Allison P. Jacob

RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis

RANK ligand (RANKL), a TNF-related molecule, is essential for osteoclast formation, function and survival through interaction with its receptor RANK. Mammary glands of RANK- and RANKL-deficient mice develop normally during sexual maturation, but fail to form lobuloalveolar structures during pregnancy because of defective proliferation and increased apoptosis of mammary epithelium. It has been shown that RANKL is responsible for the major proliferative response of mouse mammary epithelium to progesterone during mammary lactational morphogenesis, and in mouse models, manipulated to induce activation of the RANK/RANKL pathway in the absence of strict hormonal control, inappropriate mammary proliferation is observed. However, there is no evidence so far of a functional contribution of RANKL to tumorigenesis. Here we show that RANK and RANKL are expressed within normal, pre-malignant and neoplastic mammary epithelium, and using complementary gain-of-function (mouse mammary tumour virus (MMTV)-RANK transgenic mice) and loss-of function (pharmacological inhibition of RANKL) approaches, define a direct contribution of this pathway in mammary tumorigenesis. Accelerated pre-neoplasias and increased mammary tumour formation were observed in MMTV-RANK transgenic mice after multiparity or treatment with carcinogen and hormone (progesterone). Reciprocally, selective pharmacological inhibition of RANKL attenuated mammary tumour development not only in hormone- and carcinogen-treated MMTV-RANK and wild-type mice, but also in the MMTV-neu transgenic spontaneous tumour model. The reduction in tumorigenesis upon RANKL inhibition was preceded by a reduction in pre-neoplasias as well as rapid and sustained reductions in hormone- and carcinogen-induced mammary epithelial proliferation and cyclin D1 levels. Collectively, our results indicate that RANKL inhibition is acting directly on hormone-induced mammary epithelium at early stages in tumorigenesis, and the permissive contribution of progesterone to increased mammary cancer incidence is due to RANKL-dependent proliferative changes in the mammary epithelium. The current study highlights a potential role for RANKL inhibition in the management of proliferative breast disease.

Progestin effects on cell proliferation pathways in the postmenopausal mammary gland

IntroductionMenopausal hormone therapies vary widely in their effects on breast cancer risk, and the mechanisms underlying these differences are unclear. The primary goals of this study were to characterize the mammary gland transcriptional profile of estrogen + progestin therapy in comparison with estrogen-alone or tibolone and investigate pathways of cell proliferation in a postmenopausal primate model.MethodsOvariectomized female cynomolgus macaque monkeys were randomized into the following groups: placebo (Con), oral conjugated equine estrogens (CEE), CEE with medroxyprogesterone acetate (MPA) (CEE + MPA), and tibolone given at a low or high dose (Lo or Hi Tib). All study treatment doses represented human clinical dose equivalents and were administered in the diet over a period of 2 years.ResultsTreatment with CEE + MPA had the greatest effect on global mRNA profiles and markers of mammary gland proliferation compared to CEE or tibolone treatment. Changes in the transcriptional patterns resulting from the addition of MPA to CEE were related to increased growth factors and decreased estrogen receptor (ER) signaling. Specific genes induced by CEE + MPA treatment included key members of prolactin receptor (PRLR)/signal transducer and activator of transcription 5 (STAT5), epidermal growth factor receptor (EGFR), and receptor activator of nuclear factor kappa B (RANK)/receptor activator of nuclear factor kappa B ligand (RANKL) pathways that were highly associated with breast tissue proliferation. In contrast, tibolone did not affect breast tissue proliferation but did elicit a mixed pattern of ER agonist activity.ConclusionOur findings indicate that estrogen + progestin therapy results in a distinct molecular profile compared to estrogen-alone or tibolone therapy, including upregulation of key growth factor targets associated with mammary carcinogenesis in mouse models. These changes may contribute to the promotional effects of estrogen + progestin therapy on breast cancer risk.

SLC46A3 Is Required to Transport Catabolites of Noncleavable Antibody Maytansine Conjugates from the Lysosome to the Cytoplasm

Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.

RANKL inhibition decreases the incidence of mammary adenocarcinomas in wild type (WT) and MMTV-RANK transgenic mice.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #4167 Purpose: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary gland during pregnancy. Upon treatment with hormone medroxyprogesterone (MPA) and a carcinogen (DMBA), transgenic mice overexpressing RANK in the mammary gland via the MMTV promoter exhibit a higher incidence of ductular hyperplasias, mammary intraepithelial neoplasias (MIN), and mammary adenocarcinomas than WT mice. This study assessed the expression of mouse RANK and RANKL in WT and MMTV-RANK during mammary tumor progression in this model and determined whether RANKL inhibition could inhibit mammary carcinogenesis. Methods: At 6 weeks of age WT and MMTV-RANK transgenic mice were implanted with 90-day release 50mg MPA pellets that were replaced at expiration (weeks 19 and 32). DMBA was administered orally at weeks 9, 10, 12, and 13. The expression of RANK and RANKL was determined by immunohistochemistry (IHC). The specificity of the anti-mouse RANKL and anti-mouse RANK antibodies were verified by their lack of reactivity in RANKL -/- or RANK -/- mouse tissues, respectively. Simultaneous with the first DMBA treatment, mice were treated with RANK-Fc (10 mg/kg, 3x/week) or vehicle and the onset of mammary tumor formation was measured. Mammary tumor formation was determined by palpation and confirmed by histologic examination. Preneoplastic lesions were counted by whole mount analysis of the mammary gland at 4 weeks after the last DMBA treatment. Epithelial proliferation was measured by BrdU labeling of the mammary epithelium at multiple timepoints following the last DMBA treatment (2 days after the first DMBA treatment; 4 and 7 weeks following the last). Results: In the MPA/DMBA-induced mammary tumor model, MMTV-RANK mice developed palpable mammary tumors earlier than WT mice with a median onset of 86 days for the MMTV-RANK cohort compared with 132 days for the WT mice. At early timepoints, the number of preneoplastic lesions and mammary epithelial proliferation were greater in MMTV-RANK mice relative to WT. IHC demonstrated that MPA increased the number of RANKL-positive epithelial cells and that RANKL was present in preneoplastic MIN lesions and adenocarcinomas in both WT and MMTV-RANK mice. RANK expression was observed in the epithelial component of hyperplasias, MIN lesions, and adenocarcinomas in both WT and MMTV-RANK mice. Inhibition of RANKL with RANK-Fc substantially decreased the presence of preneoplastic lesions and the proliferation of mammary epithelial cells in MMTV-RANK mice. In addition, treatment of either MMTV-RANK or WT mice with RANK-Fc delayed the onset of palpable mammary tumors. Treatment of the MMTV-RANK mice with RANK-Fc decreased the incidence of mammary adenocarcinoma. Conclusion: These data suggest an operative role for RANKL in progesterone-dependent mouse mammary tumor development in both transgenic MMTV-RANK and WT mice and support additional studies to determine whether RANKL inhibition will delay tumor progression and metastases. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4167.

RANK and RANK ligand expression in primary human osteosarcoma

Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential mediator of osteoclast formation, function and survival. In patients with solid tumor metastasis to the bone, targeting the bone microenvironment by inhibition of RANKL using denosumab, a fully human monoclonal antibody (mAb) specific to RANKL, has been demonstrated to prevent tumor-induced osteolysis and subsequent skeletal complications. Recently, a prominent functional role for the RANKL pathway has emerged in the primary bone tumor giant cell tumor of bone (GCTB). Expression of both RANKL and RANK is extremely high in GCTB tumors and denosumab treatment was associated with tumor regression and reduced tumor-associated bone lysis in GCTB patients. In order to address the potential role of the RANKL pathway in another primary bone tumor, this study assessed human RANKL and RANK expression in human primary osteosarcoma (OS) using specific mAbs, validated and optimized for immunohistochemistry (IHC) or flow cytometry. Our results demonstrate RANKL expression was observed in the tumor element in 68% of human OS using IHC. However, the staining intensity was relatively low and only 37% (29/79) of samples exhibited≥10% RANKL positive tumor cells. RANK expression was not observed in OS tumor cells. In contrast, RANK expression was clearly observed in other cells within OS samples, including the myeloid osteoclast precursor compartment, osteoclasts and in giant osteoclast cells. The intensity and frequency of RANKL and RANK staining in OS samples were substantially less than that observed in GCTB samples. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response, and anti-RANKL therapy may potentially be protective against bone pathologies in OS. However, the absence of RANK expression in primary human OS cells suggests that any autocrine RANKL/RANK signaling in human OS tumor cells is not operative, and anti-RANKL therapy would not directly affect the tumor.

Aberrant Activation of the RANK Signaling Receptor Induces Murine Salivary Gland Tumors.

Unlike cancers of related exocrine tissues such as the mammary and prostate gland, diagnosis and treatment of aggressive salivary gland malignancies have not markedly advanced in decades. Effective clinical management of malignant salivary gland cancers is undercut by our limited knowledge concerning the key molecular signals that underpin the etiopathogenesis of this rare and heterogeneous head and neck cancer. Without knowledge of the critical signals that drive salivary gland tumorigenesis, tumor vulnerabilities cannot be exploited that allow for targeted molecular therapies. This knowledge insufficiency is further exacerbated by a paucity of preclinical mouse models (as compared to other cancer fields) with which to both study salivary gland pathobiology and test novel intervention strategies. Using a mouse transgenic approach, we demonstrate that deregulation of the Receptor Activator of NFkB Ligand (RANKL)/RANK signaling axis results in rapid tumor development in all three major salivary glands. In line with its established role in other exocrine gland cancers (i.e., breast cancer), the RANKL/RANK signaling axis elicits an aggressive salivary gland tumor phenotype both at the histologic and molecular level. Despite the ability of this cytokine signaling axis to drive advanced stage disease within a short latency period, early blockade of RANKL/RANK signaling markedly attenuates the development of malignant salivary gland neoplasms. Together, our findings have uncovered a tumorigenic role for RANKL/RANK in the salivary gland and suggest that targeting this pathway may represent a novel therapeutic intervention approach in the prevention and/or treatment of this understudied head and neck cancer.

P3-01-14: RANK and RANK Ligand (RANKL) Expression in Invasive Breast Carcinoma and Human Breast Cancer Cell Lines.

Purpose: RANK and its ligand (RANKL), key factors for bone remodeling and metastasis, are crucial for the development of mouse mammary gland during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mouse mammary epithelium and has a role in ovarian hormone-dependent expansion and regenerative potential of mammary stem cells (MaSC). RANKL inhibition has been shown to reduce mammary tumor formation and pulmonary metastases in mouse models. Many published expression analyses of RANK and RANKL have been performed using immunohistochemistry (IHC) without documented validation of antibody specificity. This study assessed the expression of human RANK and RANKL in human invasive breast carcinoma (IBC) and human breast cancer cell lines using specific, monoclonal antibodies validated and optimized for IHC or flow cytometry. Methods: RANK and RANKL expression was analyzed in a panel of human breast cancer cell lines representing luminal or basal breast subtypes using qPCR, flow cytometry and surface receptor quantitation. Antibodies against human RANK (N-1H8, N-2B10; Amgen) and human RANKL (M366, AMG161; Amgen) were used for flow cytometry, surface receptor quantitation or IHC staining. For human IBC, the intensity of IHC staining was scored on a semiquantitative scale (0=absent, 1=weak, 2=moderate, 3=intense). Incidence was scored as a positive IHC signal (any intensity). In vitro responses of cell lines to RANKL were also tested. Results: The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry and Western blots of positive and negative control cells and xenograft samples. Analysis of primary human IBC using IHC demonstrated that 25/114 (22%) IBC samples expressed RANK and 18/97 IBC (19%) expressed RANKL protein within the tumor epithelium. RANK protein was observed in monocytic cells infiltrating the tumor in 87/114 (76%) and within normal mammary epithelium adjacent to tumors in 35/79 (44%) of samples. RANKL was observed in infiltrating monocytic cells within the tumor in 60/115 (52%) and within normal mammary epithelium adjacent to tumors in 15/68 (22%) of samples. Both mRNA and RANK surface protein were detected in multiple breast cancer cell lines, including basal and luminal subtypes. Functional RANK expression on cell lines was confirmed in vitro by the observation of RANKL-dependent increases in mRNAs (e.g. MMP-9, IL-6 or IL-8) or proteins in conditioned media (e.g. IL-6, IL-8), despite the relatively low surface expression of RANK observed (range = 1240–9120 sites/cell). Conclusion: RANK and RANKL expression was observed in the epithelial carcinoma element in human IBC using IHC. RANK and RANKL expression was also observed in normal mammary epithelium and monocytic cells adjacent to breast tumors. Functional RANK expression was observed in human breast cancer cell lines, including both basal and luminal subtypes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-01-14.

Receptor activator of NF-kB (RANK) and RANK ligand protein expression in invasive ductal carcinoma.

Abstract #2057 Purpose: Bone metastases are commonly observed in patients with advanced breast cancer. Tumor cells interact with the bone microenvironment to induce osteoclastogenesis via local bone stromal expression of receptor activator of NF-kB ligand (RANKL), leading to bone destruction and release of growth factors. In addition to its critical role in tumor-induced osteolysis, RANKL has been demonstrated to enhance the invasive behavior of epithelial tumor cells that express RANK, and RANK over-expression in transgenic mice using the breast-specific MMTV promoter results in increased mammary carcinoma. This study assessed the expression of human RANK and its ligand (RANKL) in invasive ductal carcinoma (IDC).
 Methods: We studied a total of 57 IDC specimens. Antibodies against human RANK (AF683) and human RANKL (M366; Amgen) were used for immunohistochemistry (IHC) cell staining along with an isotype control. The specificity of the 2 antibodies was substantiated by IHC, flow cytometry and Western blot analysis of positive and negative control cells. In addition, for RANK, mass spectrometry/protein sequencing of immunoprecipitated proteins was performed. The intensity of IHC staining was scored on a semiquantitative scale (1=weak, 2=moderate, 3=intense) or a complex scale (sum of percentages of stained tumor cells x staining intensity (0-3)).
 Results: Using a complex score with a threshold of 10, 20/57 IDC (35%) expressed minimal level of RANK (range 0 to Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2057.

Abstract 3320: Functional RANK expression is observed in disaggregated primary human lung tumors

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The receptor activator of nuclear factor-κβ (RANK) and its ligand RANKL play a key role in the regulation of bone remodeling. RANK pathway activation has also been implicated in various aspects of lung tumor biology. In mouse models of NSCLC bone metastasis, RANKL inhibition led to decreased skeletal tumor progression and increased survival. By IHC, RANK expression has been reported to occur in approximately 60-70% of NSCLC cases profiled. Expression and function have been observed using human lung cancer cell line models. However, there is a lack of understanding with respect to the role RANK signaling plays in primary human lung tumor cells. To better understand RANK biology in lung cancer a novel platform was employed to measure RANK expression and pathway response to RANKL stimulation in freshly resected human lung tumors. RANK expression and function were investigated in viable, non-apoptotic disaggregated primary NSCLC samples by flow cytometry using previously described methodology. Cell surface RANK expression was evaluated using a monoclonal antibody (N2B10, Amgen). Evidence of receptor function was investigated by analysis of relevant intracellular signaling pathways using phospho-specific antibodies following ex vivo RANKL stimulation (2.5 pM - 5 µg/mL, 30 minutes). A cocktail of EGF, HGF and IGF1 was also employed as a positive control for functional pathway activation. Molecular profiling of somatic mutations is ongoing to further elucidate the molecular characteristics of tumors that have functional RANK signaling. Table 1 summarizes RANK expression and RANKL response profiles. This study demonstrates that RANK is expressed and functional in a subset of primary human lung tumors. Data presented here confirms RANK expression on tumor cells of NSCLC and suggests that RANK pathway utilization is restricted to the sub-set of tumors that express detectable RANK. View this table: Table 1 Citation Format: John M. Rossi, Yang Pan, William C. Dougall, Daniel Branstetter, Allison Jacob, Jude Canon, Robert D. Loberg. Functional RANK expression is observed in disaggregated primary human lung tumors. [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 3320. doi:10.1158/1538-7445.AM2014-3320