Steven P. Balk

Mutation of the Androgen-Receptor Gene in Metastatic Androgen-Independent Prostate Cancer

BACKGROUND Metastatic prostate cancer is a leading cause of cancer-related death in men. The rate of response to androgen ablation is high, but most patients relapse as a result of the outgrowth of androgen-independent tumor cells. The androgen receptor, which binds testosterone and stimulates the transcription of androgen-responsive genes, regulates the growth of prostate cells. We analyzed the androgen-receptor genes from samples of metastatic androgen-independent prostate cancers to determine whether mutations in the gene have a role in androgen independence. METHODS Complementary DNA was synthesized from metastatic prostate cancers in 10 patients with androgen-independent prostate cancer, and the expression of the androgen-receptor gene was estimated by amplification with the polymerase chain reaction. Exons B through H of the gene were cloned, and mutations were identified by DNA sequencing. The functional effects of the mutations were assessed in cells transfected with mutant genes. RESULTS All androgen-independent tumors expressed high levels of androgen-receptor gene transcripts, relative to the levels expressed by an androgen-independent prostate-cancer cell line (LNCaP). Point mutations in the androgen-receptor gene were identified in metastatic cells from 5 of the 10 patients examined. One mutation was in the same codon as the mutation found previously in the androgen-independent prostate-cancer cell line. The mutations were not detected in the primary tumors from of the two patients. Functional studies of two of the mutant androgen receptors demonstrated that they could be activated by progesterone and estrogen. CONCLUSIONS Most metastatic androgen-independent prostate cancers express high levels of androgen-receptor gene transcripts. Mutations in androgen-receptor genes are not uncommon and may provide a selective growth advantage after androgen ablation.

Increased Expression of Genes Converting Adrenal Androgens to Testosterone in Androgen-Independent Prostate Cancer

Androgen receptor (AR) plays a central role in prostate cancer, and most patients respond to androgen deprivation therapies, but they invariably relapse with a more aggressive prostate cancer that has been termed hormone refractory or androgen independent. To identify proteins that mediate this tumor progression, gene expression in 33 androgen-independent prostate cancer bone marrow metastases versus 22 laser capture-microdissected primary prostate cancers was compared using Affymetrix oligonucleotide microarrays. Multiple genes associated with aggressive behavior were increased in the androgen-independent metastatic tumors (MMP9, CKS2, LRRC15, WNT5A, EZH2, E2F3, SDC1, SKP2, and BIRC5), whereas a candidate tumor suppressor gene (KLF6) was decreased. Consistent with castrate androgen levels, androgen-regulated genes were reduced 2- to 3-fold in the androgen-independent tumors. Nonetheless, they were still major transcripts in these tumors, indicating that there was partial reactivation of AR transcriptional activity. This was associated with increased expression of AR (5.8-fold) and multiple genes mediating androgen metabolism (HSD3B2, AKR1C3, SRD5A1, AKR1C2, AKR1C1, and UGT2B15). The increase in aldo-keto reductase family 1, member C3 (AKR1C3), the prostatic enzyme that reduces adrenal androstenedione to testosterone, was confirmed by real-time reverse transcription-PCR and immunohistochemistry. These results indicate that enhanced intracellular conversion of adrenal androgens to testosterone and dihydrotestosterone is a mechanism by which prostate cancer cells adapt to androgen deprivation and suggest new therapeutic targets.

Extreme Th1 bias of invariant Vα24JαQ T cells in type 1 diabetes

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is a disease controlled by the major histocompatibility complex (MHC) which results from T-cell-mediated destruction of pancreatic β-cells. The incomplete concordance in identical twins and the presence of autoreactive T cells and autoantibodies in individuals who do not develop diabetes suggest that other abnormalities must occur in the immune system for disease to result,. We therefore investigated a series of at-risk non-progressors and type1 diabetic patients (including five identical twin/triplet sets discordant for disease). The diabetic siblings had lower frequencies of CD4−CD8− Vα24JαQ+ T cells compared with their non-diabetic sibling. All 56 Vα24JαQ+ clones isolated from the diabetic twins/triplets secreted only interferon (IFN)-γ upon stimulation; in contrast, 76 of 79 clones from the at-risk non-progressors and normals secreted both interleukin (IL)-4 and IFN-γ. Half of the at-risk non-progressors had high serum levels of IL-4 and IFN-γ. These results support a model for IDDM in which Th1-cell-mediated tissue damage is initially regulated by Vα24JαQ+ T cells producing both cytokines; the loss of their capacity to secrete IL-4 is correlated with IDDM.

Androgen Receptor Regulates a Distinct Transcription Program in Androgen-Independent Prostate Cancer

The evolution of prostate cancer from an androgen-dependent state to one that is androgen-independent marks its lethal progression. The androgen receptor (AR) is essential in both, though its function in androgen-independent cancers is poorly understood. We have defined the direct AR-dependent target genes in both androgen-dependent and -independent cancer cells by generating AR-dependent gene expression profiles and AR cistromes. In contrast to what is found in androgen-dependent cells, AR selectively upregulates M-phase cell-cycle genes in androgen-independent cells, including UBE2C, a gene that inactivates the M-phase checkpoint. We find that epigenetic marks at the UBE2C enhancer, notably histone H3K4 methylation and FoxA1 transcription factor binding, are present in androgen-independent cells and direct AR-enhancer binding and UBE2C activation. Thus, the role of AR in androgen-independent cancer cells is not to direct the androgen-dependent gene expression program without androgen, but rather to execute a distinct program resulting in androgen-independent growth.

Biology of Prostate-Specific Antigen

Prostate-specific antigen (PSA) is an androgen-regulated serine protease produced by both prostate epithelial cells and prostate cancer (PCa) and is the most commonly used serum marker for cancer. It is a member of the tissue kallikrein family, some of the members of which are also prostate specific. PSA is a major protein in semen, where its function is to cleave semenogelins in the seminal coagulum. PSA is secreted into prostatic ducts as an inactive 244-amino acid proenzyme (proPSA) that is activated by cleavage of seven N-terminal amino acids. PSA that enters the circulation intact is rapidly bound by protease inhibitors, primarily alpha1-antichymotrypsin, although a fraction is inactivated in the lumen by proteolysis and circulates as free PSA. This proteolytic inactivation, as well as the cleavage of proPSA to PSA, is less efficient in PCa. Serum total PSA levels are increased in PCa, and PSA screening has dramatically altered PCa presentation and management. Unfortunately, although high PSA levels are predictive of advanced PCa, a large fraction of organ-confined cancers present with much lower total PSA values that overlap those levels found in men without PCa. Measurement of free versus total PSA can increase specificity for PCa, and tests under development to measure forms of proPSA may further enhance the ability to detect early-stage PCa. PSA is also widely used to monitor responses to therapy and is under investigation as a therapeutic target. Finally, recent data indicate that there may be additional roles for PSA in the pathogenesis of PCa.

Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence.

Despite earlier detection and recent advances in surgery and radiation, prostate cancer is second only to lung cancer in male cancer deaths in the United States. Hormone therapy in the form of medical or surgical castration remains the mainstay of systemic treatment in prostate cancer. Over the last 15 years with the clinical use of prostate specific antigen (PSA), there has been a shift to using hormone therapy earlier in the disease course and for longer duration. Despite initial favorable response to hormone therapy, over a period of time these tumors will develop androgen‐independence that results in death. The androgen receptor (AR) is central to the initiation and growth of prostate cancer and to its response to hormone therapy. Analyses have shown that AR continues to be expressed in androgen‐independent tumors and AR signaling remains intact as demonstrated by the expression of the AR regulated gene, PSA. Androgen‐independent prostate cancers have demonstrated a variety of AR alterations that are either not found in hormone naïve tumors or found at lower frequency. These changes include AR amplification, AR point mutation, and changes in expression of AR co‐regulatory proteins. These AR changes result in a “super AR” that can respond to lower concentrations of androgens or to a wider variety of agonistic ligands. There is also mounting evidence that AR can be activated in a ligand independent fashion by compounds such as growth factors or cytokines working independently or in combination. These growth factors working through receptor tyrosine kinase pathways may promote AR activation and growth in low androgen environments. The clinical significance of these AR alterations in the development and progression of androgen‐independent prostate cancer remains to be determined. Understanding the changes in AR signaling in the evolution of androgen‐independent prostate cancer will be key to the development of more effective hormone therapy.

EZH2 Oncogenic Activity in Castration-Resistant Prostate Cancer Cells Is Polycomb-Independent

Alternative Role for EZH2 Epigenetic regulators are implicated in cancer progression and proposed as therapeutic targets. Xu et al. (p. 1465; see the Perspective by Cavalli) report that EZH2 (Enhancer of zeste homolog 2), a factor previously thought to exert its oncogenic function primarily as part of the polycomb repressive complex, acts through a distinct mechanism in cells of castration-resistant prostate cancer. Rather than exclusively silencing gene expression through histone methylation, EZH2 acts as a transcriptional coactivator. The activation function of EZH2 plays a critical role in the growth of castration-resistant prostate cancer cells, which could be relevant in future drug development. An epigenetic regulator positively regulates gene expression in cell-based models of hormone-resistant prostate cancer. Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

Loss of IFN-gamma production by invariant NK T cells in advanced cancer.

Invariant NK T cells express certain NK cell receptors and an invariant TCRα chain specific for the MHC class I-like CD1d protein. These invariant NK T cells can regulate diverse immune responses in mice, including antitumor responses, through mechanisms including rapid production of IL-4 and IFN-γ, but their physiological functions remain uncertain. Invariant NK T cells were markedly decreased in peripheral blood from advanced prostate cancer patients, and their ex vivo expansion with a CD1d-presented lipid Ag (α-galactosylceramide) was diminished compared with healthy donors. Invariant NK T cells from healthy donors produced high levels of both IFN-γ and IL-4. In contrast, whereas invariant NK T cells from prostate cancer patients also produced IL-4, they had diminished IFN-γ production and a striking decrease in their IFN-γ:IL-4 ratio. The IFN-γ deficit was specific to the invariant NK T cells, as bulk T cells from prostate cancer patients produced normal levels of IFN-γ and IL-4. These findings support an immunoregulatory function for invariant NK T cells in humans mediated by differential production of Th1 vs Th2 cytokines. They further indicate that antitumor responses may be suppressed by the marked Th2 bias of invariant NK T cells in advanced cancer patients.

Intratumoral De Novo Steroid Synthesis Activates Androgen Receptor in Castration Resistant Prostate Cancer and is Upregulated by Treatment with CYP17A1 Inhibitors

Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.

Hodgkin's Disease, Lymphomatoid Papulosis, and Cutaneous T-Cell Lymphoma Derived from a Common T-Cell Clone

BACKGROUND Lymphomatoid papulosis is a benign cutaneous eruption that in 10 to 20 percent of patients is associated with the development of lymphoma. The atypical cells of lymphomatoid papulosis histologically resemble the malignant cells of cutaneous T-cell lymphoma or the Reed-Sternberg cells of Hodgkins disease. We studied a patient in whom lymphomatoid papulosis developed in 1971, Hodgkins disease in 1975, and cutaneous T-cell lymphoma in 1985, to determine whether these diseases are clonally related. METHODS The T-cell-receptor alpha-chain gene was cloned and sequenced from a cell line derived from the advanced-stage cutaneous T-cell lymphoma, and the polymerase chain reaction was used to search for this rearrangement of the alpha-chain gene in tissues obtained earlier that were affected by Hodgkins disease or lymphomatoid papulosis. RESULTS The tumor-specific rearrangement of the alpha-chain gene was detected in the patients earlier tissues affected by lymphomatoid papulosis and Hodgkins disease, but not in control tissue, including uninvolved tissues from the staging laparotomy for Hodgkins disease. Cytogenetic studies revealed a translocation, t(8;9)(p22;p24), in cutaneous T-cell lymphoma lines and in a dermatopathic lymph node removed two years before the clinical onset of the cutaneous T-cell lymphoma. Immunohistochemical findings were consistent with an activated T-cell phenotype for the atypical cells of lymphomatoid papulosis, the Reed-Sternberg cells of Hodgkins disease, and the malignant cells of the T-cell lymphoma. CONCLUSIONS Lymphomatoid papulosis, Hodgkins disease, and cutaneous T-cell lymphoma can be derived from a single T-cell clone. A t(8;9) genetic translocation may be involved in the pathogenesis of lymphomatoid papulosis or its progression to malignant disease.