B-2-Microglobulin Is an Androgen-Regulated Secreted Protein Elevated in Serum of Patients with Advanced Prostate Cancer MitchellGross,IrinaTop,IsettLaux,JonathanKatz,JohnCurran,CharlesTindell,andDavidAgus Abstract Purpose: Abetterunderstandingofsecretedproteinsmayleadtothediscoveryofnewbio- markers,which,alongwithprostate-specificantigen(PSA),maybeusefulinthediagnosisand treatmentofprostatecancerpatients. Experimental Design: ConditionedmediumwascollectedfromLNCaPcellsfollowingstimu- lationwithmethyltrienolone(R1881),17h -estradiol(estradiol),orinterleukin-6andanalyzedfor differentialproteinexpressionwithsurface-enhancedlaserdesorption/ionization-timeofflight massspectrometry.Quantitativereversetranscription-PCR,immunoblots,andELISAwereused tomeasure h-2-microglobulin(B2M)messageandproteinlevelsincells,conditionedmedium, andserum. Results: Surface-enhancedlaserdesorption/ionization-timeofflightrevealedthatmanypeaks wereinducedorrepressedfollowingstimulationwithR1881orestradiol.Apeakofinterestcen- teredat11.8kDawaschosenforadditionalanalysis.Immunodepletionidentifiedthepeakofinter- estasB2M.Reversetranscription-PCRandimmunoblotsconfirmedthatPSAandB2Mwere inducedbyR1881.However,unlikePSA,B2Mwasnotincreasedonstimulationwithestradiol orinterleukin-6.HumanB2Misidentifiedintheserumofmicebearinghumanprostatecancer xenograft.B2Misexpressedinhumanprostatecancercelllinesandtissues.SerumB2Mlevels areelevatedinpatientswithmetastatic,androgen-independentprostatecancer. Conclusions: B2Misasecretedproteinexpressedinprostatecancer,whichismorespecificfor androgenstimulationthanPSAundertheconditionstested.Additionalstudiesarewarrantedto exploreifB2Misasusefulmarkerforprostatecancer.Identificationofproteinssecretedfrom cancercellsinpreclinicalmodelsmaybeausefulstrategyforbiomarkerdiscovery. Most serum biomarkers routinely used in the diagnosis and treatment of cancer are cell surface or secreted proteins expressed by both benign and malignant tissue. As these proteins are generally found at low abundance, highly sensitive tests (such as ELISAs) are required to measure biomarker levels in peripheral blood. However, the limited sensitivity and specificity of these tests often restricts their clinical utility. For example, prostate-specific antigen (PSA) is an androgen- regulated secreted protein that is expressed by both normal and malignant prostate epithelial cells, which is widely used as a serum marker for prostate cancer (1). Occult prostate cancers may be found in f25% of patients with PSA levels in the generally accepted reference range (V4 ng/mL; ref. 2). Most, but not all, patients with metastatic prostate cancer show elevated levels of PSA, which is often used as a marker to guide treatment decisions. However, the validity of PSA as a surrogate to predict overall survival in advanced prostate cancer remains controversial (3, 4). Therefore, additional biomarkers may be useful in the diagnosis and treatment of patients with prostate cancer. Serum-based protein profiling with mass spectrometry (MS) is a promising technology to improve the diagnosis and treatment for cancer patients (5). It is generally believed that changes in serum proteins reflect the complex interplay between malignant cells and host tissues and that protein profiles may elucidate molecular processes that will improve the care of patients with cancer. Surface-enhanced laser desorption/ionization-time of flight MS (SELDI-TOF MS) was one of the first MS-based profiling technologies applied to study serum from patients with cancer (6–8). SELDI-TOF uses substrate-coated chips to differentially retain species from complex protein mixtures based on chromatographic proper- ties, such as hydrophobicity and binding to anionic, cationic, or metal affinity surfaces. The retained species are then analyzed via TOF MS. Although initial experiments have confirmed the reproducibility of SELDI-TOF, a major pitfall of this approach is the relatively low resolution, as a typical Human Cancer Biology Authors’Affiliation: LouisWarschawProstateCancerCenter,Cedars-Sinai MedicalCenter,LosAngeles,California Received5/15/06;revised11/20/06;accepted1/8/07. Grant support: PhaseOneFoundation(M.GrossandD.Agus)andNIHgrant DK64379(M.Gross). Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.Thisarticlemustthereforebeherebymarked advertisement inaccordance with18U.S.C.Section1734solelytoindicatethisfact. Requests for reprints: MitchellGross,LouisWarschawProstateCancerCenter, Cedars-SinaiMedicalCenter,8631WestThirdStreet,Suite1001E,LosAngeles, CA90048.Phone:310-423-7600;Fax:310-423-1998;E-mail:mitchell.gross@ cshs.org. F 2007AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-06-1156 www.aacrjournals.org Clin Cancer Res 2007;13(7) April1, 2007 1979 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/13/7/1979/1974578/1979.pdf by guest on 19 May 2023