Vol. 10, 101–112, January 1, 2004 Clinical Cancer Research 101 Genomic Organization, Incidence, and Localization of the SPAN-X Family of Cancer-Testis Antigens in Melanoma Tumors and Cell Lines 1 1 V. Anne Westbrook, Pamela D. Schoppee, immunoreactive proteins migrated between Mr 15,000 and 1 1 Alan B. Diekman, Kenneth L. Klotz, Mr 20,000 similar to those observed in spermatozoa. Immu- Margaretta Allietta,1 Kevin T. Hogan,2 noperoxidase labeling of melanoma cells and tissue sections 2 3 demonstrated SPAN-X protein localization in the nucleus, Craig L. Slingluff, James W. Patterson, cytoplasm, or both. Ultrastructurally, in melanoma cells 3 4 Henry F. Frierson, William P. Irvin, Jr., with nuclear SPAN-X, the protein was associated with the Charles J. Flickinger,1 Michael A. Coppola,1 and nuclear envelope, a localization similar to that observed in John C. Herr1 human spermatids and spermatozoa. Significantly, the inci- 1Departments of Cell Biology, 2Surgery, 3Pathology, and 4Obstetrics dence of SPAN-X-positive immunostaining was greatest in and Gynecology, Center for Research in Contraceptive and the more aggressive skin tumors, particularly in distant, Reproductive Health, University of Virginia, Charlottesville, Virginia nonlymphatic metastatic melanomas. Conclusions: The data herein suggest that the SPAN-X protein may be a useful target in cancer immunotherapy. ABSTRACT Purpose: Members of the SPAN-X (sperm protein as- INTRODUCTION sociated with the nucleus mapped to the X chromosome) Molecular characterization of human tumor antigens rec- family of cancer-testis antigens are promising targets for ognized by the cellular or humoral immune systems of cancer tumor immunotherapy because they are normally expressed patients has generated new prospects for the development of exclusively during spermiogenesis on the adluminal side of cancer vaccines (1–3). The list of human cancer antigens de- the blood-testis barrier, an immune privileged compart- fined to date falls into three major categories: (a) differentiation ment. antigens; (b) mutational antigens; and (c) viral antigens. Cancer- Experimental Design and Results: This study analyzed testis (CT) antigens are a distinct class of differentiation anti- the human SPANX genomic organization, as well as SPAN-X gens that are viewed as attractive candidates for cancer vaccines, mRNA and protein expression in somatic and cancer cells. because the expression of CT antigens in normal tissues seems The SPANX family consists of five genes, one of which is to be restricted to the testis. Malignant transformation is asso- duplicated, all located in a gene cluster at Xq27.1. From the ciated with activation or derepression of these normally silent centromere, the arrangement of the five SPANX genes genes resulting in CT antigen gene expression in a variable mapped on one contiguous sequence is SPANXB, -C, -A1, portion of many human tumors. No tumor specimen or cell line -A2, and –D. Reverse transcription-PCR analyses demon- screened to date has been found to express all of the defined CT strated expression of SPAN-X mRNA in melanoma and antigens (1–3). ovarian cell lines, and virtual Northern analysis established CT antigens represent good targets for immunotherapy, SPANX gene expression in numerous cancer cell lines. Im- because they are widely distributed in a number of tumors and munoblot analysis using polyclonal antisera raised against are present primarily in normal testes. The testis is considered an recombinant SPAN-X confirmed the translation of SPAN-X immune privileged site due to the presence of the blood-testis proteins in melanoma and ovarian tumor cell lines. The barrier, which inhibits contact between differentiating germ-line and immune cells, and to the apparent lack of HLA class I expression on the surface of germ cells (4). These considerations suggest that only cancerous cells will be targeted by CTLs Received 4/21/03; revised 8/11/03; accepted 8/14/03. during CT antigen immunotherapy. Unfortunately, many of the Grant support: U54HD29099 and D43TW/HD00654 (J.C.H.) from the Fogarty International Center, a pilot project grant funded through the CT antigens studied to date are first expressed in spermatogonia National Cancer Institute Cancer Center, Support Grant to the Univer- or spermatocytes located in the basal compartment of the sem- sity of Virginia Cancer Center (M. A. C.), and a grant from the Cancer iniferous epithelium, the nonprotected side of the blood-testis Antigen Discovery Collaborative of the Cancer Research Institute and barrier. Thus, targeting of these antigens may reduce or elimi- the Ludwig Institute for Cancer Research (M. A. C.). nate spermatogenesis and compromise the subsequent fertility The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked of the patient. In contrast, the identification and validation of CT advertisement in accordance with 18 U.S.C. Section 1734 solely to antigens, which lie on the adluminal (protected) side of the indicate this fact. blood-testis barrier, may reduce the chances of inducing auto- Requests for reprints: John C. Herr, Department of Cell Biology, immune orchitis (5, 6) and permanent injury to the testes as a Center for Research in Contraceptive and Reproductive Health, Univer- sity of Virginia, P. O. Box 800732, 1300 Jefferson Park Avenue, result of cancer immunotherapy. Furthermore, polyvalent CT Charlottesville, Virginia 22908. Phone: (434) 924-2007; Fax: (804) 982- antigen vaccines comprised of a mixture of immunogens repre- 3912; E-mail: [email protected]. sent an important strategy for obtaining adequate therapeutic Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2004 American Association for Cancer Research. 102 Characterization of SPAN-X in Melanomas responses given the variable incidence of CT antigen expression reverse transcriptase kit (Qiagen, Valencia, CA) according to in tumors. For these reasons, it is important to identify and the protocol provided therein. An RNase inhibitor (10 units) was ␮ characterize new CT antigens, particularly those that are ex- added to the reaction and oligo(dT)18 (1 M) was used as the pressed postmeiotically and are immunogenic in humans. primer. The reactions were incubated at 37°C for 60 min and We have characterized a testis-specific protein designated then at 95°C for 5 min to inactivate the enzyme. Subsequently, SPAN-X (sperm protein associated with the nucleus on the X PCR was performed on 2 ␮l of cDNA using the HotStarTaq chromosome; Refs.7–9). Ultrastructurally, the proteins are as- DNA polymerase and master mix kit (Qiagen). The reaction sociated with the nuclear envelope of ejaculated spermatozoa. In contained specific primers published previously (10) with mod- transfected mammalian CV1 cells the SPAN-Xa protein local- ifications. The forward primers were 5Ј-CTGCCRCWGACAT- ized to the nucleus, whereas SPAN-Xb localized to the cyto- TGAAGAA-3Ј (SPAN-XA, C and D) and 5Ј-CTACTGTA- plasm. SPAN-X mRNAs were expressed exclusively after mei- GATCGAAGAA-3Ј (SPAN-XB) mixed at a 3:1 molar ratio; the osis in the testis in round and elongating spermatids (7). The reverse primer was 5Ј-TCYATGAATTCCTCCTCCTC-3Ј. Am- human SPANX gene was mapped to chromosome Xq27.1. The plification was performed over 30 cycles at 94°C for 1 min, expression of SPAN-X, an X-linked gene product, exclusively 58°C for 30 s, and 72°C for 30 s. in haploid spermatids leads to interesting questions regarding Western Blot Analysis. For SDS-PAGE, snap-frozen the transcription of sex-linked genes in a precise temporal and cell pellets were resuspended in 2ϫ Laemmli sample buffer (7), spatial sequence during spermiogenesis and transcriptional reg- vortexed, boiled for 10 min, and centrifuged at 16,000 ϫ g for ulation of this highly regulated gene during tumorigenesis. 15 min. The supernatants were transferred to clean tubes and SPAN-Xc, also designated CTp11, was identified previ- diluted with an equal volume of distilled water. One dimen- ously as a CT gene by comparing mRNA expression between a sional SDS-PAGE was performed on 15% acrylamide gels 6 human parental melanoma cell line and its metastatic variant using 2 ϫ 10 cell equivalents of protein extract per lane and the using mRNA differential display (10). A recent study demon- polypeptides transferred onto nitrocellulose (14). A 1% SDS strated the expression of SPAN-Xb in myeloma and other he- extract of sperm (40 ␮g per lane) was included as a positive matological cancers (11). Significantly, high titer antibodies control for the SPAN-X protein. All of the samples were ob- against SPAN-X were present in sera of these patients, but tained with informed consent using forms approved by the absent in healthy individuals, confirming the immunogenicity of University of Virginia Human Investigation Committee. After the SPAN-X differentiation antigen. In the present study, we transfer, the blots were washed with PBS [150 mM NaCl and 10 examined the incidence of SPAN-X expression in several tumor mM sodium phosphate (pH 7.4)], incubated with blocking buffer types and placed particular emphasis on SPAN-X protein ex- (PBS, 0.05% Tween 20, and 5.0% nonfat dry milk), and then pression in melanomas. We determined that SPAN-X protein, a with guinea pig anti-SPAN-X antiserum diluted 1:1000 in postmeiotic, germ cell marker, was present in more than half the blocking buffer. SPAN-X antiserum was produced by immuniz- melanoma tumors examined and that its