Christopher Fiore

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.

Fatty Acid Synthase Polymorphisms, Tumor Expression, Body Mass Index, Prostate Cancer Risk, and Survival

PURPOSE Fatty acid synthase (FASN) regulates de novo lipogenesis, body weight, and tumor growth. We examined whether common germline single nucleotide polymorphisms (SNPs) in the FASN gene affect prostate cancer (PCa) risk or PCa-specific mortality and whether these effects vary by body mass index (BMI). METHODS In a prospective nested case-control study of 1,331 white patients with PCa and 1,267 age-matched controls, we examined associations of five common SNPs within FASN (and 5 kb upstream/downstream, R(2) > 0.8) with PCa incidence and, among patients, PCa-specific death and tested for an interaction with BMI. Survival analyses were repeated for tumor FASN expression (n = 909). RESULTS Four of the five SNPs were associated with lethal PCa. SNP rs1127678 was significantly related to higher BMI and interacted with BMI for both PCa risk (P(interaction) = .004) and PCa mortality (P(interaction) = .056). Among overweight men (BMI > or = 25 kg/m(2)), but not leaner men, the homozygous variant allele carried a relative risk of advanced PCa of 2.49 (95% CI, 1.00 to 6.23) compared with lean men with the wild type. Overweight patients carrying the variant allele had a 2.04 (95% CI, 1.31 to 3.17) times higher risk of PCa mortality. Similarly, overweight patients with elevated tumor FASN expression had a 2.73 (95% CI, 1.05 to 7.08) times higher risk of lethal PCa (P(interaction) = .02). CONCLUSION FASN germline polymorphisms were significantly associated with risk of lethal PCa. Significant interactions of BMI with FASN polymorphisms and FASN tumor expression suggest FASN as a potential link between obesity and poor PCa outcome and raise the possibility that FASN inhibition could reduce PCa-specific mortality, particularly in overweight men.

ERG induces androgen receptor-mediated regulation of SOX9 in prostate cancer

Fusion of the androgen receptor-regulated (AR-regulated) TMPRSS2 gene with ERG in prostate cancer (PCa) causes androgen-stimulated overexpression of ERG, an ETS transcription factor, but critical downstream effectors of ERG-mediating PCa development remain to be established. Expression of the SOX9 transcription factor correlated with TMPRSS2:ERG fusion in 3 independent PCa cohorts, and ERG-dependent expression of SOX9 was confirmed by RNAi in the fusion-positive VCaP cell line. SOX9 has been shown to mediate ductal morphogenesis in fetal prostate and maintain stem/progenitor cell pools in multiple adult tissues, and has also been linked to PCa and other cancers. SOX9 overexpression resulted in neoplasia in murine prostate and stimulated tumor invasion, similarly to ERG. Moreover, SOX9 depletion in VCaP cells markedly impaired invasion and growth in vitro and in vivo, establishing SOX9 as a critical downstream effector of ERG. Finally, we found that ERG regulated SOX9 indirectly by opening a cryptic AR-regulated enhancer in the SOX9 gene. Together, these results demonstrate that ERG redirects AR to a set of genes including SOX9 that are not normally androgen stimulated, and identify SOX9 as a critical downstream effector of ERG in TMPRSS2:ERG fusion-positive PCa.

Surface Replacement Hemiarthroplasty for the Treatment of Osteonecrosis of the Femoral Head

We reviewed the results of thirty-three femoral resurfacing procedures in twenty-five patients who had stage-III or early stage-IV osteonecrosis of the femoral head according to the classification system of Ficat and Arlet. There were no perioperative complications. Thirty hip prostheses (91 percent) survived for a minimum of five years. At a mean of 10.5 years (range, four to fourteen years) postoperatively, sixteen (62 percent) of the twenty-six hips with stage-III disease had a good or excellent Harris hip score. Four of the seven hips with stage-IV disease did not have or need a total hip arthroplasty. Overall, twenty hips (61 percent) had a good or excellent result according to the scoring system of Harris, and thirteen (39 percent) had a fair or poor result and subsequently had or needed a total hip arthroplasty. The mean interval between the hemiarthroplasty and the total hip arthroplasty was sixty months (range, thirty-six to 136 months). These thirteen hips all had a successful clinical result (a Harris hip score of at least 80 points) at a mean of thirty months (range, twenty-four to seventy-two months) after the total hip arthroplasty. The results of the present study suggest that resurfacing of the femoral head can be a successful interim procedure for the management of patients who have Ficat and Arlet stage-III or early stage-IV disease with a large lesion that is not amenable to other treatment options except total hip arthroplasty.

c-Jun amplification and overexpression are oncogenic in liposarcoma but not always sufficient to inhibit the adipocytic differentiation programme

Genomic amplification of c‐Jun and its upstream kinases have been implicated as a mechanism of progression from well‐differentiated to dedifferentiated liposarcoma. To further define the role of c‐Jun in liposarcoma progression, we performed immunohistochemistry for c‐Jun and its activating kinase ASK1 on a series of liposarcomas (n = 81). We correlated the results with fluorescence in situ hybridization to detect c‐Jun amplification. We also derived new cell lines from dedifferentiated liposarcomas with c‐Jun amplification. c‐Jun protein is expressed in the majority of dedifferentiated liposarcomas (91%) and their well‐differentiated components (59%), but only in the minority of pure well‐differentiated liposarcomas (27%). When c‐Jun is amplified in dedifferentiated liposarcoma, it is interspersed with amplified MDM2 on ring and giant marker chromosomes. MDM2 amplification is one of the earliest events in liposarcoma development, and these results suggest that c‐Jun was amplified at a similar time in the evolution of the tumour. In addition, shRNA to c‐Jun in c‐Jun‐amplified liposarcoma cells reduces cell number in vitro and inhibits tumour formation in vivo without an observable effect on the differentiation state of the liposarcoma cells. Thus, c‐Jun amplification is oncogenic in liposarcomas but not always sufficient for inhibition of adipocytic differentiation. Copyright

Utility of multispectral imaging in automated quantitative scoring of immunohistochemistry.

Background Automated scanning devices and image analysis software provide a means to overcome the limitations of manual semiquantitative scoring of immunohistochemistry. Common drawbacks to automated imaging systems include an inability to classify tissue type and an inability to segregate cytoplasmic and nuclear staining. Methods Immunohistochemistry for the membranous marker α-catenin, the cytoplasmic marker stathmin and the nuclear marker Ki-67 was performed on tissue microarrays (TMA) of archival formalin-fixed paraffin-embedded tissue comprising 471 (α-catenin and stathmin) and 511 (Ki-67) cases of prostate adenocarcinoma. These TMA were quantitatively analysed using two commercially available automated image analysers, the Ariol SL-50 system and the Nuance system from CRi. Both systems use brightfield microscopy for automated, unbiased and standardised quantification of immunohistochemistry, while the Nuance system has spectral deconvolution capabilities. Results Overall concordance between scores from both systems was excellent (r=0.90; 0.83–0.95). The software associated with the multispectral imager allowed accurate automated classification of tissue type into epithelial glandular structures and stroma, and a single-step segmentation of staining into cytoplasmic or nuclear compartments allowing independent evaluation of these areas. The Nuance system, however, was not able to distinguish reliably between tumour and non-tumour tissue. In addition, variance in the labour and time required for analysis between the two systems was also noted. Conclusion Despite limitations, this study suggests some beneficial role for the use of a multispectral imaging system in automated analysis of immunohistochemistry.

Antitumor synergy with SY-1425, a selective RARα agonist, and hypomethylating agents in retinoic acid receptor pathway activated models of acute myeloid leukemia

Acute myeloid leukemia (AML) and biologically related myelodysplastic syndrome (MDS) are hematologic malignancies with poor outcomes. While recent approvals of new targeted therapies have increased options for some patients, those unfit for intensive treatment have few options, with single agent hypomethylating agents (HMAs) remaining as standard of care. The retinoic acid receptor alpha (RARα) transcription factor, encoded by the RARA gene, plays a critical role in myeloid cells and shows dysregulation in a subset of AML and MDS tumors. We recently demonstrated that the selective RARα agonist SY-1425 (tamibarotene) had biologic and clinical activity in 43% of evaluable relapsed or refractory AML and higher-risk MDS patients with activation of the RARA pathway. In this study, we sought to determine whether HMAs and SY-1425 exerted synergistic antiproliferative effects in AML models of RARA pathway activation in vitro and in vivo. Addition of HMAs and SY-1425 to RARA-high or IRF8-high, but not RARAlow, AML cell lines resulted in synergistic antiproliferative effects supported by evidence of DNA damage and apoptosis to a far greater extent than either agent alone. Studies in a patient-derived xenograft mouse model also demonstrated deeper and more durable responses with the combination than either agent alone. Furthermore, preclinical testing of various regimens determined that treating with azacitidine for one week followed by treatment with SY-1425 for three weeks maximized tumor suppression and tolerability. These findings directly support the ongoing clinical study of SY-1425 in combination with azacitidine. Both AML and MDS arise, in part, due to genetic alterations in transcription factors (i.e., RUNX1, NPM1) and epigenetic modifying genes (i.e., MLL, DNMT3A) leading to inactivation of tumor suppressor genes, thus enabling proliferation of immature cells. Alterations in DNA methyltransferases (DNMTs) specifically result in DNA hypermethylation which contributes to gene silencing through promoter inactivation, and can be targeted by HMAs that mimic native nucleoside residues and incorporate into DNA. Once incorporated, HMAs are recognized by DNMT1 as a cytosine, however this interaction creates an irreversible DNA-DNMT1 adduct that requires DNA damage repair to resolve. This then results in loss of DNMT1, as the DNA-protein adduct is degraded by the DNA damage response pathway. After loss of

Abstract P6-11-18: A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment

Endocrine-resistance remains a major challenge for treatment of breast cancer. Multiple mechanisms for endocrine resistance have been proposed, including altered expression of ER co-regulators such as Retinoic Acid Receptor Alpha (RARα). Furthermore, crosstalk between estradiol and RA signaling is known and upregulation of RARα has been observed in tamoxifen resistance. We propose a novel treatment paradigm for a newly-defined subset of HR+ patients based on our discovery of a super-enhancer (SE) associated with the RARA locus. SEs are large, highly active chromatin regions that pinpoint cancer vulnerabilities. The RARA SE-identified vulnerability can be targeted using the potent, selective, and metabolically stable RARα agonist SY-1425 (tamibarotene). SY-1425 is approved in Japan to treat Acute Promyelocytic Leukemia, has a well-established efficacy and safety profile, and may enhance response to hormonal therapy (HT) in this newly-defined subset of HR+ patients potentially delaying the need for alternate treatment. Tumor samples from 42 breast cancer patients were analyzed across a range of molecular subtypes. We identified an SE linked to the RARA gene in 54.5% of the hormone positive patient samples. RARA SEs predicted sensitivity to SY-1425 in 12 breast cancer cell lines confirming their functional role, and showed a correlation with RARA gene expression. A panel of 37 breast cancer cell lines was tested for SY-1425 anti-proliferative activity and gene expression levels, and identified RARA as the single best predictor of response. Proliferation of RARA-high cells was inhibited by SY-1425 with low nanomolar EC50s. Transcriptional profiling was performed on 4 HR+ and 3 HER2+/HR- breast cancer cell lines and analyzed by GSEA to examine the molecular response to SY-1425. Signatures for growth including E2F, MYC, DNA replication, and cell cycle were significantly downregulated while retinol metabolism and luminal signaling were upregulated. Estrogen signaling was also significantly altered by SY-1425, supporting known crosstalk between RARα and ER. Consistent with differentiation, CYP26A1 and VE-Cadherin were induced and Actin and Ki67 were diminished at relevant concentrations of SY-1425 and could serve as pharmacodynamic markers of response. To test responses to SY-1425 in vivo , two cell line-derived models and two patient-derived breast cancer models (one RARA-high, and one RARA-low each) were treated with SY-1425. SY-1425 inhibited tumor growth in the RARA-high models, but not the RARA-low models (43% versus 0% TGI). Consistent with the observed changes in transcription, SY-1425 in combination with tamoxifen synergistically inhibited proliferation of RARA-high breast cancer cell lines. Although a few clinical studies have investigated the use of ATRA in HR+ breast cancer without success, our results suggest that patient selection based on the RARA SE may predict which HR+ breast cancer patients could derive benefit by adding an RARα agonist to HT. The potential to prolong or increase the clinical effect of anti-estrogen therapy with SY-1425, which has improved potency, selectivity, and PK stability versus ATRA, would be an attractive strategy to explore. Citation Format: McKeown MR, Fiore C, Lee E, Eaton ML, Orlando D, Guenther MG, Collins C, Chen MW, Fritz CC, di Tomaso E. A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-18.

Abstract 2167: Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy

Deregulation of the MYC oncoprotein promotes tumorigenesis in most, if not all, cancers and is often associated with poor prognosis. However, targeting MYC has long been considered impossible based on the assumption that it would cause catastrophic side effects in normal tissues. Despite this general preconceived notion, we showed that MYC inhibition exerts extraordinary therapeutic impact in various genetic mouse models of cancer, and causes only mild, well-tolerated and reversible side effects. For these studies we employed the systemic and conditional expression of a dominant negative of MYC, called Omomyc, which we designed and validated, and that can inhibit MYC transactivation function both in vitro and in vivo. To date, Omomyc has only been considered a proof of principle, with any potential clinical application limited to gene therapy. Here we actually show that the 11 kDa Omomyc polypeptide spontaneously transduces into cancer cells, demonstrating unexpected cell-penetrating ability. Once inside the nuclei, the polypeptide effectively blocks MYC binding to its target DNA sites, interfering with MYC transcriptional regulation and halting cell proliferation. Moreover, intranasal (i.n.) administration of the Omomyc polypeptide in mice results in its rapid and persistent distribution to lungs, as well as to other organs (i.e. intestine, liver, kidneys and brain). Importantly, i.n. treatment of mice bearing either Non-Small-Cell-Lung-Cancer (NSCLC) or glioblastoma (GBM) with the Omomyc cell-penetrating peptide (OmomycCPP) significantly reduces tumor burden compared to their control counterparts. Notably, tumor regression is accompanied by significant reprogramming of the tumor microenvironment and tumor immune response. In summary, our data indicate that this novel generation of polypeptides represents a new opportunity to potentially inhibit MYC pharmacologically in a variety of malignant diseases. Citation Format: Marie-eve Beaulieu, Toni Jauset, Daniel Masso-Valles, Peter Rahl, Sandra Martinez-Martin, Loika Maltais, Mariano F. Zacarias-Fluck, Silvia Casacuberta, Erika Serrano del Pozo, Christopher Fiore, Laia Foradada, Matthew Guenther, Eduardo Romero Sanz, Marta Oteo Vives, Cynthia Tremblay, Martin Montagne, Miguel Angel Morcillo Alonso, Jonathan R. Whitfield, Pierre Lavigne, Laura Soucek. Preclinical validation of an Omomyc cell-penetrating peptide as a viable anti-Myc therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2167. doi:10.1158/1538-7445.AM2017-2167

Abstract PR-01: Vitamin D receptor expression is inversely associated with prostate cancer progression

Background: Vitamin D is inversely associated with risk of several malignancies. Circulating vitamin D interacts with the vitamin D receptor (VDR) at the cellular level to inhibit proliferation, increase apoptosis and decrease angiogenesis. Thus, although vitamin D levels appear to be unrelated to total prostate cancer incidence, VDR levels in tumor tissue may influence prostate cancer prognosis. Methods: We examined the immunohistochemical expression of VDR on archival tumor tissue from 841 prostate cancer cases diagnosed between 1982 and 2004 within two ongoing, prospective cohorts: the Physicians9 Health Study and Health Professionals Follow‐up Study. VDR expression was measured quantitatively using the CRi Vectra™ system. We used Cox proportional hazards regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association of VDR expression with lethal prostate cancer (N=73) through 2008. On a subset of cases, we also examined correlation of tumor VDR expression with circulating 25(OH)D3 and 1alpha,25(OH)2D3 measured at baseline (N=84) in 1982 and two SNPs in VDR: Fok1 and bsm1 (N=140). Results: Men with high tumor VDR expression had significantly lower Gleason score, lower prostate specific antigen (PSA) levels at diagnosis, and were less likely to have advanced tumor stage (p=0.008, p=0.012, p=0.001, respectively). Compared to the lowest quartile, men in the highest quartile of VDR expression were at significantly lower risk of developing lethal prostate cancer (age‐adjusted HRs across quartiles Q2=0.95, 95% CI: 0.51–1.79; Q3=0.93, 95% CI: 0.49–1.76; Q4 = 0.23, 95% CI:0.09–0.55). This association was attenuated (HRQ4vsQ1 = 0.42, 95% CI: 0.16–1.09) after further adjustment for pathological tumor stage, Gleason grade, and PSA level at diagnosis. Tumors expressing high levels of VDR had modest downregulation of cell proliferation as measured by Ki67 (r=‐0.11). Moreover, expression of estrogen receptor alpha (r=0.40, p Conclusion: In this large prospective study, men with tumors that demonstrated upregulation of VDR had significantly improved clinical features and reduced risk of lethal prostate cancer. In line with experimental studies, the positive correlation between VDR expression, and androgen receptor and estrogen receptor provides evidence that that VDR acts in an androgen‐ and/or estrogen‐dependent manner. These data highlight the potential role of the vitamin D system in preventing prostate cancer progression. Citation Information: Cancer Prev Res 2010;3(1 Suppl):PR-01.