Jose M. Larios

Myofibroblast Differentiation by Transforming Growth Factor-β1 Is Dependent on Cell Adhesion and Integrin Signaling via Focal Adhesion Kinase

Myofibroblast differentiation and activation by transforming growth factor-β1 (TGF-β1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-β1and adhesion-dependent signals. TGF-β1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-β receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-β1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-β1-induced α-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of α-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-β1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-β1/Smad signaling.

Genes regulated by estrogen in breast tumor cells in vitro are similarly regulated in vivo in tumor xenografts and human breast tumors.

BackgroundEstrogen plays a central role in breast cancer pathogenesis. Although many studies have characterized the estrogen regulation of genes using in vitro cell culture models by global mRNA expression profiling, it is not clear whether these genes are similarly regulated in vivo or how they might be coordinately expressed in primary human tumors.ResultsWe generated DNA microarray-based gene expression profiles from three estrogen receptor α (ERα)-positive breast cancer cell lines stimulated by 17β-estradiol (E2) in vitro over a time course, as well as from MCF-7 cells grown as xenografts in ovariectomized athymic nude mice with E2 supplementation and after its withdrawal. When the patterns of genes regulated by E2 in vitro were compared to those obtained from xenografts, we found a remarkable overlap (over 40%) of genes regulated by E2 in both contexts. These patterns were compared to those obtained from published clinical data sets. We show that, as a group, E2-regulated genes from our preclinical models were co-expressed with ERα in a panel of ERα+ breast tumor mRNA profiles, when corrections were made for patient age, as well as with progesterone receptor. Furthermore, the E2-regulated genes were significantly enriched for transcriptional targets of the myc oncogene and were found to be coordinately expressed with Myc in human tumors.ConclusionOur results provide significant validation of a widely used in vitro model of estrogen signaling as being pathologically relevant to breast cancers in vivo.

The Androgen Metabolite 5α-androstane-3β,17β-diol (3βAdiol) Induces Breast Cancer Growth via Estrogen Receptor: Implications for Aromatase Inhibitor Resistance

The aromatase inhibitors (AIs) are used to treat estrogen receptor-positive (ER+) breast tumors in post-menopausal women, and function by blocking the conversion of adrenal androgens to estrogens by the enzyme CYP19 aromatase. Breast cancer patients receiving AI therapy have circulating estrogen levels below the level of detection; however, androgen concentrations remain unchanged. We were interested in studying the effects of androgens on breast cancer cell proliferation under profound estrogen-deprived conditions. Using in vitro models of estrogen-dependent breast cancer cell growth we show that the androgens testosterone and 5α-dihydrotestosterone induce the growth of MCF-7, T47D and BT-474 cells in the absence of estrogen. Furthermore, we demonstrate that under profound estrogen-deprived conditions these breast cancer cells up-regulate steroidogenic enzymes that can metabolize androgens to estrogens. Lastly, we found that the downstream metabolite of 5α-dihydrotestosterone, 5α-androstane-3β,17β-diol (3βAdiol), is estrogenic in breast cancer cells, and induces growth and ER-signaling via activation of ERα. In conclusion, our results show that breast cancer cells deprived of estrogen up-regulate steroidogenic enzymes and metabolize androgens to estrogen-like steroids. The generation of estrogen-like steroids represents a potential mechanism of resistance to aromatase inhibitors.

Expression levels and activation of a PXR variant are directly related to drug resistance in osteosarcoma cell lines

Approximately 30% to 40% of all patients with osteosarcomas ultimately experience recurrence. The study investigated the hypothesis that the resistance of osteosarcoma to chemotherapy may be related to the expression of a pregnane xenobiotic receptor (PXR) variant protein and its role as the major inducer of P450 3A4 in these tumors.

Heterogeneous estrogen receptor expression in circulating tumor cells suggests diverse mechanisms of fulvestrant resistance

Fulvestrant is a dose dependent selective estrogen receptor (ER) down‐regulator (SERD) used in ER‐positive metastatic breast cancer (MBC). Nearly all patients develop resistance. We performed molecular analysis of circulating tumor cells (CTC) to gain insight into fulvestrant resistance.

The CYP17A1 inhibitor abiraterone exhibits estrogen receptor agonist activity in breast cancer

Abstract Cytochrome P450 17A1 (CYP17A1) is the requisite enzyme for synthesis of sex steroids, including estrogens and androgens. As such, inhibition of CYP17A1 is a target for inhibiting the growth of hormone-dependent cancers including prostate and breast cancer. Abiraterone, is a first in class potent and selective CYP17A1 inhibitor that has been approved for the treatment of castration-resistant prostate cancer. Given that, androgens are the precursors for estrogen production, it has been proposed that abiraterone could be an effective form of treatment for estrogen receptor (ER)-positive breast cancer, though its utility in this context has yet to be established. Abiraterone has a core steroid-like chemical structure, and so we hypothesized that it may bind to nuclear steroid receptors including ER and have estrogenic activity. We tested this hypothesis by investigating abiraterone’s ability to directly modulate ER signaling in breast cancer cell line models. We show that abiraterone directly activates ER, induces ER-target gene expression, and elicits estrogen-response-element reporter activity in the ER-positive cell lines MCF-7 and T47D. Abiraterone also induced cell proliferation by ~2.5-fold over vehicle in both MCF-7 and T47D cells. Importantly, abiraterone-induced cell proliferation and ER-activity was blocked by the selective estrogen receptor downregulator (SERD) fulvestrant, confirming that abiraterone directly acts at the ER. These data suggest that abiraterone should be combined with other ER antagonists when used for the clinical management of ER-positive breast cancer.

Comprehensive mutation and copy number profiling in archived circulating breast cancer tumor cells documents heterogeneous resistance mechanisms

Addressing drug resistance is a core challenge in cancer research, but the degree of heterogeneity in resistance mechanisms in cancer is unclear. In this study, we conducted next-generation sequencing (NGS) of circulating tumor cells (CTC) from patients with advanced cancer to assess mechanisms of resistance to targeted therapy and reveal opportunities for precision medicine. Comparison of the genomic landscapes of CTCs and tissue metastases is complicated by challenges in comprehensive CTC genomic profiling and paired tissue acquisition, particularly in patients who progress after targeted therapy. Thus, we assessed by NGS somatic mutations and copy number alterations (CNA) in archived CTCs isolated from patients with metastatic breast cancer who were enrolled in concurrent clinical trials that collected and analyzed CTCs and metastatic tissues. In 76 individual and pooled informative CTCs from 12 patients, we observed 85% concordance in at least one or more prioritized somatic mutations and CNA between paired CTCs and tissue metastases. Potentially actionable genomic alterations were identified in tissue but not CTCs, and vice versa. CTC profiling identified diverse intra- and interpatient molecular mechanisms of endocrine therapy resistance, including loss of heterozygosity in individual CTCs. For example, in one patient, we observed CTCs that were either wild type for ESR1 (n = 5/32), harbored the known activating ESR1 p.Y537S mutation (n = 26/32), or harbored a novel ESR1 p.A569S (n = 1/32). ESR1 p.A569S was modestly activating in vitro, consistent with its presence as a minority circulating subclone. Our results demonstrate the feasibility and potential clinical utility of comprehensive profiling of archived fixed CTCs. Tissue and CTC genomic assessment are complementary, and precise combination therapies will likely be required for effective targeting in advanced breast cancer patients.Significance: These findings demonstrate the complementary nature of genomic profiling from paired tissue metastasis and circulating tumor cells from patients with metastatic breast cancer. Cancer Res; 78(4); 1110-22. ©2017 AACR.

Abstract 5568: CYP17A1 and abiraterone: Implications for breast cancer endocrine therapy

The cytochrome P450 17A1 (CYP17A1) inhibitor abiraterone, which blocks synthesis of all steroid hormones including androgens and estrogens, is an effective treatment for castrate resistant prostate cancer. Its utility for treating hormone receptor-positive breast cancer has yet to be established. In prostate cancer, the anti-proliferative effects of abiraterone are mediated by inhibiting CYP17A1 activity resulting in a significant reduction in circulating concentrations of the sex steroids dehydroepiandrosterone (DHEA), androstenedione, testosterone, and dihydrotestosterone (DHT). Recently, it has been proposed that the reduction in aromatizable androgens by abiraterone may make this drug useful for the management of hormone-dependent breast cancer. However, data from our lab suggests that abiraterone has estrogenic properties and can induce proliferation in breast cancer cells expressing the estrogen receptor (ER). Crystal violet assays were used to assess abiraterone-induced proliferation in the ER-positive, estrogen-dependent breast cancer cell lines MCF-7 and T47D. We show that abiraterone induced a dose dependent increase in cell proliferation with an IC50 of 3.7 μM and maximal stimulation (200%) observed at 8μM, compared to vehicle treated cells. Abiraterone also induced the expression of the ER response gene, GREB1. Abiraterone-induced proliferation and gene expression was blocked by increasing doses of the selective estrogen receptor down-regulator (SERD) fulvestrant. Abiraterone induced the dose-dependent expression of luciferase in MCF-7 cells transfected with an estrogen responsive luciferase reporter construct. In conclusion, our data suggest that abiraterone can induce ER-positive breast cancer cell proliferation in vitro by acting as a weak ER agonist. Further studies are underway to determine whether estrogenic effects of abiraterone are also observed using in vivo models of ER response. If abiraterone exhibits estrogenic effects in vivo then these data would suggest that abiraterone should be combined with an ER antagonist if used for the clinical management of women with ER+ tumors. Citation Format: Cameron P. Capper, Michael D. Johnson, Jose M. Larios, James M. Rae. CYP17A1 and abiraterone: Implications for breast cancer endocrine therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5568. doi:10.1158/1538-7445.AM2014-5568

Functional characterization of the G162R and D216H genetic variants of human CYP17A1

Cytochrome P450 17A1 (CYP17A1) is a dual-function enzyme catalyzing reactions necessary for cortisol and androgen biosynthesis. CYP17A1 is a validated drug target for prostate cancer as CYP17A1 inhibition significantly reduces circulating androgens and improves survival in castration-resistant prostate cancer. Germline CYP17A1 genetic variants with altered CYP17A1 activity manifesting as various endocrinopathies are extremely rare; however, characterizing these variants provides critical insights into CYP17A1 protein structure and function. By querying the dbSNP online database and publically available data from the 1000 genomes project (http://browser.1000genomes.org), we identified two CYP17A1 nonsynonymous genetic variants with unknown consequences for enzymatic activity and stability. We hypothesized that the resultant amino acid changes would alter CYP17A1 stability or activity. To test this hypothesis, we utilized a HEK-293T cell-based expression system to characterize the functional consequences of two CYP17A1 variants, D216H (rs200063521) and G162R (rs141821705). Cells transiently expressing the D216H variant demonstrate a selective impairment of 16α-hydroxyprogesterone synthesis by 2.1-fold compared to wild-type (WT) CYP17A1, while no effect on 17α-hydroxyprogesterone synthesis was observed. These data suggest that substrate orientations in the active site might be altered with this amino acid substitution. In contrast, the G162R substitution exhibits decreased CYP17A1 protein stability compared to WT with a near 70% reduction in protein levels as determined by immunoblot analysis. This variant is preferentially ubiquitinated and degraded prematurely, with an enzyme half-life calculated to be ∼2.5 h, and proteasome inhibitor treatment recovers G162R protein expression to WT levels. Together, these data provide new insights into CYP17A1 structure-function and stability mechanisms.

Abstract P2-02-19: Somatic genetic profiling of circulating tumor cells (CTC) in metastatic breast cancer (MBC) patients

Introduction: Somatic mutations, including those in TP53 , PIK3CA , and estrogen receptor alpha ( ESR1 ), are key to the biology of cancer and response to therapy. Recently, somatic cancer-associated mutations have been identified in circulating cell free plasma tumor DNA (ptDNA). Less is known about the mutation profile of DNA extracted from CTC (CTC-DNA). Since CTC-DNA provides mutational information of single cells, we hypothesize CTC-DNA will complement ptDNA to give greater insight into tumor heterogeneity. Methods: Patients with ER positive MBC who were enrolled in the Mi CTC-ONCOSEQ, a companion trial to Mi-ONCOSEQ (the Michigan Oncology Sequencing Program), and who had ≥5CTC/7.5 ml whole blood were included. CTC were enriched from white blood cells (WBC) with CellSearch