CD133 as a Biomarker for an Autoantibody-to-ImmunoPET Paradigm for the Early Detection of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a deadly neuroendocrine tumor for which there are no screening methods sensitive enough to facilitate early, effective intervention. We propose targeting the neuroendocrine tumor neoantigen CD133 via antibody-based early detection and PET (immunoPET) to facilitate earlier and more accurate detection of SCLC. Methods: RNA sequencing datasets, immunohistochemistry, flow cytometry, and Western blots were used to quantify CD133 expression in healthy and SCLC patients. CD133 was imaged in vivo using near-infrared fluorescence (NIRF) immunoimaging, and 89Zr immunoPET. Anti(α)-CD133 autoantibody levels were measured in SCLC patient plasma using antibody microarrays. Results: Across 6 publicly available datasets, CD133 messenger RNA was found to be higher in SCLC tumors than in other tissues, including healthy or normal adjacent lung and non-SCLC samples. Critically, the upregulation of CD133 messenger RNA in SCLC was associated with a significant increase (hazard ratio, 2.62) in death. CD133 protein was expressed in primary human SCLC, in SCLC patient-derived xenografts, and in both SCLC cell lines tested (H82 and H69). Using an H82 xenograft mouse model, we first imaged CD133 expression with NIRF. Both in vivo and ex vivo NIRF clearly showed that a fluorophore-tagged αCD133 homed to lung tumors. Next, we validated the noninvasive visualization of subcutaneous and orthotopic H82 xenografts via immunoPET. An αCD133 antibody labeled with the positron-emitting radiometal 89Zr demonstrated significant accumulation in tumor tissue while producing minimal uptake in healthy organs. Finally, plasma αCD133 autoantibodies were found in subjects from cohort studies up to 1 year before SCLC diagnosis. Conclusion: In light of these findings, we conclude that the presence of αCD133 autoantibodies in a blood sample followed by CD133-targeted 89Zr-immunoPET could be an effective early detection screening strategy for SCLC.

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.

Bone-Specific Metabolism of Dietary Polyphenols in Resorptive Bone Diseases.

SCOPE: Curcumin prevents bone loss in resorptive bone diseases and inhibits osteoclast formation, a key process driving bone loss. Curcumin circulates as an inactive glucuronide that can be deconjugated in situ by bone's high ß-glucuronidase (GUSB) content, forming the active aglycone. Because curcumin is a common remedy for musculoskeletal disease, effects of microenvironmental changes consequent to skeletal development or disease on bone curcumin metabolism are explored. METHODS AND RESULTS: Across sexual/skeletal development or between sexes in C57BL/6 mice ingesting curcumin (500 mg kg-1 ), bone curcumin metabolism and GUSB enzyme activity are unchanged, except for >twofold higher (p < 0.05) bone curcumin-glucuronide substrate levels in immature (4-6-week-old) mice. In ovariectomized (OVX) or bone metastasis-bearing female mice, bone substrate levels are also >twofold higher. Aglycone curcumin levels tend to increase proportional to substrate such that the majority of glucuronide distributing to bone is deconjugated, including OVX mice where GUSB decreases by 24% (p < 0.01). GUSB also catalyzes deconjugation of resveratrol and quercetin glucuronides by bone, and a requirement for the aglycones for anti-osteoclastogenic bioactivity, analogous to curcumin, is confirmed. CONCLUSION: Dietary polyphenols circulating as glucuronides may require in situ deconjugation for bone-protective effects, a process influenced by bone microenvironmental changes.

Mechanistic Differences in the Inhibition of NF-κB by Turmeric and Its Curcuminoid Constituents.

Turmeric extract, a mixture of curcumin and its demethoxy (DMC) and bisdemethoxy (BDMC) isomers, is used as an anti-inflammatory preparation in traditional Asian medicine. Curcumin is considered to be the major bioactive compound in turmeric but less is known about the relative anti-inflammatory potency and mechanism of the other components, their mixture, or the reduced in vivo metabolites. We quantified inhibition of the NF-κB pathway in cells, adduction to a peptide mimicking IκB kinase ß, and the role of cellular glutathione as a scavenger of electrophilic curcuminoid oxidation products, suggested to be the active metabolites. Turmeric extracts (IC50 14.5 ± 2.9 µM), DMC (IC50 12.1 ± 7.2 µM), and BDMC (IC50 8.3 ± 1.6 µM), but not reduced curcumin, inhibited NF-κB similar to curcumin (IC50 18.2 ± 3.9 µM). Peptide adduction was formed with turmeric and DMC but not with BDMC, and this correlated with their oxidative degradation. Inhibition of glutathione biosynthesis enhanced the activity of DMC but not BDMC in the cellular assay. These findings suggest that NF-κB inhibition by curcumin and DMC involves their oxidation to reactive electrophiles, whereas BDMC does not require oxidation. Because it has not been established whether curcumin undergoes oxidative transformation in vivo, oxidation-independent BDMC may be a promising alternative to test in clinical trials.

Incomplete Hydrolysis of Curcumin Conjugates by ß-Glucuronidase: Detection of Complex Conjugates in Plasma.

SCOPE: The diphenol curcumin from turmeric is rapidly metabolized into phase II conjugates following oral administration, resulting in negligible plasma concentration of the free compound, which is considered the bioactive form. Total plasma concentration of curcumin is often quantified after treatment with ß-glucuronidase to hydrolyze curcumin-glucuronide, the most abundant conjugate in vivo. The efficiency of enzymatic hydrolysis has not been tested. METHODS AND RESULTS: Using liquid chromatography-mass spectrometry (LC-MS) analyses the efficiency of ß-glucuronidase and sulfatase from Helix pomatia is compared to hydrolyze curcumin conjugates in human and mouse plasma after oral administration of turmeric. Both ß-glucuronidase and sulfatase completely hydrolyze curcumin-glucuronide. Unexpectedly, ß-glucuronidase hydrolysis is incomplete, affording a large amount of curcumin-sulfate, whereas sulfatase hydrolyzed both glucuronide and sulfate conjugates. With sulfatase, the concentration of free curcumin is doubled in human and increased in mouse plasma compared to ß-glucuronidase treatment. Incomplete hydrolysis by ß-glucuronidase suggests the presence of mixed glucuronide-sulfate conjugates. LC-MS based searches detect diglucuronide, disulfate, and mixed sulfate-glucuronide and sulfate-diglucuronide conjugates in plasma that likely contribute to the increase of free curcumin upon sulfatase treatment. CONCLUSION: ß-Glucuronidase incompletely hydrolyzes complex sulfate-containing conjugates that appear to be major metabolites, resulting in an underestimation of the total plasma concentration of curcumin.

Beta-Glucuronidase Catalyzes Deconjugation and Activation of Curcumin-Glucuronide in Bone.

The biological basis for documented in vivo bone-protective effects of turmeric-derived curcumin is unclear since curcumin is barely detectable in serum, being rapidly conjugated to form what is thought to be an inactive glucuronide. Studies were therefore undertaken to test the postulate that antiresorptive effects of curcumin require deconjugation within bone to form the bioactive aglycone and that ß-glucuronidase (GUSB), a deconjugating enzyme expressed by hematopoietic marrow cells, facilitates this site-specific transformation. Consistent with this postulate, aglycone, but not glucuronidated, curcumin inhibited RANKL-stimulated osteoclastogenesis, a key curcumin target in bone. Aglycone curcumin, expressed relative to total curcumin, was higher in bone marrow than in serum of curcumin-treated C57BL/6J mice, while remaining a minor component. Ex vivo, under conditions preventing further metabolism of the unstable aglycone, the majority of curcumin-glucuronide delivered to marrow in vivo was hydrolyzed to the aglycone, a process that was inhibited by treatment with saccharolactone, a GUSB inhibitor, or in mice having reduced (C3H/HeJ) or absent (mps/mps) GUSB activity. These findings suggest that curcumin, despite low systemic bioavailability, may be enzymatically activated (deconjugated) within GUSB-enriched bone to exert protective effects, a metabolic process that could also contribute to bone-protective effects of other highly glucuronidated dietary polyphenols.

Curcumin, but not curcumin-glucuronide, inhibits Smad signaling in TGFß-dependent bone metastatic breast cancer cells and is enriched in bone compared to other tissues.

Breast cancer (BCa) bone metastases (BMETs) drive osteolysis via a feed-forward loop involving tumoral secretion of osteolytic factors (e.g., PTHrP) induced by bone-matrix-derived growth factors (e.g., TGFß). In prior experiments, turmeric-derived curcumin inhibited in vivo BMET progression and in vitro TGFß/Smad-signaling in a TGFß-stimulated PTHrP-dependent human xenograft BCa BMET model (MDA-SA cells). However, it is unclear whether curcumin or curcumin-glucuronide mediates in vivo protection since curcumin-glucuronide is the primary circulating metabolite in rodents and in humans. Thus, effects of curcumin vs. curcumin-glucuronide on Smad-dependent TGFß signaling were compared in a series of BCa cell lines forming TGFß-dependent BMET in murine models, and tissue-specific metabolism of curcumin in mice was examined by LC-MS. While curcumin inhibited TGFß-receptor-mediated Smad2/3 phosphorylation in all BCa cells studied (human MDA-SA, MDA-1833, MDA-2287 and murine 4T1 cells), curcumin-glucuronide did not. Similarly, curcumin, but not curcumin-glucuronide, blocked TGFß-stimulated secretion of PTHrP from MDA-SA and 4T1 cells. Because the predominant serum metabolite, curcumin-glucuronide, lacked bioactivity, we examined tissue-specific metabolism of curcumin in mice. Compared to serum and other organs, free curcumin (both absolute and percentage of total) was significantly increased in bone, which was also a rich source of enzymatic deglucuronidation activity. Thus, curcumin, and not curcumin-glucuronide, appears to inhibit bone-tropic BCa cell TGFß-signaling and to undergo site-specific activation (deconjugation) within the bone microenvironment. These findings suggest that circulating curcumin-glucuronide may act as a prodrug that preferentially targets bone, a process that may contribute to the bone-protective effects of curcumin and other highly glucuronidated dietary polyphenols.

Vitamin D and Colorectal, Breast, and Prostate Cancers: A Review of the Epidemiological Evidence.

Over the past two decades, the question of whether vitamin D has a role in cancer incidence, progression, and mortality has been studied in detail. Colorectal, breast, and prostate cancers have been a particular area of focus; together, these three malignancies account for approximately 35% of cancer cases and 20% of cancer deaths in the United States, and as such are a major public health concern. Herein, we review and synthesize the epidemiological research regarding vitamin D, as measured by the biomarker 25-hydroxycholecalciferol [25(OH)D], and the incidence, progression, and mortality of these cancers. Overall, the results of observational studies of the relationship between 25(OH)D and colorectal cancer have revealed a consistent inverse association for incidence and mortality; while for breast cancer, results have generally demonstrated a relationship between higher 25(OH)D and lower risk for progression and mortality. In contrast, randomized, double-blind clinical trials conducted to date have generally failed to support these findings. For prostate cancer, there is no convincing evidence of an association between 25(OH)D and incidence, and inconsistent data for progression and mortality, though results of one open label clinical trial suggest that supplementation with 4000 IU/d of vitamin D3 may inhibit progression of the disease. Nonetheless, until the results of additional ongoing randomized, double-blind clinical trials are reported, it will be difficult to ascertain if vitamin D itself is related to a reduction in risk for some cancer endpoints, or whether high concentrations of the vitamin D biomarker 25(OH)D may instead serve as a marker for an overall beneficial risk factor profile.