Bryan M. Wittmann

The Homeodomain Protein HOXB13 Regulates the Cellular Response to Androgens

HOXB13 is a member of the homeodomain family of sequence-specific transcription factors and, together with the androgen receptor (AR), plays a critical role in the normal development of the prostate gland. We demonstrate here that, in prostate cancer cells, HOXB13 is a key determinant of the response to androgens. Specifically, it was determined that HOXB13 interacts with the DNA-binding domain of AR and inhibits the transcription of genes that contain an androgen-response element (ARE). In contrast, the AR:HOXB13 complex confers androgen responsiveness to promoters that contain a specific HOXB13-response element. Further, HOXB13 and AR synergize to enhance the transcription of genes that contain a HOX element juxtaposed to an ARE. The profound effects of HOXB13 knockdown on androgen-regulated proliferation, migration, and lipogenesis in prostate cancer cells highlight the importance of the observed changes in gene expression.

Definition of functionally important mechanistic differences among selective estrogen receptor down-regulators.

One subclass of antiestrogens, the selective estrogen receptor down-regulators (SERDs), have received considerable attention of late as they competitively inhibit estrogen binding and induce a rapid, proteasome-dependent degradation of the receptor. Contained within this class of molecules is the steroidal antiestrogen ICI182,780 (faslodex), recently approved for the treatment of metastatic cancer, and GW5638/DPC974, a SERD that is currently being evaluated in the clinic. Given that mechanistic differences between different selective estrogen receptor modulators have been translated into important clinical profiles, it was of interest to determine if the SERD subclass of ligands were likewise functionally or mechanistically distinguishable. In this study, we show that although the steroidal and nonsteroidal SERDs target ERalpha for degradation, the underlying mechanism(s) are different. Of note was the identification of a specific protein-protein interaction surface presented on ERalpha in the presence of the ICI182,780-activated receptor which is required for degradation. Interestingly, this surface is also presented on ERalpha in the presence of RU58,668, a SERD that is chemically distinct from ICI182,780. This surface is not required for GW5638-mediated degradation, and thus, this SERD seems to affect ERalpha down-regulation by a different mechanism. These data suggest that sequencing of therapies using drugs of this class is likely to be possible. Finally, because of the unmet need for orally active SERDS that function similarly to ICI182,780, we have used the insights from these mechanistic studies to develop and validate a high-throughput screen for compounds of this class with improved pharmaceutical properties.

The breast cell growth inhibitor, estrogen down regulated gene 1, modulates a novel functional interaction between estrogen receptor alpha and transcriptional elongation factor cyclin T1.

Estrogen receptor alpha (ERα) regulates transcription of specific genes and is believed to play a major role in breast tumorigenesis. We previously identified estrogen down regulated gene 1 (EDG1 (also known as HEXIM1)) using the C-terminus of ERα (E/F domain) as bait in yeast two-hybrid screenings. Here we report on the role of EDG1 as a coregulator of ERα transcriptional activity. We observe an interaction between EDG1 and ERα. EDG1 inhibits the transcriptional activity of ERα and this is dependent upon the C-terminus of EDG1. The C-terminus of EDG1/HEXIM1 was recently shown to inhibit the positive transcription elongation factor b (P-TEFb) by interacting with the cyclin T1 subunit. Here we show that ERα interacts with cyclin T1, cyclin T1 and ER co-occupancy on the promoter region of an ER target gene, and that this interaction plays an important role in ERα-induced gene expression. The interaction of ERα with cyclin T1 also allows ERα to compete with EDG1 for cyclin T1, and may release cyclin T1 from EDG1 repression. Conversely, increased EDG1 expression results in inhibition of cyclin T1 recruitment and ERα DNA binding. Our results support a novel functional interaction between ERα and cyclin T1 that is modulated by EDG1.

CaM Kinase Kinase β-Mediated Activation of the Growth Regulatory Kinase AMPK Is Required for Androgen-Dependent Migration of Prostate Cancer Cells

While patients with advanced prostate cancer initially respond favorably to androgen ablation therapy, most experience a relapse of the disease within 1-2 years. Although hormone-refractory disease is unresponsive to androgen-deprivation, androgen receptor (AR)-regulated signaling pathways remain active and are necessary for cancer progression. Thus, both AR itself and the processes downstream of the receptor remain viable targets for therapeutic intervention. Microarray analysis of multiple clinical cohorts showed that the serine/threonine kinase Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) is both highly expressed in the prostate and further elevated in prostate cancers. Using cellular models of prostate cancer, we have determined that androgens (a) directly increase the expression of a CaMKKβ splice variant and (b) increase functional CaMKKβ protein levels as determined by the phosphorylation of both CaMKI and AMP-activated protein kinase (AMPK), two of CaMKKβs primary substrates. Importantly, inhibition of the CaMKKβ-AMPK, but not CaMKI, signaling axis in prostate cancer cells by pharmacological inhibitors or siRNA-mediated knockdown blocks androgen-mediated migration and invasion. Conversely, overexpression of CaMKKβ alone leads to both increased AMPK phosphorylation and cell migration. Given the key roles of CaMKKβ and AMPK in the biology of prostate cancer cells, we propose that these enzymes are potential therapeutic targets in prostate cancer.

Induction of Krüppel-Like Factor 5 Expression by Androgens Results in Increased CXCR4-Dependent Migration of Prostate Cancer Cells in Vitro

Advanced prostate cancers preferentially metastasize to bone, suggesting that this tissue produces factors that provide a suitable microenvironment for prostate cancer cells. Recently, it has become clear that even in antiandrogen-resistant cancers, the androgen receptor (AR)-signaling axis is required for prostate cancer progression. Therefore, we hypothesized that AR may be involved in the regulation of pathways that are responsible for the homing of prostate cancer cells to select microenvironments. In support of this hypothesis, we have determined that chemokine (C-X-C motif) receptor 4 (CXCR4), the receptor for the chemokine CXCL12, is up-regulated in prostate cancer cells in response to androgens. Given that the levels of CXCL12 are elevated at sites of known prostate cancer metastases such as bone, these results suggest that androgens may influence prostate cancer metastasis. Specifically, we demonstrate that androgens increase the levels of both CXCR4 mRNA and functional protein in LNCaP prostate cancer cells. Importantly, androgens enhanced the migration of LNCaP cells toward a CXCL12 gradient, an effect that could be blocked by the specific CXCR4 antagonist AMD3100. Interestingly, CXCR4 is not directly regulated by androgens but rather is positively up-regulated by Krüppel-like factor 5 (KLF5), a transcription factor that we have shown to be an early, direct target of AR. Further, KLF5 is both required and sufficient for androgen-mediated CXCR4 expression and migration toward CXCL12. Taken together, these findings demonstrate that AR can utilize the CXCL12/CXCR4 axis through induction of KLF5 expression to promote prostate cancer progression and highlight the potential utility of CXCR4 antagonists as prostate cancer therapeutics.

Regulation of Aryl Hydrocarbon Receptor Function by Selective Estrogen Receptor Modulators

Selective estrogen receptor modulators (SERMs), such as tamoxifen (TAM), have been used extensively for the treatment and prevention of breast cancer and other pathologies associated with aberrant estrogen receptor (ER) signaling. These compounds exhibit cell-selective agonist/antagonist activities as a consequence of their ability to induce different conformational changes in ER, thereby enabling it to recruit functionally distinct transcriptional coregulators. However, the observation that SERMs can also regulate aspects of calcium signaling and apoptosis in an ER-independent manner in some systems suggests that some of the activity of drugs within this class may also arise as a consequence of their ability to interact with targets other than ER. In this study, we demonstrate that 4-hydroxy-TAM (4OHT), an active metabolite of TAM, directly binds to and modulates the transcriptional activity of the aryl hydrocarbon receptor (AHR). Of specific interest was the observation, that in the absence of ER, 4OHT can induce the expression of AHR target genes involved in estradiol metabolism, cellular proliferation, and metastasis in cellular models of breast cancer. The potential role for AHR in SERM pharmacology was further underscored by the ability of 4OHT to suppress osteoclast differentiation in vitro in part through AHR. Cumulatively, these findings provide evidence that it is necessary to reevaluate the relative roles of ER and AHR in manifesting the pharmacological actions and therapeutic efficacy of TAM and other SERMs.

Identification and Characterization of a Novel Factor That Regulates Quinone Reductase Gene Transcriptional Activity

The regulation of the quinone reductase (QR) gene as well as other genes involved in detoxification is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE). We have previously observed that QR is up-regulated by the antiestrogen trans-hydroxytamoxifen in breast cancer cells. QR gene regulation by the antiestrogen-occupied estrogen receptor (ER) is mediated by the EpRE-containing region of the human QR gene, and the ER is one of the complex of proteins that binds to the EpRE. In an effort to further understand the mechanism for ER regulation of QR gene we identified other protein factors that regulate QR gene transcriptional activity in breast cancer cells. One of these protein factors, hPMC2 (human homolog of Xenopus gene whichprevents mitotic catastrophe), directly binds to the EpRE and interacts with the ER in yeast genetic screening and in vitro assays. Interestingly hPMC2 interacts more strongly to ERβ when compared with ERα. In transient transfection assays using reporter constructs containing the EpRE, hPMC2 alone can slightly activate reporter in ER-negative MDA-MB-231 breast cancer cells. The activation of QR gene activity by hPMC2 is enhanced in the presence of ERβ.

Inhibition of prostate cancer cell growth by second-site androgen receptor antagonists

The impact of ligand binding on nuclear receptor (NR) structure and the ability of target cells to distinguish between different receptor-ligand complexes are key determinants of the pharmacological activity of NR ligands. However, until relatively recently, these mechanistic insights have not been used in a prospective manner to develop screens for NR modulators with specific therapeutic activities. Driven by the need for unique androgen receptor (AR) antagonists that retain activity in hormone-refractory prostate cancer, we developed and applied a conformation-based screen to identify AR antagonists that were mechanistically distinct from existing drugs of this class. Two molecules were identified by using this approach, D36 and D80, which interact with AR in a unique manner and allosterically inhibit AR agonist activity. Unlike the clinically important antiandrogens, casodex and hydroxyflutamide, both D36 and D80 block androgen action in cellular models of hormone-refractory prostate cancer. Mechanistically, these compounds further distinguish themselves from classical AR antagonists in that they do not promote AR nuclear translocation and quantitatively inhibit the association of AR with DNA even under conditions of overexpression. Although the therapeutic potential of these antiandrogens is apparent, it is the demonstration that it is possible, to modulate the interaction of cofactors with agonist-activated AR, using second-site modulators, that has the greatest potential with respect to the therapeutic exploitation of AR and other NRs.

Mechanisms of Progesterone Receptor Inhibition of Inflammatory Responses in Cellular Models of Breast Cancer

Both pro- and antimitogenic activities have been ascribed to progesterone receptor (PR) agonists and antagonists in breast cancer cells; however, the transcriptional responses that underlie these paradoxical functions are not apparent. Using nontransformed, normal human mammary epithelial cells engineered to express PR and standard microarray technology, we defined 2370 genes that were significantly regulated by the PR agonist R5020. Gene ontology (GO) analysis revealed that GO terms involved in inflammation and nuclear factor-κB (NF-κB) signaling were among the most significantly regulated. Interestingly, on those NF-κB responsive genes that were inhibited by agonist-activated PR, antagonists either 1) mimicked the actions of agonists or 2) reversed the inhibitory actions of agonists. This difference in pharmacological response could be attributed to the fact that although agonist- and antagonist-activated PR is recruited to NF-κB-responsive promoters, the physical presence of PR tethered to the promoter of some genes is sufficient for transcriptional inhibition, whereas on others, an agonist-activated PR conformation is required for inhibition of NF-κB signaling. Importantly, the actions of PR on the latter class of genes were reversed by an activation function-2-inhibiting, LXXLL-containing peptide. Consideration of the relative activities of these distinct antiinflammatory pathways in breast cancer may be instructive with respect to the likely therapeutic activity of PR agonists or antagonists in the treatment of breast cancer.

Estrogen receptor regulation of quinone reductase in breast cancer: implications for estrogen-induced breast tumor growth and therapeutic uses of tamoxifen.

Antiestrogens have found widespread use in the treatment of breast cancer (reviewed in ref. 1). There is also interest in the use of tamoxifen as a preventive agent for breast cancer. However, the mechanism for the antitumor effects of antiestrogens is relatively unknown. For the most part it is thought that the basis for the anticancer action of antiestrogens is the inhibition of estradiol (E2)-stimulated tumor growth. We have observed however that antiestrogens can activate detoxifying enzymes, like quinone reductase (NQO1), which protect cells against the toxic and tumor-promoting effects of carcinogens (2). Studies characterizing the molecular mechanisms behind the regulation of NQO1 by the Estrogen Receptor (ER) support an important role of the ER in pathways regulating antioxidant defenses. Moreover these findings represent a novel mechanism through which antiestrogens function. The activation of NQO1 may contribute to the beneficial anticancer and antioxidant activity of antiestrogens in breast cancer and possibly other estrogen target tissues. It is possible that the basis for some of the anticancer action of antiestrogens is that the induction of NQO1 inhibits the genotoxic effects induced by the oxidative metabolism of estrogens.