Expression In Bone Metastases Research Articles

Lymphotoxin-β promotes breast cancer bone metastasis colonization and osteolytic outgrowth.

Bone metastasis is a lethal consequence of breast cancer. Here we used single-cell transcriptomics to investigate the molecular mechanisms underlying bone metastasis colonization-the rate-limiting step in the metastatic cascade. We identified that lymphotoxin-β (LTβ) is highly expressed in tumour cells within the bone microenvironment and this expression is associated with poor bone metastasis-free survival. LTβ promotes tumour cell colonization and outgrowth in multiple breast cancer models. Mechanistically, tumour-derived LTβ activates osteoblasts through nuclear factor-κB2 signalling to secrete CCL2/5, which facilitates tumour cell adhesion to osteoblasts and accelerates osteoclastogenesis, leading to bone metastasis progression. Blocking LTβ signalling with a decoy receptor significantly suppressed bone metastasis in vivo, whereas clinical sample analysis revealed significantly higher LTβ expression in bone metastases than in primary tumours. Our findings highlight LTβ as a bone niche-induced factor that promotes tumour cell colonization and osteolytic outgrowth and underscore its potential as a therapeutic target for patients with bone metastatic disease.

Abstract 2787: Heme oxygenase 1 pulls the brake on stemness-induced properties by prostate cancer-bone crosstalk

Abstract Prostate cancer (PCa) is the second most diagnosed cancer in men globally, and it is positioned as the fifth leading cause of cancer-related death in males. PCa aggressiveness and mortality is intricately linked to the metastatic disease, which is propelled by PCa stem cells (PCSCs). We have previously showcased the strong antitumoral role heme oxygenase-1 (HO-1) exerts in PCa. However, little is known about the association of HO-1 with the stemness capacity of PCa cells. In this work, we first performed clonogenic assays in PCa cells (PC3 and C4-2B) to assess colony formation, which evidenced a reduction on the stem-like properties of tumor cells treated with hemin (FDA approved drug and specific HO-1 inducer and activator). Next, we employed an indirect co-culture system of PCa cells (PC3) grown with bone progenitors (MC3T3) to emulate the dynamic PCa-bone crosstalk. PC3 cells were pre-treated or not with hemin. We performed comprehensive RNA-seq analyses and assessed the transcriptome of PC3 cells focusing on genes associated with stemness (CD44, CD133 and other pluripotency markers), metastasis and a stem-like signature previously identified by our group (ADAM15, BCL2L1, LTBR, MBNL2, SPINT1). PC3 cells that were co-cultured with MC3T3 displayed upregulated PCSC and pluripotency markers, as well as BCL2L1 and LTBR, when compared with PC3 cells cultured alone. Intriguingly, pre-treatment of PC3 cells with hemin prior to the co-culture impaired the upregulation of these genes, including the PCSC marker CD44, underscoring the protective effect of HO-1 induction against the pro-stemness effect triggered by bone progenitors secreted factors. Furthermore, we extended our analysis using PCa patients’ survival and transcriptomic publicly available data (TCGA-PRAD (n=565), SU2C-PRAD (n=444), FHCRC-PRAD (n=176)), which revealed that ADAM15, BCL2L1, LTBR and SPINT1 expression was higher in bone metastases vs. primary PCa (p<0.001). Moreover, POU5F1, ALDH1L2, ADAM15, BCL2L1, LTBR and SPINT1 presented higher expression in bone metastases vs. other sites of metastasis (p<0.01). We also performed multiple survival analyses including progression-free, metastasis-free, biochemical relapse-free and overall survival (TCGA-PRAD, GSE116918, GSE70770) which revealed that CD44 expression is associated with increased risk of metastasis (p<0.05), while patients with high expression of SOX2, ALDH1B1 and ALDH1L2 showed shorter times to biochemical relapse (p<0.05) Altogether, HO-1 expression modulates stemness and metastasis-related genes in PCa cells, steering tumor cells towards a more differentiated state and counteracting the pro-stemness effect inherent to the communication with bone progenitors. These findings support the antitumoral role of HO-1 in PCa and underscores CD44 as an HO-1-modulated compelling candidate for further investigations. Citation Format: Agustina Ayelen Sabater, Ines Achinelli, Pablo Sanchis, Nicolas Anselmino, Juan Bizzotto, Gaston Pascual, Rocio Seniuk, Javier Cotignola, Elba Vazquez, Geraldine Gueron, Ayelen Toro. Heme oxygenase 1 pulls the brake on stemness-induced properties by prostate cancer-bone crosstalk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2787.

NFκB-Mediated Mechanisms Drive PEDF Expression and Function in Pre- and Post-Menopausal Oestrogen Levels in Breast Cancer

Pigment epithelium-derived factor (PEDF) protein regulates normal bone, with anti-tumour roles in bone and breast cancer (BC). Pre- and post-menopausal oestrogen levels may regulate PEDF expression and function in BC, though the mechanisms behind this remain unknown. In this study, in vitro models simulating pre- and post-menopausal bone microenvironments were used to evaluate if PEDF regulates pro-metastatic biomarker expression and downstream functional effects on BC cells. PEDF treatment reduced phosphorylated-nuclear factor-κB p65 subunit (p-NFκB-p65), tumour necrosis factor-α (TNFα), C-X-C chemokine receptor type-4 (CXCR4), and urokinase plasminogen activator receptor (uPAR) in oestrogen receptor (ER)+/human epidermal growth factor receptor-2 (HER2)- BC cells under post-menopausal oestrogen conditions. In triple negative BC (TNBC) cells, PEDF treatment reduced pNFκB-p65 and uPAR expression under pre-menopausal oestrogen conditions. A potential reciprocal regulatory axis between p-NFκB-65 and PEDF in BC was identified, which was BC subtype-specific and differentially regulated by menopausal oestrogen conditions. The effects of PEDF treatment and NFκB inhibition on BC cell function under menopausal conditions were also compared. PEDF treatment exhibited superior anti-viability effects, while combined PEDF and NFκB-p65 inhibitor treatment was superior in reducing BC cell colony formation in a subtype-specific manner. Lastly, immunohistochemical evaluation of p-NFκB-p65 and PEDF expression in human BC and bone metastases specimens revealed an inverse correlation between nuclear PEDF and NFκB expression in bone metastases. We propose that menopausal status is associated with a PEDF/NFκB reciprocal regulatory axis, which drives PEDF expression and anti-metastatic function in a subtype-specific manner. Altogether, our findings identify pre-menopausal TNBC and post-menopausal ER+/HER2- BC patients as target populations for future PEDF research.

Incidence and prognostic significance of receptor discordance between primary breast cancer and paired bone metastases.

ER, PgR and HER-2 status are the cornerstones of choosing systemic therapy for breast cancer, but can change during the disease course. Guidelines recommended the biopsy of the metastatic tumor to reassess receptor status. Bone is the most frequent metastatic site of breast cancer but remained technically difficult to biopsy. Our study aimed to evaluate the incidence and prognostic significance of receptor discordance between primary breast cancer and paired bone metastases. One hundred and fifty-five breast cancer patients were diagnosed with pathology-confirmed bone metastasis at Fudan University Shanghai Cancer Center. Ninety-three patients with receptor status available on both primary tumor and bone metastases were included in our study. ER, PgR and HER-2 status converted from positive to negative in 10.8% (10/93), 28.0% (26/93) and 8.6% (8/93) of the patients, while ER, PgR and HER-2 status converted from negative to positive in 3.2% (3/93), 4.3% (4/93) and 1.1% (1/93) of the patients, respectively. 40.4% (17/42) of the HER2-0 tumors converted to HER2-low, which enabled them to receive the treatment of new antibody-drug conjugates (ADCs). Prior endocrine and anti-HER2 therapy were the independent risk factors for receptor conversion. Loss of HR expression in bone metastases was significantly associated with worse first-line PFS (adjusted hazard ratio=3.271, P-value=.039) and OS (adjusted hazard ratio=6.09, P-value=.011). In conclusion, our study confirmed that patients may experience receptor conversion between primary breast cancer and bone metastases, possibly influenced by prior treatments, which significantly influenced prognosis. The rebiopsy of bone metastases in patients with primary HER2-0 tumors may benefit from the new ADC drugs.

ERRα Expression in Bone Metastases Leads to an Exacerbated Antitumor Immune Response.

Bone is the most common metastatic site for breast cancer. Although the estrogen-related receptor alpha (ERRα) has been implicated in breast cancer cell dissemination to the bone from the primary tumor, its role after tumor cell anchorage in the bone microenvironment remains elusive. Here, we reveal that ERRα inhibits the progression of bone metastases of breast cancer cells by increasing the immune activity of the bone microenvironment. Overexpression of ERRα in breast cancer bone metastases induced expression of chemokines CCL17 and CCL20 and repressed production of TGFβ3. Subsequently, CD8+ T lymphocytes recruited to bone metastases escaped TGFβ signaling control and were endowed with exacerbated cytotoxic features, resulting in significant reduction in metastases. The clinical relevance of our findings in mice was confirmed in over 240 patients with breast cancer. Thus, this study reveals that ERRα regulates immune properties in the bone microenvironment that contributes to decreasing metastatic growth. SIGNIFICANCE: This study places ERRα at the interplay between the immune response and bone metastases of breast cancer, highlighting a potential target for intervention in advanced disease.

Bone decalcification to assess programmed cell death ligand 1 expression in bone metastases of non-small cell lung cancers

As for molecular alterations of lung adenocarcinoma, it is critical that pathologists are able to give PD-L1 expression status before first-line of treatment. The present study compared PD-L1 expression (clone 22-C3) in decalcified using EDTA or formic acid and non-decalcified lung cancer metastases bone samples. Amongst the 84 bone samples analysed for PD-L1 expression, and independently of decalcification, TPS ≥ 1% was 25.0% and ≥ 50% was 11.4%. There was no significant difference between decalcified samples (n = 45) and non-decalcified samples (n = 39) for both TPS ≥ 1% (p = 0.32) and TPS ≥ 50% (p = 1). To conclude, we confirm decalcified bone metastasis specimens may be used for PD-L1 IHC in routine practice. These results also highlight potentially interesting specificities of the bone microenvironment that should be further studied

Immunohistochemical evaluation of bone metastases

Introduction. Metastases are the most common type of malignancy involving the bone, while bone is the third most frequent site for metastases, after the lung and liver. In some patients, medical history, physical and laboratory exami­nation are not conclusive to identify the primary tumor site. In such cases a bone biopsy and immunohistochemical analysis may contribute to the diagnosis, determination of appropriate treatment and evaluation of prognosis. In this study, we tried to evaluate the imunochistochemical expression in bone metastases. Material and methods. We reviewed 125 patients, with a mean age of 63 years, treated for bone metastases in our institution. All patients received palliative orthopaedic surgery for bone metastatic carcinoma. Fifty-eight patients had already an established diagnosis of the primary tumor, while 67 patients presented metastases with an unknown primary tumor origin. Immunohistochemical analysis was performed to intra-operative bone biopsy specimens. The expression of cytokeratine 7, cytokeratin 20 and the expression of a panel of other organ-specific markers were re­corded. In patients with a known primary tumor, we examined the relationship between the origin of metastases, as suggested by the cytokeratin phenotype, compared with the one indicated by the initial histological diagnosis. We also recorded the efficacy of organ-specific markers to identify the primary tumor origin in epithelial bone metastases and we evaluated the prognosis between patients with a immunohistologically determined primary tumor origin, with those with an undetermined one. Results. Associations of cytokeratine 7 and cytokeratine 20 expression confirmed diagnosis in 51 out of the 58 patients (88%) with a known primary tumor (Cohen’s K test 0.79 SE 0.80, P < 0.0005). Immunohistochemical analysis also contributed to establish the diagnosis of patients with an unknown primary tumor, yielding diagnosis in 35 out of the 67 cases (52%). Patients with an immunochistologically undetermined primary tumor site presented a statisti­cally significant poorer prognosis. Conclusions. Cytokeratine 7 and cytokeratine20 are useful immunochistochemical markers in determining a pre­liminary evaluation of bone metastases. Organ-specific immunohistochemical markers have a reliable role in either suggesting or confirming the possible origin of metastases. An indeterminate immunohistochemical phenotype seems to relate to a less differentiated lesion, with a worse prognosis.

Abstract A39: Expression of DNA methylation-related proteins in metastatic breast cancer

Abstract Purpose: To investigate the expressions of methylation-related proteins (5-methylcytosine [5-meC] and DNA [cytosine-5-]-methyltransferase 1 [DNMT1]) in metastatic breast cancers of variable sites and their associations with clinicopathologic factors. Methods: A total of 126 metastatic breast cancers (31, 36, 11, and 48 bone, brain, liver, and lung metastases, respectively) were made into tissue microarrays, and immunohistochemical staining was performed for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor-2 (HER-2), Ki-67, 5-meC, and DNMT1. Molecular classification was made on the basis of the immunohistochemical staining results of ER, PR, HER-2, and Ki-67, as luminal A, luminal B, HER-2-positive, and triple-negative breast cancers. Results: Methylation-related proteins were differentially expressed based on the metastatic sites. Tumoral and stromal 5-meC showed the lowest expression in bone metastases (P &amp;lt; 0.001), while tumoral DNMT1 showed the lowest expression in bone metastases and the highest expression in brain metastases (P &amp;lt; 0.001). Expression of DNMT1 correlated with ER-negativity (P = 0.004), PR-negativity (P = 0.011), HER-2-positivity (P = 0.016), higher Ki-67 labeling indices (P = 0.016), and non-luminal A type (P = 0.017). Univariate analysis revealed that DNMT1-positivity was associated with shorter overall survival in patients with bone (P = 0.017) and lung metastases (P = 0.028). Conclusion: In metastatic breast cancer, methylation-related proteins are differentially expressed according to the metastatic sites. Tumoral and stromal 5-meC showed the lowest expression in bone metastases, while tumoral DNMT1 expression was low in bone metastases and the highest in brain metastases. Citation Format: Lee Yukyung, Kim Eun-Sol, Koo Ja Seung. Expression of DNA methylation-related proteins in metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A39.

Abstract P2-04-23: A monoclonal antibody against hypo-glycosylated bone sialoprotein II has application for diagnostic purposes in samples of breast cancer patients and for treatment of skeletal metastasis caused by MDA-MB-231 breast cancer cells in rats

Abstract The SIBLING protein bone sialoprotein II (BSP) has been implicated in lytic skeletal metastasis as it is expressed in a subset of primary breast cancers and can be detected at elevated levels in the serum of patients with increased risk to develop skeletal metastasis. The aim of this study was to investigate the potential application of a rat monoclonal antibody against hypo-glycosylated BSP (IDK1) for diagnostic and therapeutic purposes. The diagnostic part of this study was based on breast cancer specimens from the biobank / repository of the Institute of Pathology of the Municipal Hospital Kassel, Germany. Immune-histochemical analyses were performed with IDK1 for comparing BSP expression between ten human primary breast tumor sections and their corresponding bone metastatic tissue samples. The therapeutic part of this study was based on a model in nude rats, in which the rats were implanted with human MDA-MB-231 breast cancer cells for selective and orthotopic appearance of osteolytic skeletal lesions. Tumor bearing rats were treated with IDK1 starting at two or four weeks after tumor cell inoculation into the femoral artery of one hind leg. Tumor growth was monitored by light emission, caused by luciferase mediated metabolism of luciferin. Photon emission was recorded at regular intervals by a Xenogen IVIS 100 imaging system. After sacrifice, samples of lesions and apparently healthy tissues were investigated by H&amp;E staining as well as by immune-histological staining for BSP. BSP staining was found within the cytoplasm of tumor cells. Increased expression of BSP was also detected in healthy bone cells, e.g. osteoblasts, as soon as breast tumor cells invaded bone tissue. An elevation of BSP expression near necrotic centers was also found. Expression of BSP in primary breast tumors was positively correlated with BSP expression in bone metastases. Furthermore, bone metastases showed higher and more intensive expression of BSP than their respective primary breast tumors (p&amp;lt;0.0039). In the experimental treatment part, all but one untreated tumor bearing rats showed rapid tumor growth accompanied with lytic destruction of femur and tibia of the respective hind leg (18/19; tumor take rate 95%). In contrast, rats treated with the anti-BSP antibody did not show a significant increase in light emission nor a clinical deterioration. In fact, 8 of 10 rats receiving the antibody at a dose of 10 mg/kg/week starting at two weeks after tumor implantation did not show any light emission after 4 to 6 weeks (p = 0.01 versus control) as well as 6 of 10 rats receiving the antibody at the same dose starting at four weeks after tumor implantation (p &amp;lt; 0.05). Radiological and histological examination confirmed that animals without light emission were free of tumor growth, corresponding to a complete remission. In conclusion, the rat monoclonal antibody directed against BSP is a powerful tool with potential for diagnostic and therapeutic applications in breast cancer skeletal metastasis and warrants further development. Citation Format: Berger MR, Zepp M, Westphal G, Berger I, Armbruster FP. A monoclonal antibody against hypo-glycosylated bone sialoprotein II has application for diagnostic purposes in samples of breast cancer patients and for treatment of skeletal metastasis caused by MDA-MB-231 breast cancer cells in rats [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 P2-04-23.

EpCAM Expression in Lymph Node and Bone Metastases of Prostate Carcinoma: A Pilot Study.

There is an urgent need for new imaging modalities in prostate carcinoma staging. A non-invasive modality that can assess lymph node and bone metastases simultaneously is preferred. Epithelial cell adhesion molecule (EpCAM) is a membranous protein of interest as an imaging target since it is overexpressed in prostatic carcinoma compared with benign prostate epithelium and compared with stroma. However, EpCAM expression in lymph node metastases is sparsely available in the literature and EpCAM expression in bone metastases is yet unknown. The current study evaluates the expression of EpCAM in prostate carcinoma lymph nodes, in matched normal lymph nodes, in prostate carcinoma bone metastases, and in normal bone by immunohistochemistry. EpCAM was expressed in 100% of lymph node metastases (21 out of 21), in 0% of normal lymph nodes (0 out of 21), in 95% of bone metastases (19 out of 20), and in 0% of normal bone (0 out of 14). Based on these results, EpCAM may be a feasible imaging target in prostate carcinoma lymph node and bone metastases. Prospective clinical trials are needed to confirm current results. Preoperative visualization of prostate carcinoma metastases will improve disease staging and will prevent unnecessary invasive surgery.

Abstract 2198: Integrin αvβ3-targeted lipase-labile docetaxel-prodrug micelles preferentially treat breast cancer bone metastases

Abstract Background: Bone metastases occur in 70% of metastatic breast cancer patients and are a leading cause of morbidity. Current therapies are often palliative, in part due to a lack of specificity for tumor targets within the bone. Integrin αvβ3 is overexpressed on neo-angiogenic blood vessels, tumor-promoting macrophages, osteoclasts, and more aggressive breast cancer cells, making it an attractive therapeutic target. Objective: The goal of this study was to use Sn2 lipase-labile docetaxel-prodrug nanoparticle, targeted against activated integrin αvβ3, to attenuate breast cancer metastases. Methods: A novel phospholipid-based micelle (∼12.5 nm) was functionalized with a peptidomimetic for activated integrin αvβ3, and designed to carry either rhodamine for fluorescent labeling or Sn2 lipase-labile prodrug of docetaxel (DTX-PD) for drug delivery. For microscopic localization studies, fluorescently labeled micelles were prepared with or without integrin αvβ3-targeting. C57BL/6 female mice received MMTV-PyMT breast cancer cell line (luciferase-labeled) via intracardiac (IC) injection to achieve tumor metastasis in all major organs. On day 8 post-IC injection, micelle preparations were administered i.v. and circulated within C57BL/6 mice for 3 hours prior to sacrifice and tissue collection. For drug efficacy studies, Sn2 lipase-labile docetaxel-prodrug was incorporated into αvβ3-micelles (αvβ3-DTX-PD). C57BL/6 female mice IC injected with MMTV-PyMT cells (luciferase-labeled) were treated with αvβ3-DTX-PD, or molar equivalent dose of free-DTX, or saline. Beginning on day 4 post-IC injection, mice were treated 3 times, once every 3 days (1.85mg/kg DTX per treatment). On day 12 post-IC injection, metastatic burden in the major organs was analyzed via ex vivo bioluminescent imaging. Results: Fluorescent histological analysis of the tibiofemoral bone region showed significant colocalization of αvβ3-micelles with breast cancer bone metastases, as compared with non-targeted micelles (6.5-fold increase, p&amp;lt;0.001). αvβ3-micelles did not localize within tumor-free bones. In drug efficacy analysis, αvβ3-DTX-PD micelles significantly attenuated bone metastases (3-fold, p = 0.007) and significantly reduced osteolytic bone destruction as assessed by x-ray analysis (3.5-fold, p = 0.021). Serum chemistry analysis of enzymes indicative of liver damage were elevated in the DTX treated group, but not in αvβ3-DTX-PD or saline treated groups. Interestingly, αvβ3-DTX-PD micelles did not attenuate breast cancer metastases within the liver, lungs, or kidneys. Histological examination of metastatic tumor tissue demonstrated elevated integrin β3 expression in bone metastases, as compared to the other sites. Conclusion: These findings suggest that the unique elevated expression of integrin αvβ3 within breast cancer bone metastases could be exploited with αvβ3-DTX-PD micelles for effective therapy. Citation Format: Michael H. Ross, Alison K. Esser, Anne H. Schmieder, Grace Cui, Xiaoxia Yang, Xinming Su, Dipanjan Pan, Gregory M. Lanza, Katherine N. Weilbaecher. Integrin αvβ3-targeted lipase-labile docetaxel-prodrug micelles preferentially treat breast cancer bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2198.

Extratumoral Heme Oxygenase-1 (HO-1) Expressing Macrophages Likely Promote Primary and Metastatic Prostate Tumor Growth.

Aggressive tumors induce tumor-supporting changes in the benign parts of the prostate. One factor that has increased expression outside prostate tumors is hemoxygenase-1 (HO-1). To investigate HO-1 expression in more detail, we analyzed samples of tumor tissue and peritumoral normal prostate tissue from rats carrying cancers with different metastatic capacity, and human prostate cancer tissue samples from primary tumors and bone metastases. In rat prostate tumor samples, immunohistochemistry and quantitative RT-PCR showed that the main site of HO-1 synthesis was HO-1+ macrophages that accumulated in the tumor-bearing organ, and at the tumor-invasive front. Small metastatic tumors were considerably more effective in attracting HO-1+ macrophages than larger non-metastatic ones. In clinical samples, accumulation of HO-1+ macrophages was seen at the tumor invasive front, almost exclusively in high-grade tumors, and it correlated with the presence of bone metastases. HO-1+ macrophages, located at the tumor invasive front, were more abundant in bone metastases than in primary tumors. HO-1 expression in bone metastases was variable, and positively correlated with the expression of macrophage markers but negatively correlated with androgen receptor expression, suggesting that elevated HO-1 could be a marker for a subgroup of bone metastases. Together with another recent observation showing that selective knockout of HO-1 in macrophages reduced prostate tumor growth and metastatic capacity in animals, the results of this study suggest that extratumoral HO-1+ macrophages may have an important role in prostate cancer.

High neuropeptide Y release associates with Ewing sarcoma bone dissemination - in vivo model of site-specific metastases.

Ewing sarcoma (ES) develops in bones or soft tissues of children and adolescents. The presence of bone metastases is one of the most adverse prognostic factors, yet the mechanisms governing their formation remain unclear. As a transcriptional target of EWS-FLI1, the fusion protein driving ES transformation, neuropeptide Y (NPY) is highly expressed and released from ES tumors. Hypoxia up-regulates NPY and activates its pro-metastatic functions. To test the impact of NPY on ES metastatic pattern, ES cell lines, SK-ES1 and TC71, with high and low peptide release, respectively, were used in an orthotopic xenograft model. ES cells were injected into gastrocnemius muscles of SCID/beige mice, the primary tumors excised, and mice monitored for the presence of metastases. SK-ES1 xenografts resulted in thoracic extra-osseous metastases (67%) and dissemination to bone (50%) and brain (25%), while TC71 tumors metastasized to the lungs (70%). Bone dissemination in SK-ES1 xenografts associated with increased NPY expression in bone metastases and its accumulation in bone invasion areas. The genetic silencing of NPY in SK-ES1 cells reduced bone degradation. Our study supports the role for NPY in ES bone invasion and provides new models for identifying pathways driving ES metastases to specific niches and testing anti-metastatic therapeutics.

Abstract 4599: Negative feedback loop between serum response factor and androgen receptor in castrate-resistant prostate cancer

Abstract Despite initial response to androgen ablation, patients with advanced disease relapse to develop castrate-resistant prostate cancer (CRPC) which is difficult to treat. We previously identified Serum Response Factor (SRF) as an important transcription factor in an in vitro model of CRPC. Since CRPC is associated with androgen receptor (AR) hypersensitivity and previous studies linked SRF with AR, we investigated the relationship between SRF and AR. DHT stimulation increased SRF transcriptional activity in both parental LNCaP cells and their castration-resistant Abl subline. To investigate AR involvement in this process, AR expression was down-regulated using siRNA, resulting in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in the AR-negative PC346 DCC cells. However, when AR was over-expressed, SRF induction post-DHT was restored. To explore whether this was reciprocal, SRF was down-regulated by siRNA and AR transcriptional activity and protein expression were assessed. These experiments showed AR increased activity and expression following SRF knock-down, suggesting a negative feedback loop, which was confirmed by immunohistochemistry (IHC) in CRPC bone metastases (n=42), showing a negative association between SRF and AR positivity. This negative feedback was also supported by a computational model based on our experimental data. We next assessed cell proliferation following SRF inhibition and demonstrated that SRF inhibition by siRNA or CCG-1423 was as effective as AR inhibition in decreasing cell proliferation. Moreover concomitant inhibition of AR and SRF showed a synergistic effect. To confirm SRF relevance in clinical samples, we performed IHC staining for SRF on 94 primary tumours and 21 castration-resistant patients who received a transurethral resection of the prostate. This analysis showed that SRF nuclear staining was associated with castration-resistance with 95% of castration-resistant patients showing positive SRF staining versus only 50% of primary tumours. In addition SRF expression in bone metastases in a cohort of 38 patients who died of CRPC, was an independent predictor of survival from (a) prostate cancer diagnosis and (b) diagnosis with CRPC. Overall our data highlight the importance of SRF in CRPC and support SRF as a promising therapeutic target alone or in combination with current treatments. Citation Format: Maria Prencipe, Gillian O'Hurley, Amanda O'Neill, Dara Lundon, Lan K. Nguyen, Susie Boyce, Colm Morrissey, Helmut Klocker, Elaine W. Kay, William Gallagher, William Watson. Negative feedback loop between serum response factor and androgen receptor in castrate-resistant prostate cancer. [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 4599. doi:10.1158/1538-7445.AM2014-4599

The analysis of serum response factor expression in bone and soft tissue prostate cancer metastases

Castration-resistant prostate cancer (CRPC) represents a challenge to treat with no effective treatment options available. We recently identified serum response factor (SRF) as a key transcription factor in an in vitro model of castration resistance where we showed that SRF inhibition resulted in reduced cellular proliferation. We also demonstrated an association between SRF protein expression and CRPC in a cohort of castrate-resistant transurethral resections of the prostate (TURPS). The mechanisms regulating the growth of CRPC bone and visceral metastases have not been explored in depth due to the paucity of patient-related material available for analysis. In this study, we aim to evaluate SRF protein expression in prostate cancer (PCa) metastases, which has not previously been reported. We evaluated the nuclear tissue expression profile of SRF by immunohistochemistry in 151 metastatic sites from 42 patients who died of advanced PCa. No relationship between SRF nuclear expression and the site of metastasis was observed (P = 0.824). However, a negative association between SRF nuclear expression in bone metastases and survival from (a) diagnosis with PCa (P = 0.005) and (b) diagnosis with CRPC (P = 0.029) was seen. These results demonstrate that SRF nuclear expression in bone metastases is associated with survival, with patients with the shortest survival showing high SRF nuclear expression and patients with the longest survival having low SRF nuclear expression. Our study indicates that SRF is a key factor determining patients' survival in metastatic CRPC and therefore may represent a promising target for future therapies.

Tumour cell survival mechanisms in lethal metastatic prostate cancer differ between bone and soft tissue metastases

The complexity of survival mechanisms in cancer cells from patients remains poorly understood. To obtain a comprehensive picture of tumour cell survival in lethal prostate cancer metastases, we examined five survival proteins that operate within three survival pathways in a cohort of 185 lethal metastatic prostate metastases obtained from 44 patients. The expression levels of BCL-2, BCL-XL, MCL-1, cytoplasmic survivin, nuclear survivin, and stathmin were measured by immunohistochemistry in a tissue microarray. Simultaneous expression of three or more proteins occurred in 81% of lethal prostate cancer metastases and BCL-2, cytoplasmic survivin and MCL-1 were co-expressed in 71% of metastatic sites. An unsupervised cluster analysis separated bone and soft tissue metastases according to patterns of survival protein expression. BCL-2, cytoplasmic survivin and MCL-1 had significantly higher expression in bone metastases (p < 10(-5)), while nuclear survivin was significantly higher in soft tissue metastases (p = 3 × 10(-14)). BCL-XL overexpression in soft tissue metastases almost reached significance (p = 0.09), while stathmin expression did not (p = 0.28). In addition, the expression of MCL-1 was significantly higher in AR-positive tumours. Neuroendocrine differentiation was not associated with specific survival pathways. These studies show that bone and soft tissue metastases from the same patient differ significantly in expression of a panel of survival proteins and that with regard to survival protein expression, expression is associated with the metastatic site and not the patient. Altogether, this suggests that optimal therapeutic inhibition may require combinations of drugs that target both bone and soft tissue-specific survival pathways.

Abstract A11: Categorization of castration-resistant prostate cancer bone metastases according to expression of steroid-converting enzymes and constitutively active androgen receptor variants

Abstract Patients with advanced prostate cancer (PC) are given androgen ablation therapy. After a period of initial remission tumors eventually relapse, predominantly within the bone, and are then termed castration-resistant prostate cancer (CRPC). The majority of CRPC tumors express the androgen receptor (AR) and AR regulated genes. Possible mechanisms behind AR activity in CRPC include steroid synthesis by tumor cells and expression of constitutively active AR variants. We have previously found high levels of cholesterol in prostate cancer bone metastases that theoretically could be the substrate for CYP11 and intra-tumoral synthesis of steroids (Thysell et al, 2010). Steroid-converting enzymes have, as far as we know, not been thoroughly examined in PC bone metastases, and the aim of this study was therefore to analyze the expression of the main enzymes involved in the conversion of cholesterol into testosterone (T) and dihydrotestosterone (DHT) in hormone-naïve (HN) and CRPC bone metastases in comparison to primary tumors. We have previously also shown that the constitutively active AR variant AR-V7 is expressed at high levels in CRPC bone metastases in patients with particularly poor prognosis (Hörnberg et al, 2011). Another aim of this study was therefore to evaluate if CRPC bone metastases could be differentiated according to expression of steroid-converting enzymes and AR-V7 into sub-groups with potentially different mechanisms behind castration-resistance. Expression levels of the enzymes included in the classical and backdoor steroidogenesis pathways were analyzed by applying an Illumina whole-genome array to total RNA extracted from frozen biopsies of prostate tissue obtained at radical prostatectomy and metastasis surgery, respectively. Levels were compared between HN bone metastases (n=10), CRPC bone metastases (n=30), and non-malignant (n=12) and malignant (n=13) areas of the primary tumors. Results were verified using RT-PCR and immunohistochemistry in an extended series of samples. Significant increased gene expression levels of HSD17B10 were found in CRPC compared to HN bone metastases, which possibly indicated enhanced conversion of androstanediol to DHT in CRPC metastases. The CRPC metastases did however not show higher levels of CYP11, CYP17, AKR1C3, HSD3B2, or SRD5A1/2 than the HN metastases, indicating no general induction of the classical steroidogenesis pathway in CRPC bone metastases. The expression levels of AKR1C3 and AKR1C2 were significantly higher, and of SRD5A2 significantly lower, in bone metastases than in primary tumors, with highly variable levels found for AKR1C3. About 20% of the CRPC metastases expressed extremely high levels of AKR1C3 which suggested the likelihood of androstenedione conversion into T in those tumors. Another 20% of the CRPC metastases expressed extreme values of the constitutively active AR-V7. In conclusion, AKR1C3 and AR-V7 expression in bone metastases were able to categorize CRPC patients according to potentially different mechanisms behind castration-resistance, and may furthermore also influence patient response to different types of anti-androgen therapies. Citation Format: Emma Jernberg, Anders Widmark, Sead Crnalic, Anders Bergh, Pernilla Wikström. Categorization of castration-resistant prostate cancer bone metastases according to expression of steroid-converting enzymes and constitutively active androgen receptor variants [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A11.

The a3 Isoform Vacuolar Type H+-ATPase Promotes Distant Metastasis in the Mouse B16 Melanoma Cells

Accumulating evidence indicates that the acidic microenvironments critically influence malignant behaviors of cancer including invasiveness, metastasis, and chemoresistance. Because the vacuolar-type H(+)-ATPase (V-ATPase) has been shown to cause extracellular acidification by pumping protons, we studied the role of V-ATPase in distant metastasis. Real-time PCR analysis revealed that the high-metastatic B16-F10 melanoma cells strongly expressed the a3 isoform V-ATPase compared to the low-metastatic B16 parental cells. Consistent with this, B16-F10 cells created acidic environments in lung metastases by acridine orange staining and strong a3 V-ATPase expression in bone metastases by immunohistochemistry. Immunocytochemical analysis showed B16-F10 cells expressed a3 V-ATPase not only in cytoplasm but also plasma membrane, whereas B16 parental cells exhibited its expression only in cytoplasm. Of note, knockdown of a3 V-ATPase suppressed invasiveness and migration with reduced MMP-2 and MMP-9 expression in B16-F10 cells and significantly decreased lung and bone metastases, despite that tumor growth was not altered. Importantly, administration of a specific V-ATPase a3 inhibitor FR167356 reduced bone metastasis of B16-F10 cells. These results suggest that a3 V-ATPase promotes distant metastasis of B16-F10 cells by creating acidic environments via proton secretion. Our results also suggest that inhibition of the development of cancer-associated acidic environments by suppressing a3 V-ATPase could be a novel therapeutic approach for the treatment of cancer metastasis.

135 CLINICAL SIGNIFICANCE OF CXCL16/CXCR6 EXPRESSION IN PATIENTS WITH PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: We hypothesized that the CXCL16-CXCR6 ligand-receptor system may play an important role in prostate cancer progression. METHODS: Levels of CXCL16 and CXCR6 expression were evaluated in prostate cancer cell lines (PC-3 and LNCaP) and in normal prostate epithelial cells (PrEC) as well as tissues from 354 patients. RESULTS: The immunohistochemical expression of CXCL16/ CXCR6 was greater in PC-3/LNCaP than in PrEC. The expression of CXCL16/CXCR6 was significantly higher in prostate cancer than benign prostatic hypertrophy. Using RT-PCR, the expression of CXCL16/ CXCR6 was greater in PC-3/LNCaP than in PrEC. CXCL16/CXCR6 was weakly detected in lung and liver tissue, whereas CXCL16 was highly expressed in bone metastases. CXCL16 immunostaining was related to the Gleason score, T stage, tumor volume, perineural invasion, and lymph node metastasis. However, biochemical PSA recurrence was not related to the expression of CXCL16/CXCR6. CONCLUSIONS: High CXCL16/CXCR6 expression may be related to aggressive cancer behavior and high CXCL16 expression in bone metastases.

Abstract P1-13-04: Immunohistochemical Evaluation of RANK/RANKL/OPG Axis and CXCR4 in Primary Breast Cancer and Their Role in Bone Metastasization

Abstract Background: The RANK/RANKL/OPG axis plays an important role in the bone metastasization process. CXCL12 is overexpressed in bone and, like its receptor, CXCR4, is a determinant of organ tropism. The objective of the present study was to evaluate whether the expression of these markers in primary breast cancer can predict the onset of bone metastases. Methods: Marker expression was evaluated by immunohistochemical staining in paraffin-embedded sections of primary breast cancers from 40 individuals: 10 patients (median age 64 years, range 48-78) showed no evidence of disease (NED — control group) at a median of 9.8 (range 6.9-11.5) years, while 30 (median age 67 years, range 42-87) had relapsed. In the latter group, 10 (median age 66 years, range 42-87) had visceral metastases (VM — control group) and 20 (median age 69 years, range 42-87) had bone metastases (BM — case group), both confirmed radiologically. The 3 patient subgroups (NED, visceral lesions, bone lesions) were matched for age classes (≥60 years, &amp;gt; 60 years). The study design was reviewed and approved by the local ethics committee. Results: In the overall series, 17.5% of tumors were positive for RANK, 22.5% for OPG and 25% for CXCR4. None of the patients expressed RANKL. RANK and OPG resulted equally expressed in 20% of NED patients, while CXCR4 was expressed in only 10% of this group. OPG was expressed in 20% of VM patients, whereas RANK or CXCR4 were not detected. RANK and OPG positivity was present in 25% of BM patients, while CXCR4 was expressed in 45% of cases. CXCR4 was the only marker with a significantly higher frequency of expression in bone metastases than in visceral lesions (p=0.013). Taking into consideration all the patients without bone involvement (NED plus VM patients), it was observed that CXCR4 expression, alone and in combination with RANK, significantly discriminated between BM patients and the control group (p=0.008 and P&amp;lt;0.001, respectively). ER-positivity and HER2-negative status were observed in 80% and 95% of BM patients, respectively, but neither discriminated between cases and controls. Conclusions: In our study, ER-positivity and HER2- negativity identified a high percentage of bone relapse patients, but also highlighted patients who did not have bone involvement. RANK and CXCR4 expression, on the other hand, would appear to be a more accurate predictor of bone relapse. Such evidence could help to identify patients with a high probability of developing bone metastases, which can be treated with bisphosphonates or other bone-targeted therapies, such as denosumab, in an attempt to prevent distant metastases. Our results are currently being validated in a larger series of breast cancer patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-13-04.