Interrogating Estrogen Signaling Pathways in Human ER-Positive Breast Cancer Cells Forming Bone Metastases in Mice.

Breast cancer bone metastases (BMET) are incurable, primarily osteolytic, and occur most commonly in estrogen receptor-α positive (ER+) breast cancer. ER+ human breast cancer BMET modeling in mice has demonstrated an estrogen (E2)-dependent increase in tumor-associated osteolysis and bone-resorbing osteoclasts, independent of estrogenic effects on tumor proliferation or bone turnover, suggesting a possible mechanistic link between tumoral ERα-driven osteolysis and ER+ bone progression. To explore this question, inducible secretion of the osteolytic factor, parathyroid hormone-related protein (PTHrP), was utilized as an in vitro screening bioassay to query the osteolytic potential of estrogen receptor- and signaling pathway-specific ligands in BMET-forming ER+ human breast cancer cells expressing ERα, ERß, and G protein-coupled ER. After identifying genomic ERα signaling, also responsibility for estrogen's proliferative effects, as necessary and sufficient for osteolytic PTHrP secretion, in vivo effects of a genomic-only ER agonist, estetrol (E4), on osteolytic ER+ BMET progression were examined. Surprisingly, while pharmacologic effects of E4 on estrogen-dependent tissues, including bone, were evident, E4 did not support osteolytic BMET progression (vs robust E2 effects), suggesting an important role for nongenomic ER signaling in ER+ metastatic progression at this site. Because bone effects of E4 did not completely recapitulate those of E2, the relative importance of nongenomic ER signaling in tumor vs bone cannot be ascertained here. Nonetheless, these intriguing findings suggest that targeted manipulation of estrogen signaling to mitigate ER+ metastatic progression in bone may require a nuanced approach, considering genomic and nongenomic effects of ER signaling on both sides of the tumor/bone interface.

Curcumin Inhibition of TGFß signaling in bone metastatic breast cancer cells and the possible role of oxidative metabolites.

TGFß signaling promotes progression of bone-metastatic (BMET) breast cancer (BCa) cells by driving tumor-associated osteolysis, a hallmark of BCa BMETs, thus allowing for tumor expansion within bone. Turmeric-derived bioactive curcumin, enriched in bone via local enzymatic deconjugation of inactive circulating curcumin-glucuronides, inhibits osteolysis and BMET progression in human xenograft BCa BMET models by blocking tumoral TGFß signaling pathways mediating osteolysis. This is a unique antiosteolytic mechanism in contrast to current osteoclast-targeting therapeutics. Therefore, experiments were undertaken to elucidate the mechanism for curcumin inhibition of BCa TGFß signaling and the application of this finding across multiple BCa cell lines forming TGFß-dependent BMETs, including a possible role for bioactive curcumin metabolites in mediating these effects. Immunoblot analysis of TGFß signaling proteins in bone tropic human (MDA-SA, MDA-1833, MDA-2287) and murine (4T1) BCa cells revealed uniform curcumin blockade of TGFß-induced Smad activation due to down-regulation of plasma membrane associated TGFßR2 and cellular receptor Smad proteins that propagate Smad-mediated gene expression, resulting in downregulation of PTHrP expression, the osteolytic factor driving in vivo BMET progression. With the exception of early decreases in TGFßR2, inhibitory effects appeared to be mediated by oxidative metabolites of curcumin and involved inhibition of gene expression. Interestingly, while not contributing to changes in Smad-mediated TGFß signaling, curcumin caused early activation of MAPK signaling in all cell lines, including JNK, an effect possibly involving interactions with TGFßR2 within lipid rafts. Treatment with curcumin or oxidizable analogs of curcumin may have clinical relevancy in the management of TGFß-dependent BCa BMETs.

Osteolytic effects of tumoral estrogen signaling in an estrogen receptor-positive breast cancer bone metastasis model.

AIM: Estrogen receptor α-positive (ER+) subtypes of breast cancer have the greatest predilection for forming osteolytic bone metastases (BMETs). Because tumor-derived factors mediate osteolysis, a possible role for tumoral ERα signaling in driving ER+ BMET osteolysis was queried using an estrogen (E2)-dependent ER+ breast cancer BMET model. METHODS: Female athymic Foxn1nu mice were inoculated with human ER+ MCF-7 breast cancer cells via the left cardiac ventricle post-E2 pellet placement, and age- and dose-dependent E2 effects on osteolytic ER+ BMET progression, as well as direct bone effects of E2, were determined. RESULTS: Osteolytic BMETs, which did not form in the absence of E2 supplementation, occurred with the same frequency in young (5-week-old) vs. skeletally mature (16-week-old) E2 (0.72 mg)-treated mice, but were larger in young mice where anabolic bone effects of E2 were greater. However, in mice of a single age and across a range of E2 doses, anabolic E2 bone effects were constant, while osteolytic ER+ BMET lesion incidence and size increased in an E2-dose-dependent fashion. Osteoclasts in ER+ tumor-bearing (but not tumor-naive) mice increased in an E2-dose dependent fashion at the bone-tumor interface, while histologic tumor size and proliferation did not vary with E2 dose. E2-inducible tumoral secretion of the osteolytic factor parathyroid hormone-related protein (PTHrP) was dose-dependent and mediated by ERα, with significantly greater levels of secretion from ER+ BMET-derived tumor cells. CONCLUSION: These results suggest that tumoral ERα signaling may contribute to ER+ BMET-associated osteolysis, potentially explaining the greater predilection for ER+ tumors to form clinically-evident osteolytic BMETs.

A Role for TGFß Signaling in Preclinical Osteolytic Estrogen Receptor-Positive Breast Cancer Bone Metastases Progression.

While tumoral Smad-mediated transforming growth factor ß (TGFß) signaling drives osteolytic estrogen receptor α-negative (ER-) breast cancer bone metastases (BMETs) in preclinical models, its role in ER+ BMETs, representing the majority of clinical BMETs, has not been documented. Experiments were undertaken to examine Smad-mediated TGFß signaling in human ER+ cells and bone-tropic behavior following intracardiac inoculation of estrogen (E2)-supplemented female nude mice. While all ER+ tumor cells tested (ZR-75-1, T47D, and MCF-7-derived) expressed TGFß receptors II and I, only cells with TGFß-inducible Smad signaling (MCF-7) formed osteolytic BMETs in vivo. Regulated secretion of PTHrP, an osteolytic factor expressed in >90% of clinical BMETs, also tracked with osteolytic potential; TGFß and E2 each induced PTHrP in bone-tropic or BMET-derived MCF-7 cells, with the combination yielding additive effects, while in cells not forming BMETs, PTHrP was not induced. In vivo treatment with 1D11, a pan-TGFß neutralizing antibody, significantly decreased osteolytic ER+ BMETs in association with a decrease in bone-resorbing osteoclasts at the tumor-bone interface. Thus, TGFß may also be a driver of ER+ BMET osteolysis. Moreover, additive pro-osteolytic effects of tumoral E2 and TGFß signaling could at least partially explain the greater propensity for ER+ tumors to form BMETs, which are primarily osteolytic.

Skeletal impact of 17ß-estradiol in T cell-deficient mice: age-dependent bone effects and osteosarcoma formation.

Estrogen (E2)-dependent ER+ breast cancer, the most common breast cancer subtype, is also the most likely to metastasize to bone and form osteolytic lesions. However, ER+ breast cancer bone metastasis human xenograft models in nude mice are rarely studied due to complexities associated with distinguishing possible tumoral vs. bone microenvironmental effects of E2. To address this knowledge gap, we systematically examined bone effects of E2 in developing young (4-week-old) vs. skeletally mature (15-week-old) female Foxn1nu nude mice supplemented with commercial 60-day slow-release E2 pellets and doses commonly used for ER+ xenograft models. E2 pellets (0.05-0.72 mg) were implanted subcutaneously and longitudinal changes in hind limb bones (vs. age-matched controls) were determined over 6 weeks by dual-energy X-ray absorptiometry (DXA), microCT, radiographic imaging, and histology, concurrent with assessment of serum levels of E2 and bone turnover markers. All E2 doses tested induced significant and identical increases in bone density (BMD) and volume (BV/TV) in 4-week-old mice with high bone turnover, increasing bone mineral content (BMC) while suppressing increases in bone area (BA). E2 supplementation, which caused dose-dependent changes in circulating E2 that were not sustained, also led to more modest increases in BMD and BV/TV in skeletally mature 15-week-old mice. Notably, E2-supplementation induced osteolytic osteosarcomas in a subset of mice independent of age. These results demonstrate that bone effects of E2 supplementation should be accounted for when assessing ER+ human xenograft bone metastases models.

Pre-diagnostic dynamic HPV16 IgG seropositivity and risk of oropharyngeal cancer.

OBJECTIVE: The aim of this study was to determine the association of HPV16 antibodies (Abs) and oropharyngeal cancer (OPC) risk in sera obtained prior to clinical diagnosis. METHODS: We identified 92 participants with incident OPC and 460 matched controls from the Janus Serum Bank Cohort in Norway. Archived tumor specimens were requested for a subset of the cases. Serum samples were collected from cases, on average, 9.3years before diagnosis (range, 0.1-14.9years). Ten cases had serum samples from multiple time points. IgG seropositivity to 8 HPV16 antigens was determined, and a logistic regression classifier of a panel of all early-antigen (EA) Abs for the predictive diagnosis of OPC was applied. RESULTS: HPV16 EA seropositivity was present in 25.0% of patients with OPC and 7.6% of controls (odds ratio (OR), 4.1; 95% CI, 2.3-7.2, p<0.0001). Abs to E2 were strongly associated with cases 0-2years pre- diagnosis (OR, 150.1; 95% CI, 27.4-1040.0, p<0.0001), and the probability of seropositivity was inversely associated with time to diagnosis (OR, 0.7 per additional year; 95% CI, 0.6-0.9, p=0.0002). Abs to E2 were also strongly associated with tumor HPV status (OR, 35.6; 95% CI, 8.7-200.0, p<0.0001). A positive score on the binary classifier was associated with an overall OR of 15.8 (95% CI, 5.6-53.4) compared with controls (p<0.05), and was strongly associated with tumor HPV status (OR, 27.4; 95% CI, 8.6-99.6, p<0.001). CONCLUSIONS: HPV16 Abs are detectable years prior to diagnosis of OPC, and the probability of seropositivity increases closer to diagnosis.

HPV Serum Antibodies as Predictors of Survival and Disease Progression in Patients with HPV-Positive Squamous Cell Carcinoma of the Oropharynx.

PURPOSE: Oropharyngeal carcinoma positive for human papillomavirus type 16 (HPV16) has a significantly better prognosis than oropharyngeal carcinoma unrelated to HPV. Within HPV16-positive oropharyngeal carcinoma, biomarkers of prognosis are urgently needed to individualize care. We hypothesized that serum antibodies specific to HPV16, the major HPV type causing oropharyngeal carcinoma, have biologic relevance and are potential biomarkers for improved prognosis among patients with HPV16-positive oropharyngeal carcinoma. EXPERIMENTAL DESIGN: IgG antibodies to the HPV16 antigens E1, E4-E7, L1, L2, and the N-terminal and C-terminal fragments of E2 (NE2, CE2) were quantified using a custom programmable enzyme-linked immunosorbent assay. Sera were obtained at diagnosis from 209 oropharyngeal carcinoma patients (96 HPV16-positive). The ratios of median fluorescent intensity (MFI) for each antigen to MFI for control GST protein were determined. Kaplan-Meier survival curves and Cox proportional hazards regression were used to determine survival differences between groups. ROC curves were used to determine the best combination of E antibodies to predict disease recurrence. RESULTS: E1, NE2, and E6 antibody positivity were all strongly associated with improved overall and progression-free survival in the entire cohort and in patients with known HPV16-positive tumors (P < 0.05). For both overall and progression-free survival among HPV-positive patients, hazard ratios were 0.2 for NE2, 0.3 for E1, and 0.3 for E6 antibody positivity. CONCLUSIONS: We identified three HPV16-specific antibodies that are associated with improved overall and progression-free survival in patients with HPV-related oropharyngeal carcinoma. These results suggest that differential serologic responses in patients may reflect differential biologic processes within the host and tumor and may have prognostic value.

Biologic predictors of serologic responses to HPV in oropharyngeal cancer: The HOTSPOT study.

OBJECTIVES: We hypothesized that viral and host factors impact the serologic responses to HPV early antigens in HPV-positive oropharyngeal cancer (HPVOPC). MATERIALS AND METHODS: We conducted a multicenter study to measure HPV16-specific IgG among patients with HPVOPC, their long-term sexual partners, and healthy volunteers. Risk factor surveys and rinse and gargle specimens were collected. Peripheral blood samples at diagnosis were evaluated for IgG Abs to HPV16 antigens using a programmable ELISA assay. Predictors for HPV16 serologic responses were evaluated using univariate and multivariable linear regression. RESULTS: 116 patients with HPVOPC, 43 partners, and 81 healthy volunteers were enrolled and had baseline sera for analysis. Cases were primarily male (90%), with a median age of 56 years. Abs to E1, E2, E6 or E7 antigens were detected more often in HPVOPC compared with volunteers or partner sera (p<0.0001). HPV16 Abs to at least one early protein (E1, E2, E4, E5, E6, or E7) were detected in the sera of 90.6% of cases, 0% of partners and 7.4% of healthy volunteers. Gender, race, sexual behavior, and viral integration were not associated with antibody response. Younger age and higher oral HPV16 copy number were associated with higher HPV16 E6 and NE2 antibody levels. CONCLUSIONS: HPV16 seroreactivity is commonly detected among patients with HPVOPC at diagnosis, but not among partners or healthy volunteers. Seroreactivity among cases are correlated with viral load and stage and not with other demographic or behavioral factors. Positive HPV16 serology was strongly associated with HPV 16 oropharyngeal cancer.

HPV16 antibodies as risk factors for oropharyngeal cancer and their association with tumor HPV and smoking status.

BACKGROUND: Antibodies (Abs) to the HPV16 proteome increase risk for HPV-associated OPC (HPVOPC). The goal of this study was to investigate the association of a panel of HPV16 Abs with risk for OPC as well as the association of these Abs with tumor HPV and smoking status among patients with OPC. METHODS: IgG Abs to the HPV16 antigens E1, E2, E4, E5, E6, E7, L1, L2 were quantified using a programmable ELISA assay. Sera were obtained from 258 OPC patients at diagnosis and 250 healthy controls. HPV16 tumor status was measured by PCR for 137 cases. Multivariable logistic regression was used to calculate odds ratios for the association of HPV16 Abs with risk for OPC. RESULTS: HPV16 E1, E2, E4, E5, E6, E7 and L1-specific IgG levels were elevated in OPC patients compared to healthy controls (p<0.05). After multivariable adjustment, Ab positivity for NE2, CE2, E6, and/or E7 was associated with OPC risk (OR [95% CI], 249.1 [99.3-624.9]). Among patients with OPC, Ab positivity for these antigens was associated with tumor HPV status, especially among never or light smokers (OR [95% CI], 6.5 [2.1-20.1] and OR [95% CI], 17.5 [4.0-77.2], respectively). CONCLUSIONS: Antibodies to HPV16 proteins are associated with increased risk for HPVOPC. Among patients with OPC, HPV16 Abs are associated with tumor HPV status, in particular among HPV positive patients with no or little smoking history.