP-1c or not, and exposed to 10 nM R881 or vehicle for 40 h. Lysates were analyzed by Western blot. C and D, Same experiment analyzed for FAS mRNA and protein. [Reproduced with permission from P. Martel, C. Bingham, C. McGraw, C. Baker, P. Morganelli, M. Meng, J. M. Armstrong, J. Moncur, W. B. Kinlaw: Exp Cell Res 312:278–288, 2005 (28 ). © Elsevier Publishing.]

The synergistic induction of S14 and FAS gene expression that we observe in breast cancer cells stimulated with progestin and active SREBP-1c similarly cannot be explained by enhanced SREBP-1c processing because the mutant that we delivered is fully processed and requires no activation. Carbohydrate response element-binding protein, a transcription factor that communicates the rate of glucose metabolism to lipogenic genes, is required, along with SREBP-1c, to elicit S14 and FAS gene expression in liver (47). We demonstrated carbohydrate response element-binding protein expression in lipogenic cancer cells (28) and considered it as a candidate progestin-induced lipogenic amplification signal in breast cancer. Levels of glucose that maximally activate lipogenesis-related genes in hepatocytes, however, do not affect S14 gene expression in breast cancer cells. The mediator of the synergy thus remains elusive.

S14 and breast cancer cell growth
Gene amplification suggested that S14 confers a growth advantage to breast cancer (5). Experiments using both S14 overexpression and knockdown support this idea. S14 overexpression in lipogenic human breast cancer cells (T47D, MCF7) accelerated growth, whereas knockdown with short inhibitory RNA or antisense abrogated growth and fostered apoptosis (28). Moreover, inhibition of S14 induction impaired progestin-induced lipogenesis and FAS gene expression. Thus, S14 is an intermediary of the lipogenic actions of progestin in breast cancer cells, analogous to its transduction of thyroid hormone signaling for lipogenesis in liver (8). Overall, targeting of S14 inhibits cell growth and survival, as occurs if FAS activity is inhibited pharmacologically.

Paradoxically, Sanchez-Rodriguez (48) reported that enforced S14 overexpression in breast cancer cells exerted a tumor suppressor-like effect. This finding is not consonant with those from other cell culture systems or the observed apoptotic effect of S14 knockdown discussed above. Most importantly, observations in actual human breast tumors strongly suggest the opposite conclusion. It appears that the tissue culture system employed by Sanchez et al. may not faithfully model human breast cancer. Cell culture models employing lower levels of S14 overexpression will be instructive in this regard.

	Mouse Models to Elucidate the Role of S14 in Breast Cancer

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 
Inconsistent S14 knockout mouse phenotypes
Our attempt to produce a S14 knockout mouse revealed the homozygous mutation to be preimplantation embryonic lethal (summarized in Fig. 8). We removed the proximal promoter and entire coding region of the S14 gene. Colleagues in Minnesota (49) produced a less disruptive S14 knockout, with the gene promoter and part of the N-terminal coding sequence intact, and obtained viable mice with a phenotype of deficient milk fat production. This mouse has been quite instructive, as detailed by Dr. Mariash in an accompanying paper (58). The different phenotypes, however, remain unexplained. They could be related to strain differences or to the specific mutations employed. It is also possible that the Minnesota phenotype is partially compensated by expression of truncated S14 and/or expression of S14-related protein elicited during embryonic development. The absence of S14 in all tissues could produce a complex phenotype given the multiple hormonal and metabolic interactions between S14-expressing tissues (liver, adipose, mammary) (10, 50). Indeed, the Minnesota mouse paradoxically exhibited increased lipogenesis in the liver (51), in stark contrast to effects of antisense-mediated S14 knockdown in hepatocytes (7). In view of the breast cancer metastasis model shown in Fig. 5, enhanced secretion of TG-rich lipoproteins by the liver could confound breast cancer experiments.

View larger version (41K): [in this window] [in a new window]   FIG. 8. Constitutive knockout of the S14 gene. A, Diagram of the wild-type (upper) and knockout (lower) alleles. The SalI-XhoI fragment of the S14 gene from an approximately 100-kb P1 clone (Genome Systems, Wilmington, DE) containing the 129 mouse S14 gene was cloned into vector pSL1180. The ClaI-EcoRI fragment containing the proximal promoter and entire coding sequence was replaced with neo and transfected into ES129 cells. B, Six of 123 ES129 clones showed targeted insertion, and one was used to generate mice with germline transmission, shown on Southern blot of SpeI-cut tail DNA using the probe shown in A. Left lane, Tail DNA from an f1 generation mouse with germline transmission of the knockout allele. Middle lane, Original P1 bacteriophage containing the wild-type 129 S14 genomic clone. Right lane, Wild-type mouse tail DNA. C, Compiled genotypes of live-born mice resulting from crossing S14 +/– mice. Analysis of midgestation embryos and preimplantation blastocysts (by PCR) similarly revealed no homozygote knockouts. At each developmental stage, the distribution of genotypes approximated that expected for a lethal homozygous mutation (wild-type:heterozygote:homozygote, approximately 1:2:0).

Prospects for targeting S14
Therapeutic inhibition of S14 will be desirable if GEM models further validate S14 as a target in lipogenic breast cancer. Although most protein structure-based, rationally designed drugs interfere with enzyme active sites or kinase/phosphatases (54), protein-protein interactions have also been successfully targeted by small molecules. IL-2/IL-2R (55), the SH3 domain (56), and vascular endothelial growth factor (57) are a few examples. Precise identification of residues critical for stabilization of the S14 multimer (3) will facilitate the design of soluble molecules to prevent its assembly. Additional potentially druggable surfaces of S14 may be identified for interactions between S14 and other peptides. Such attempts will rest on x-ray crystallographic structure determination in the future.

	Acknowledgments

 
We thank Drs. Steven Fiering, Murray Korc, and Mark Schneider for critically reading the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grant RO1 DK 058961 (to W.B.K.), by Department of Defense Grant DAMD 17-03-1-0544 (to W.B.K.), and by Department of Defense Medical Research and Materiel Command/U.S. Army Medical Research Acquisition Activity CBCP Grant W81XWH-05-2-0053 (P.I. Col. Craig D. Shriver, to J.T.M.).

Present address for C.R.-J.: The University of Pennsylvania School of Medicine, 295 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104.

The views expressed in this article are those of the authors and do not reflect the official policy of the Departments of the Army or Defense or the U.S. Government.

All authors have nothing to disclose.

First Published Online June 29, 2006

Abbreviations: FAS, Fatty acid synthase; GEM, genetically engineered mouse; LPL, lipoprotein lipase; SREBP-1c, sterol response element-binding protein-1c; TG, triglyceride.

Received April 11, 2006.

Accepted for publication May 23, 2006.

	References

Top Abstract Introduction What Is S14? S14 in Mammary Physiology Breast Cancers and Other... S14 in Human Breast... S14 Is a Key... Mouse Models to Elucidate... References

 

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