Characterization and Clinical Relevance of ALDHbright Populations in Prostate Cancer

PURPOSE High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDH(bright)) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDH(bright) populations in clinical prostate cancer specimens. EXPERIMENTAL DESIGN We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDH(bright) populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. RESULTS ALDH(bright) cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM((+)) and Trop2((+)) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. CONCLUSIONS Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance.

Characterization and optimization of antigen-specific T cell responses during ex vivo expansion of melanoma tumor-infiltrating lymphocytes

Treatment of metastatic melanoma with tumor reactive T cells (adoptive T cell therapy, ACT) is a promising approach associated with a high clinical response rate. However, further optimization of this treatment modality is required to increase the clinical response after this therapy. ACT in melanoma involves an initial phase (pre-REP) of tumor-infiltrating lymphocyte (TIL) expansion ex vivo from tumor isolates followed by a second phase, “rapid expansion protocol” (REP) generating the billions of cells used as the TIL infusion product. The main question addressed in this thesis was how the currently used REP affected the responsiveness of the CD8+ T cells to defined melanoma antigens. We hypothesized that the REP drives the TIL to further differentiate and become hyporesponsive to antigen restimulation, therefore, proper cytokine treatment or other ways to expand TIL is required to improve upon this outcome. We evaluated the response of CD8+ TIL to melanoma antigen restimulation using MART-1 peptide-pulsed mature DC in vitro. Post-REP TILs were mostly hypo-responsive with poor proliferation and higher apoptosis. Phenotypic analysis revealed that the expression of CD28 was significantly reduced in post-REP TILs. By sorting experiment and microarray analysis, we confirmed that the few CD28+ post-REP TILs had superior survival capacity and proliferated after restimulation. We then went on to investigate methods to maintain CD28 expression during the REP and improve TIL responsiveness. Firstly, IL-15 and IL-21 were found to synergize in maintaining TIL CD28 expression and antigenic responsiveness during REP. Secondly, we found IL-15 was superior as compared to IL-2 in supporting the long-term expansion of antigen-specific CD8+ TIL after restimulation. These results suggest that current expansion protocols used for adoptive T-cell therapy in melanoma yield largely hyporesponsive products containing CD8+ T cells unable to respond in vivo to re-stimulation with antigen. A modification of our current approaches by using IL-15+IL-21 as supporting cytokines in the REP, or/and administration of IL-15 instead of IL-2 after TIL infusion, may enhance the anti-tumor efficacy and long-term persistence of infused T cells in vivo.

Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

Characterization and function of murine Thy-1$\sp+$ dendritic epidermal cells

The skin immune system is believed to be a crucial site of contact between immunocompetent cells and invading organisms. A novel T cell component of murine epidermis is the Thy-1$\sp+$ dendritic epidermal cell (Tdec). To assess the immunocompetence of Tdec, the ability of Tdec to induce immune responses was tested. Tdec were unable to induce positive immune responses in three models of immunocompetence. Subsequent studies were designed to test the hypothesis that Tdec are involved in the down-regulation of cell-mediated immunity against cutaneous antigens. Cultured Tdec lines were conjugated in vitro with the hapten, fluorescein isothiocyanate (FITC). The intrafootpad (ifp.) or intravenous (i.v.) injection of FTIC-conjugated Tdec induced immunologic tolerance to subsequent epicutaneous sensitization with FITC. This induction of tolerance was antigen-specific, and injection of unconjugated Tdec had no effect on the contact hypersensitivity response to FITC. Tolerance was not H-2-restricted, since it could be induced in both syngeneic and allogeneic recipients of FITC-conjugated Tdec. No suppressive activity could be detected in lymphoid organs of animals tolerized by the ifp. injection of hapten-conjugated Tdec. In contrast, suppressor T cells were present in the spleens of mice injected i.v. with hapten-conjugated Tdec. These results indicate that Ts cells are not involved in the induction of tolerance by the ifp. injection of hapten-conjugated Tdec. To investigate the mechanism by which the ifp. injection of hapten-conjugated Tdec induced tolerance to contact sensitization, the activity of these cells was measured in vitro. The addition of hapten-conjugated Tdec inhibited the proliferation of Con A-stimulated lymphocytes. In addition, FITC-conjugated Tdec abrogated the proliferation of normal lymphocytes in response to FITC-labeled stimulator cells. These studies suggest that specific T cell-mediated immunity is the target of the inhibitory effect of Tdec in vitro. In summary, these results demonstrate that while Tdec are unable to induce positive immune responses, they can produce a state of specific immunologic tolerance when injected ifp. or i.v. These results also suggest that the induction of immunologic tolerance by hapten-conjugated Tdec may occur through the inactivation or elimination of activated T lymphocytes resulting in down-regulation of cell-mediated immunity against cutaneous antigens. ^

Isolation, characterization and transcriptional regulation of {\it Xenopus myod\/}

I have cloned cDNAs corresponding to two distinct genes, Xlmf1 and Xlmf25, which encode skeletal muscle-specific, transcriptional regulatory proteins. These proteins are members of the helix-loop-helix family of DNA binding factors, and are most homologous to MyoD1. These two genes have disparate temporal expression patterns during early embryogenesis; although, both transcripts are present exclusively in skeletal muscle of the adult. Xlmf1 is first detected 7 hours after fertilization, shortly after the midblastula transition. Xlmf25 is detected in maternal stores of mRNA, during early cleavage stages of the embryo and throughout later development. Both Xlmf1 and Xlmf25 transcripts are detected prior to the expression of other, previously characterized, muscle-specific genes. The ability of Xlmf1 and Xlmf25 to convert mouse 10T1/2 fibroblasts to a myogenic phenotype demonstrates their activity as myogenic regulatory factors. Additionally, Xlmf1 and Xlmf25 can directly transactivate a reporter gene linked to the muscle-specific, muscle creatine kinase (MCK) enhancer. The functional properties of Xlmf1 and Xlmf25 proteins were further explored by investigating their interactions with the binding site in the MCK enhancer. Analysis of dissociation rates revealed that Xlmf25-E12 dimers had a two-fold lower avidity for this site than did Xlmf1-E12 dimers. Clones containing genomic sequence of Xlmf1 and Xlmf25 have been isolated. Reporter gene constructs containing a lac-z gene driven by Xlmf1 regulatory sequences were analyzed by embryo injections and transfections into cultured muscle cells. Elements within $-$200 bp of the transcription start site can promote high levels of muscle specific expression. Embryo injections show that 3500 bp of upstream sequence is sufficient to drive somite specific expression. EMSAs and DNAse I footprint analysis has shown the discrete interaction of factors with several cis-elements within 200 bp of the transcription start site. Mutation of several of these elements shows a positive requirement for two CCAAT boxes and two E boxes. It is evident from the work performed with this promoter that Xlmf1 is tightly regulated during muscle cell differentiation. This is not surprising given the fact that its gene product is crucial to the determination of cell fate choices. ^

Characterization and subchromosomal location of a novel tumor suppressor gene on human chromosome 7

Alterations in oncogenes and tumor suppressor genes (TSGs) are considered to be critical steps in oncogenesis. Consistent deletions and loss of heterozygosity (LOH) of polymorphic markers in a determinate chromosomal fragment are known to be indicative of a closely mapping TSG. Deletion of the long arm of chromosome 7 (hchr 7) is a frequent trait in many kinds of human primary tumors. LOH was studied with an extensive set of markers on chromosome 7q in several types of human neoplasias (primary breast, prostate, colon, ovarian and head and neck carcinomas) to determine the location of a putative TSG. The extent of LOH varied depending the type of tumor studied but all the LOH curves we obtained had a peak at (C-A)$\sb{\rm n}$ microsatellite repeat D7S522 at 7q31.1 and showed a Gaussian distribution. The high incidence of LOH in all tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on the 7q31.1. To investigate whether the putative TSG is conserved in the syntenic mouse locus, we studied LOH of 30 markers along mouse chromosome 6 (mchr 6) in chemically induced squamous cell carcinomas (SCCs). Tumors were obtained from SENCAR and C57BL/6 x DBA/2 F1 females by a two-stage carcinogenesis protocol. The high incidence of LOH in the tumor types studied suggests that a TSG relevant to the development of epithelial cancers is present on mchr 6 A1. Since this segment is syntenic with the hchr 7q31, these data indicate that the putative TSG is conserved in both species. Functional evidence for the existence of a TSG in hchr 7 was obtained by microcell fusion transfer of a single hchr 7 into a murine SCC-derived cell line. Five out of seven hybrids had two to three-fold longer latency periods for in vivo tumorigenicity assays than parental cells. One of the unrepressed hybrids had a deletion in the introduced chromosome 7 involving q31.1-q31.3, confirming the LOH data. ^

Isolation, characterization and functional analysis of the {\it xnf7\/} gene from {\it Xenopus laevis\/}

A fundamental problem in developmental biology concerns the mechanisms involved in the establishment of the embryonic axis. We are studying Xenopus nuclear factor 7 (xnf7) which we believe to be involved in dorsal-ventral patterning in Xenopus laevis. Xnf7 is a maternal gene product that is retained in the cytoplasm during early embryogenesis until the mid-blastula transition (MBT) when it reenters the nuclei. It is a member of a novel zinc finger proteins, the B-box family, consisting mainly of transcription factors and protooncogenes.^ The xnf7 gene is reexpressed during embryogenesis at the gastrula-neurula stage of development, with its zygotic expression limited to the central nervous system (CNS). In this study we showed that there are two different cDNAs coding for xnf7, xnf7-O and xnf7-B. They differ by 39 amino acid changes scattered throughout the cDNA. The expression of both forms of xnf7 is limited primarily to the central nervous system (CNS) and dorsal axial structures during later stages of embryogenesis.^ In order to study the spatial and temporal regulation of the gene, we screened a Xenopus genomic library using part of xnf7 cDNA as a probe. A genomic clone corresponding to the xnf7-O type was isolated, its 5$\sp\prime$ putative regulatory region sequenced, and its transcriptional initiation site mapped. The putative promoter region contained binding sites for Sp1, E2F, USF, a Pu box and AP1. CAT/xnf7 fusion genes were constructed containing various 5$\sp\prime$ deleted regions of the xnf7 promoter linked to a CAT (Chloramphenicol Acetyl Transferase) reporter vector. These constructs were injected into Xenopus oocytes and embryos to study the regions of the xnf7 promoter responsible for basal, temporal and spatial regulation of the gene. The activity of the fusion genes was measured by the conversion of chloramphenicol to its acetylated forms, and the spatial distribution of the transcripts by whole mount in situ hybridization. We showed that the elements involved in basal regulation of xnf7 lie within 121 basepairs upstream of the transcriptional inititiation site. A DNase I footprint analysis performed using oocyte extract showed that a E2F and 2 Sp1 sites were protected. During development, the fusion genes were expressed following the MBT, in accordance with the timing of the endogenous xnf7 gene. Spatially, the expression of the fusion gene containing 421 basepairs of the promoter was localized to the dorsal region of the embryo in a pattern that was almost identical to that detected with the endogenous transcripts. Therefore, the elements involved in spatial and temporal regulation of the xnf7 gene during development were contained within 421 basepairs upstream of the transcriptional initiation site. Future work will further define the elements involved in the spatial and temporal regulation and the trans-factors that interact with them. ^