Afadin cooperates with Claudin-2 to promote breast cancer metastasis.

Claudin-2 promotes breast cancer liver metastasis by enabling seeding and early cancer cell survival. We now demonstrate that the PDZ-binding motif of Claudin-2 is necessary for anchorage-independent growth of cancer cells and is required for liver metastasis. Several PDZ domain-containing proteins were identified that interact with the PDZ-binding motif of Claudin-2 in liver metastatic breast cancer cells, including Afadin, Arhgap21, Pdlim2, Pdlim7, Rims2, Scrib, and ZO-1. We specifically examined the role of Afadin as a potential Claudin-2-interacting partner that promotes breast cancer liver metastasis. Afadin associates with Claudin-2, an interaction that requires the PDZ-binding motif of Claudin-2. Loss of Afadin also impairs the ability of breast cancer cells to form colonies in soft agar and metastasize to the lungs or liver. Immunohistochemical analysis of Claudin-2 and/or Afadin expression in 206 metastatic breast cancer tumors revealed that high levels of both Claudin-2 and Afadin in primary tumors were associated with poor disease-specific survival, relapse-free survival, lung-specific relapse, and liver-specific relapse. Our findings indicate that signaling downstream from a Claudin-2/Afadin complex enables the efficient formation of breast cancer metastases. Moreover, combining Claudin-2 and Afadin as prognostic markers better predicts the potential of breast cancer to metastasize to soft tissues.

Comparing thermal conditions inside and outside lactating dairy cattle barns in Canada.

The health, longevity, and performance of dairy cattle can be adversely affected by heat stress. This study evaluated the in-barn condition [i.e., temperature, relative humidity, and resulting temperature-humidity index (THI)] at 9 dairy barns with various climates and farm design-management combinations. Hourly and daily indoor and outdoor conditions were compared at each farm, including both mechanically and naturally ventilated barns. On-site conditions were compared with on-farm outdoor conditions, meteorological stations up to 125 km away, and NASA Power data. Canadian dairy cattle face periods of extreme cold and periods of high THI, dependent on the regional climate and season. The northernmost location (53°N) experienced about 75% fewer hours of THI >68 compared with the southernmost location (42°N). Milking parlors had higher THI than the rest of the barn during milking times. The THI conditions inside dairy barns were well correlated with THI conditions measured outside the barns. Naturally ventilated barns with metal roofs and without sprinklers fit a linear relationship (hourly and daily means) with a slope <1, indicating that in-barn THI exceeded outdoor THI more at lower THI and reached equality at higher THI. Mechanically ventilated barns fit nonlinear relationships, which showed the in-barn THI exceeded outdoor THI more at lower THI (e.g., 55-65) and approached equality at higher THI. In-barn THI exceedance was greater in the evening and overnight due to factors such as decreased wind speed and latent heat retention. Eight regression equations were developed (4 hourly, 4 daily) to predict in-barn conditions based on outdoor conditions, considering different barn designs and management systems. Correlations between in-barn and outdoor THI were best when using the on-site weather data from the study, but publicly available weather data from stations within 50 km provided reasonable estimates. Climate stations 75 to 125 km away and NASA Power ensemble data gave poorer fit statistics. For studies involving many dairy barns, the use of NASA Power data with equations for estimating average in-barn conditions in a population is likely appropriate especially when public stations have incomplete data. Results from this study show the importance of adapting recommendation on heat stress to the barn design and guide the selection of appropriate weather data depending on the aim of the study.

CCN3/Nephroblastoma Overexpressed Is a Functional Mediator of Prostate Cancer Bone Metastasis That Is Associated with Poor Patient Prognosis.

Prostate cancer (PC) commonly metastasizes to the bone, resulting in pathologic fractures and poor prognosis. CCN3/nephroblastoma overexpressed is a secreted protein with a known role in promoting breast cancer metastasis to bone. However, in PC, CCN3 has been ascribed conflicting roles; some studies suggest that CCN3 promotes PC metastasis, whereas others argue a tumor suppressor role for CCN3 in this disease. Indeed, in the latter context, CCN3 has been shown to sequester the androgen receptor (AR) and suppress AR signaling. In the present study, we demonstrate that CCN3 functions as a bone-metastatic mediator, which is dependent on its C-terminal domain for this function. Analysis of tissue microarrays comprising >1500 primary PC patient radical prostatectomy specimens reveals that CCN3 expression correlates with aggressive disease and is negatively correlated with the expression of prostate-specific antigen, a marker of AR signaling. Together, these findings point to CCN3 as a biomarker to predict PC aggressiveness while providing clarity on its role as a functional mediator of PC bone metastasis.

Validation of the prognostic value of NF-κB p65 in prostate cancer: A retrospective study using a large multi-institutional cohort of the Canadian Prostate Cancer Biomarker Network.

BACKGROUND: The identification of patients with high-risk prostate cancer (PC) is a major challenge for clinicians, and the improvement of current prognostic parameters is an unmet clinical need. We and others have identified an association between the nuclear localization of NF-κB p65 and biochemical recurrence (BCR) in PC in small and/or single-centre cohorts of patients. METHODS AND FINDINGS: In this study, we accessed 2 different multi-centre tissue microarrays (TMAs) representing cohorts of patients (Test-TMA and Validation-TMA series) of the Canadian Prostate Cancer Biomarker Network (CPCBN) to validate the association between p65 nuclear frequency and PC outcomes. Immunohistochemical staining of p65 was performed on the Test-TMA and Validation-TMA series, which include PC tissues from patients treated by first-line radical prostatectomy (n = 250 and n = 1,262, respectively). Two independent observers evaluated the p65 nuclear frequency in digital images of cancer tissue and benign adjacent gland tissue. Kaplan-Meier curves coupled with a log-rank test and univariate and multivariate Cox regression models were used for statistical analyses of continuous values and dichotomized data (cutoff of 3%). Multivariate analysis of the Validation-TMA cohort showed that p65 nuclear frequency in cancer cells was an independent predictor of BCR using continuous (hazard ratio [HR] 1.02 [95% CI 1.00-1.03], p = 0.004) and dichotomized data (HR 1.33 [95% CI 1.09-1.62], p = 0.005). Using a cutoff of 3%, we found that this biomarker was also associated with the development of bone metastases (HR 1.82 [95% CI 1.05-3.16], p = 0.033) and PC-specific mortality (HR 2.63 [95% CI 1.30-5.31], p = 0.004), independent of clinical parameters. BCR-free survival, bone-metastasis-free survival, and PC-specific survival were shorter for patients with higher p65 nuclear frequency (p < 0.005). As the small cores on TMAs are a limitation of the study, a backward validation of whole PC tissue section will be necessary for the implementation of p65 nuclear frequency as a PC biomarker in the clinical workflow. CONCLUSIONS: We report the first study using the pan-Canadian multi-centre cohorts of CPCBN and validate the association between increased frequency of nuclear p65 frequency and a risk of disease progression.

Integrin-uPAR signaling leads to FRA-1 phosphorylation and enhanced breast cancer invasion.

BACKGROUND: The Fos-related antigen 1 (FRA-1) transcription factor promotes tumor cell growth, invasion and metastasis. Phosphorylation of FRA-1 increases protein stability and function. We identify a novel signaling axis that leads to increased phosphorylation of FRA-1, increased extracellular matrix (ECM)-induced breast cancer cell invasion and is prognostic of poor outcome in patients with breast cancer. METHODS: While characterizing five breast cancer cell lines derived from primary human breast tumors, we identified BRC-31 as a novel basal-like cell model that expresses elevated FRA-1 levels. We interrogated the functional contribution of FRA-1 and an upstream signaling axis in breast cancer cell invasion. We extended this analysis to determine the prognostic significance of this signaling axis in samples derived from patients with breast cancer. RESULTS: BRC-31 cells display elevated focal adhesion kinase (FAK), SRC and extracellular signal-regulated (ERK2) phosphorylation relative to luminal breast cancer models. Inhibition of this signaling axis, with pharmacological inhibitors, reduces the phosphorylation and stabilization of FRA-1. Elevated integrin αVß3 and uPAR expression in these cells suggested that integrin receptors might activate this FAK-SRC-ERK2 signaling. Transient knockdown of urokinase/plasminogen activator urokinase receptor (uPAR) in basal-like breast cancer cells grown on vitronectin reduces FRA-1 phosphorylation and stabilization; and uPAR and FRA-1 are required for vitronectin-induced cell invasion. In clinical samples, a molecular component signature consisting of vitronectin-uPAR-uPA-FRA-1 predicts poor overall survival in patients with breast cancer and correlates with an FRA-1 transcriptional signature. CONCLUSIONS: We have identified a novel signaling axis that leads to phosphorylation and enhanced activity of FRA-1, a transcription factor that is emerging as an important modulator of breast cancer progression and metastasis.

The Terry Fox Research Institute Canadian Prostate Cancer Biomarker Network: an analysis of a pan-Canadian multi-center cohort for biomarker validation.

BACKGROUND: Refinement of parameters defining prostate cancer (PC) prognosis are urgently needed to identify patients with indolent versus aggressive disease. The Canadian Prostate Cancer Biomaker Network (CPCBN) consists of researchers from four Canadian provinces to create a validation cohort to address issues dealing with PC diagnosis and management. METHODS: A total of 1512 radical prostatectomy (RP) specimens from five different biorepositories affiliated with teaching hospitals were selected to constitute the cohort. Tumoral and adjacent benign tissues were arrayed on tissue microarrays (TMAs). A patient clinical database was developed and includes data on diagnosis, treatment and clinical outcome. RESULTS: Mean age at diagnosis of patients in the cohort was 61 years. Of these patients, 31% had a low grade (≤6) Gleason score (GS), 55% had GS 7 (40% of 3 + 4 and 15% of 4 + 3) and 14% had high GS (≥8) PC. The median follow-up of the cohort was 113 months. A total of 34% had a biochemical relapse, 4% developed bone metastasis and 3% of patients died from PC while 9% died of other causes. Pathological review of the TMAs confirmed the presence of tumor and benign tissue cores for > 94% of patients. Immunohistochemistry and FISH analyses, performed on a small set of specimens, showed high quality results and no biorepository-specific bias. CONCLUSIONS: The CPCBN RP cohort is representative of real world PC disease observed in the Canadian population. The frequency of biochemical relapse and bone metastasis as events allows for a precise assessment of the prognostic value of biomarkers. This resource is available, in a step-wise manner, for researchers who intend to validate prognostic biomarkers in PC. Combining multiple biomarkers with clinical and pathologic parameters that are predictive of outcome will aid in clinical decision-making for patients treated for PC.

Describing perspectives of health care professionals on active surveillance for the management of prostate cancer.

BACKGROUND: Over the last decade, active surveillance has proven to be a safe approach for patients with low-risk prostate cancer. Although active surveillance presents several advantages for both patients and the health care system, all eligible patients do not adopt this approach. Our goal was to evaluate the factors that influence physicians to recommend active surveillance and the barriers that impact adherence to this approach. METHODS: Focus groups (n = 5) were held with physicians who provided care for men with low-risk prostate cancer and had engaged in conversations with men and their families about active surveillance. The experience of health care professionals (HCPs) was captured to understand their decisions in proposing active surveillance and to reveal the barriers and facilitators that affect the adherence to this approach. A content analysis was performed on the verbatim transcripts from the sessions. RESULTS: Although physicians agreed that active surveillance is a suitable approach for low-risk prostate cancer patients, they were concerned about the rapidly evolving and non-standardized guidelines for patient follow-up. They pointed out the need for additional tools to appropriately identify proper patients for whom active surveillance is the best option. Urologists and radiation-oncologists were keen to collaborate with each other, but the role of general practitioner remained controversial once patients were referred to a specialist. CONCLUSIONS: Integration of more reliable tools and/or markers in addition to more specific guidelines for patient follow-up would increase the confidence of both patients and physicians in the choice of active surveillance.

Biopsy Based Proteomic Assay Predicts Risk of Biochemical Recurrence after Radical Prostatectomy.

PURPOSE: Current clinicopathological parameters are insufficient to predict the likelihood of biochemical recurrence in patients with prostate cancer after radical prostatectomy. Such information may help identify patients who would likely benefit from adjuvant radiotherapy rather than active surveillance. A multiplex proteomic assay, previously tested on biopsies and found to be predictive of favorable or unfavorable pathology at radical prostatectomy, was assessed for its predictive value to identify patients at higher risk for biochemical relapse. MATERIALS AND METHODS: Proteomic assays from core needle biopsies of 288 men who subsequently underwent radical prostatectomy at CHUM (Centre hospitalier de l'Université de Montréal) were evaluated for the prediction of subsequent biochemical recurrence. RESULTS: Of the 288 men, biochemical relapse was observed in 47 (16.3%) and metastases were found in 5 (1.7%). Median followup was 68.5 months. The proteomic assay clearly separated patients into 3 categories, including those at low, intermediate and high risk for biochemical relapse (p = 0.0007). Assay scores predicted biochemical relapse on univariate analysis (HR 1.724, p = 0.0002 per 20% change in score), significantly better than other preoperative prognostic parameters. Additionally, the assay score had a significantly higher p value when combined with clinical National Comprehensive Cancer Network® stage compared to stage alone (HR 1.579, p = 0.0017 per 20% change in score). CONCLUSIONS: A protein based assay score derived from diagnostic needle biopsy has strong predictive ability for biochemical relapse after surgery. These results suggest that this assay score can be used at the diagnostic stage to identify patients in whom prostate cancer is potentially more biologically aggressive and active treatment should be considered.

Analysis of active surveillance uptake for low-risk localized prostate cancer in Canada: a Canadian multi-institutional study.

PURPOSE: Although the uptake of active surveillance (AS) appears to be increasing in published series, the uptake in most geographic regions remains largely unknown. Our aim was to examine practice patterns around the use of AS in low-risk prostate cancer in Canada. In addition, we examined regional variations in AS uptake, predictors of AS uptake, and persistent use for 12 months. METHODS: This is a retrospective multicentre review of low-risk patients who underwent a prostate biopsy in 2010 in six centres in four provinces (BC, QC, MB and ON). AS was identified based on chart review and required a minimum of 6 months of follow-up after diagnosis without any active treatment. RESULTS: Of 986 patients, 781 patients (mean age 64 years) were incident cases and over three-quarters (77.3 %) chose AS at diagnosis. There were significant differences in uptake of AS by centre (range 65.0-98.0 %, p ≤ 0.05). Key multivariate predictors of pursuing AS included older age (OR 1.34, p = 0.044), centre (p = 0.021), lower number of cores (OR 1.09, p = 0.025), lower number of positive biopsy cores (OR 0.52, p < 0.001), and lower percent core involvement (OR 0.84, p < 0.001). In total, 516 (85.4 %) men remained on AS over 12 months. Maintenance with AS over 12 months differed by centre, ranging from 64.1 to 93.9 % (p = 0.001). Predictors of maintenance with AS over 12 months included older age, centre, and lower number of positive cores. CONCLUSIONS: Active surveillance is widely practiced across Canada, but important regional differences were observed. Further analyses are required to understand the root causes of differences and to determine whether AS uptake is changing over time.

Canadian Men's perspectives about active surveillance in prostate cancer: need for guidance and resources.

BACKGROUND: In prostate cancer, men diagnosed with low risk disease may be monitored through an active surveillance. This research explored the perspectives of men with prostate cancer regarding their decision-making process for active surveillance to identify factors that influence their decision and assist health professionals in having conversations about this option. METHODS: Focus group interviews (n = 7) were held in several Canadian cities with men (N = 52) diagnosed with prostate cancer and eligible for active surveillance. The men's viewpoints were captured regarding their understanding of active surveillance, the factors that influenced their decision, and their experience with the approach. A content and theme analysis was performed on the verbatim transcripts from the sessions. RESULTS: Patients described their concerns of living with their disease without intervention, but were reassured by the close monitoring under AS while avoiding harmful side effects associated with treatments. Conversations with their doctor and how AS was described were cited as key influences in their decision, in addition to availability of information on treatment options, distrust in the health system, personality, experiences and opinions of others, and personal perspectives on quality of life. CONCLUSIONS: Men require a thorough explanation on AS as a safe and valid option, as well as guidance towards supportive resources in their decision-making.

Granulocytic immune infiltrates are essential for the efficient formation of breast cancer liver metastases.

INTRODUCTION: Breast cancer cells display preferences for specific metastatic sites including the bone, lung and liver. Metastasis is a complex process that relies, in part, on interactions between disseminated cancer cells and resident/infiltrating stromal cells that constitute the metastatic microenvironment. Distinct immune infiltrates can either impair the metastatic process or conversely, assist in the seeding, colonization and growth of disseminated cancer cells. METHODS: Using in vivo selection approaches, we previously isolated 4T1-derived breast cancer cells that preferentially metastasize to these organs and tissues. In this study, we examined whether the propensity of breast cancer cells to metastasize to the lung, liver or bone is associated with and dependent on distinct patterns of immune cell infiltration. Immunohistocytochemistry and immunohistofluorescence approaches were used to quantify innate immune cell infiltrates within distinct metastases and depletion of Gr1+ (Ly-6C and Ly-6G) or specifically Ly-6G+ cells was performed to functionally interrogate the role of Ly-6G+ infiltrates in promoting metastasis to these organs. RESULTS: We show that T lymphocytes (CD3+), myeloid-derived (Gr-1+) cells and neutrophils (Ly-6G+ or NE+) exhibit the most pronounced recruitment in lung and liver metastases, with markedly less recruitment within bone metastatic lesions. Interestingly, these infiltrating cell populations display different patterns of localization within soft tissue metastases. T lymphocytes and granulocytic immune infiltrates are localized around the periphery of liver metastases whereas they were dispersed throughout the lung metastases. Furthermore, Gr-1+ cell-depletion studies demonstrate that infiltrating myeloid-derived cells are essential for the formation of breast cancer liver metastases but dispensable for metastasis to the lung and bone. A specific role for the granulocytic component of the innate immune infiltrate was revealed through Ly-6G+ cell-depletion experiments, which resulted in significantly impaired formation of liver metastases. Finally, we demonstrate that the CD11b+/Ly-6G+ neutrophils that infiltrate and surround the liver metastases are polarized toward an N2 phenotype, which have previously been shown to enhance tumor growth and metastasis. CONCLUSIONS: Our results demonstrate that the liver-metastatic potential of breast cancer cells is heavily reliant on interactions with infiltrating Ly-6G+ cells within the liver microenvironment.

DNA damage signaling and apoptosis in preinvasive tubal lesions of ovarian carcinoma.

OBJECTIVE: High-grade serous ovarian cancer (HGSC) is the most life-threatening gynecological malignancy despite surgery and chemotherapy. A better understanding of the molecular basis of the preinvasive stages might be helpful in early detection and diagnosis. Genetic instability is 1 of the characteristics shared by most human cancers, and its level is variable through precancerous lesions to advanced cancer. Because DNA damage response (DDR) has been described as 1 of the first phases in genomic instability, we investigated the level of DDR activation and the apoptosis pathway in serous tubal intraepithelial carcinoma (STIC), the potential precursor of HGSC. METHODS/MATERIALS: A tissue microarray including 21 benign fallopian tubes, 21 STICs, 17 HGSCs from patients with STICs (associated ovarian cancer [AOC]) from the same individuals, and 30 HGSCs without STICs (non-AOC) was used in this study.Immunohistochemistry was performed to evaluate the level of DDR proteins (pATM, pChk2, γH2AX, 53BP1, and TRF2), apoptosis proteins (Bcl2, BAX, and BIM), and cyclin E. RESULTS: The expression of all DDR proteins increased from benign fallopian tubes to STICs. The level of expression of pATM, pChk2, γH2AX, and TRF2 was also increased in STICs in comparison with AOC. BAX, BIM, and cyclin E expressions were high in STICs, whereas Bcl2 expression was low. Immunohistochemical profiles of AOC and non-AOC were also different. CONCLUSIONS: These results suggest an activation of the DDR and apoptosis pathways in STICs, indicating that genomic instability may occur early in the precancerous lesions of HGSC.

The potential of in ovo-fed amino acids to alleviate the effects of heat stress on broiler chickens: effect on performance, body temperature, and oxidative status during the finisher phase.

The aim of the current study was to investigate the potential of in ovo-fed amino acids (AA) to reduce the effects of heat stress on finishing broiler chickens. To achieve this, a total of 1,400 fertile hatching eggs were randomly distributed into 5 groups (n = 280/group) and injected with one of the following in ovo treatments on embryonic day 18: 52 µL of sterile diluent/egg (CTRL), CTRL + 1.0 mg of L-Leucine (T1), CTRL + 0.45 mg of leucine + 1.15 mg of methionine (T2), CTRL + 3.0 mg of methionine + 2.0 mg of cysteine (T3), and CTRL + 0.40 mg of leucine + 1.60 mg of methionine + 1.60 mg of cysteine (T4). After hatch, chicks were allocated according to a complete randomized block design comprising 2 thermal conditions: thermoneutral (24°C, 45% RH) and heat stress (34°C, 55-60% RH) with 5 pens/group/condition. The cyclical heat stress regimen (10 h/d) was then applied from d 29 to d 34. Compared to the CTRL group, T3 and T4 exhibited a higher BW during the starter phase (P < 0.001). T4 also had a lower feed conversion ratio (FCR) than CTRL during this same phase (P = 0.03). During the grower phase, males of all treatment groups consistently exhibited higher BW compared to the CTRL group, which was not observed among female birds (PSex × TRT = 0.005). During the finisher phase, the in ovo treatment effect on performance was not significant. However, heat-stressed birds from treatment group T3 and T4 exhibited lower facial temperatures (Pday × TRT < 0.001) as well as lower plasma (Pcondition x TRT = 0.039) and liver (Pcondition x TRT < 0.001) malonaldehyde concentrations compared to the CTRL group. In conclusion, in ovo-fed AA have the potential to modulate the effects of heat stress on finishing broiler chickens by limiting its detrimental consequences, including increased body temperature and oxidative damage.

UGT2B28 accelerates prostate cancer progression through stabilization of the endocytic adaptor protein HIP1 regulating AR and EGFR pathways.

The androgen inactivating UGT2B28 pathway emerges as a predictor of progression in prostate cancer (PCa). However, the clinical significance of UGT2B28 tumoral expression and its contribution to PCa progression remain unclear. Using the Canadian Prostate Cancer Biomarker Network biobank (CPCBN; n = 1512), we analyzed UGT2B28 tumor expression in relation to clinical outcomes in men with localized PCa. UGT2B28 was overexpressed in tumors compared to paired normal adjacent prostatic tissue and was associated with inferior outcomes. Functional analyses indicated that UGT2B28 promoted cell proliferation, and its expression was regulated by the androgen receptor (AR)/ARv7. Mechanistically, UGT2B28 was shown to be a protein partner of the endocytic adaptor protein huntingtin-interacting protein 1 (HIP1), increasing its stability and priming AR/epidermal growth factor receptor (EGFR) pathways, leading to ERK1/2 activation triggering cell proliferation and epithelial-to-mesenchymal transition (EMT). HIP1 knockdown in UGT2B28 positive cells, and dual pharmacological targeting of AR and EGFR pathways, abolished cell proliferative advantages conferred by UGT2B28. In conclusion, UGT2B28 is a prognosticator of progression in localized PCa, regulates both AR and EGFR oncogenic signaling pathways via HIP1, and therefore can be therapeutically targeted by using combination of existing AR/EGFR inhibitors.

ABCC5 supports osteoclast formation and promotes breast cancer metastasis to bone.

INTRODUCTION: Bone is the most common site of breast cancer metastasis, and complications associated with bone metastases can lead to a significantly decreased patient quality of life. Thus, it is essential to gain a better understanding of the molecular mechanisms that underlie the emergence and growth of breast cancer skeletal metastases. METHODS: To search for novel molecular mediators that influence breast cancer bone metastasis, we generated gene-expression profiles from laser-capture microdissected trephine biopsies of both breast cancer bone metastases and independent primary breast tumors that metastasized to bone. Bioinformatics analysis identified genes that are differentially expressed in breast cancer bone metastases compared with primary, bone-metastatic breast tumors. RESULTS: ABCC5, an ATP-dependent transporter, was found to be overexpressed in breast cancer osseous metastases relative to primary breast tumors. In addition, ABCC5 was significantly upregulated in human and mouse breast cancer cell lines with high bone-metastatic potential. Stable knockdown of ABCC5 substantially reduced bone metastatic burden and osteolytic bone destruction in mice. The decrease in osteolysis was further associated with diminished osteoclast numbers in vivo. Finally, conditioned media from breast cancer cells with reduced ABCC5 expression failed to induce in vitro osteoclastogenesis to the same extent as conditioned media from breast cancer cells expressing ABCC5. CONCLUSIONS: Our data suggest that ABCC5 functions as a mediator of breast cancer skeletal metastasis. ABCC5 expression in breast cancer cells is important for efficient osteoclast-mediated bone resorption. Hence, ABCC5 may be a potential therapeutic target for breast cancer bone metastasis.

CCN3 impairs osteoblast and stimulates osteoclast differentiation to favor breast cancer metastasis to bone.

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

In vitro effects of 5-Hydroxy-L-tryptophan supplementation on primary bovine mammary epithelial cell gene expression under thermoneutral or heat shock conditions.

Serotonin (5-HT) is an autocrine-paracrine molecule within the mammary gland regulating homeostasis during lactation and triggering involution after milk stasis. Exposure of dairy cows to hyperthermia during the dry period alters mammary gland involution processes leading to reduced subsequent yields. Herein, primary bovine mammary epithelial cells (pBMEC) under thermoneutral (TN, 37 °C) or heat shock (HS, 41.5 °C) conditions were cultured with either 0, 50, 200, or 500 µM 5-Hydroxy-L-tryptophan (5-HTP; 5-HT precursor) for 8-, 12- or 24-h. Expression of 95 genes involved in 5-HT signaling, involution and tight junction regulation were evaluated using a Multiplex RT-qPCR BioMark Dynamic Array Circuit. Different sets of genes were impacted by 5-HTP or temperature, or by their interaction. All 5-HT signaling genes were downregulated after 8-h of HS and then upregulated after 12-h, relative to TN. After 24-h, apoptosis related gene, FASLG, was upregulated by all doses except TN-200 µM 5-HTP, and cell survival gene, FOXO3, was upregulated by HS-50, 200 and 500 µM 5-HTP, suggesting 5-HTP involvement in cell turnover under HS. Supplementing 5-HTP at various concentrations in vitro to pBMEC modulates the expression of genes that might aid in promoting epithelial cell turn-over during involution in dairy cattle under hyperthermia.

High Keratin-7 Expression in Benign Peri-Tumoral Prostatic Glands Is Predictive of Bone Metastasis Onset and Prostate Cancer-Specific Mortality.

BACKGROUND: New predictive biomarkers are needed to accurately predict metastasis-free survival (MFS) and cancer-specific survival (CSS) in localized prostate cancer (PC). Keratin-7 (KRT7) overexpression has been associated with poor prognosis in several cancers and is described as a novel prostate progenitor marker in the mouse prostate. METHODS: KRT7 expression was evaluated in prostatic cell lines and in human tissue by immunohistochemistry (IHC, on advanced PC, n = 91) and immunofluorescence (IF, on localized PC, n = 285). The KRT7 mean fluorescence intensity (MFI) was quantified in different compartments by digital analysis and correlated to clinical endpoints in the localized PC cohort. RESULTS: KRT7 is expressed in prostatic cell lines and found in the basal and supra-basal compartment from healthy prostatic glands and benign peri-tumoral glands from localized PC. The KRT7 staining is lost in luminal cells from localized tumors and found as an aberrant sporadic staining (2.2%) in advanced PC. In the localized PC cohort, high KRT7 MFI above the 80th percentile in the basal compartment was significantly and independently correlated with MFS and CSS, and with hypertrophic basal cell phenotype. CONCLUSION: High KRT7 expression in benign glands is an independent biomarker of MFS and CSS, and its expression is lost in tumoral cells. These results require further validation on larger cohorts.

The Movember Global Action Plan 1 (GAP1): Unique Prostate Cancer Tissue Microarray Resource.

BACKGROUND: The need to better understand the molecular underpinnings of the heterogeneous outcomes of patients with prostate cancer is a pressing global problem and a key research priority for Movember. To address this, the Movember Global Action Plan 1 Unique tissue microarray (GAP1-UTMA) project constructed a set of unique and richly annotated tissue microarrays (TMA) from prostate cancer samples obtained from multiple institutions across several global locations. METHODS: Three separate TMA sets were built that differ by purpose and disease state. RESULTS: The intended use of TMA1 (Primary Matched LN) is to validate biomarkers that help determine which clinically localized prostate cancers with associated lymph node metastasis have a high risk of progression to lethal castration-resistant metastatic disease, and to compare molecular properties of high-risk index lesions within the prostate to regional lymph node metastases resected at the time of prostatectomy. TMA2 (Pre vs. Post ADT) was designed to address questions regarding risk of castration-resistant prostate cancer (CRPC) and response to suppression of the androgen receptor/androgen axis, and characterization of the castration-resistant phenotype. TMA3 (CRPC Met Heterogeneity)'s intended use is to assess the heterogeneity of molecular markers across different anatomic sites in lethal prostate cancer metastases. CONCLUSIONS: The GAP1-UTMA project has succeeded in combining a large set of tissue specimens from 501 patients with prostate cancer with rich clinical annotation. IMPACT: This resource is now available to the prostate cancer community as a tool for biomarker validation to address important unanswered clinical questions around disease progression and response to treatment.

Expression of ERBB Family Members as Predictive Markers of Prostate Cancer Progression and Mortality.

BACKGROUND: EGFR, ERBB2, ERBB3, and ERBB4 are growth receptors of the ERBB family implicated in the development of epithelial cancers. Studies have suggested a role for EGFR and ERBB3 in the development of prostate cancer (PC), while the involvement of ERBB2 and ERBB4 remains unclear. In this study, we evaluated the expression of all members of the ERBB family in PC tissue from a large cohort and determined their contribution, alone or in combination, as prognostic markers. METHODS: Using immunofluorescence coupled with digital image analyses, we quantified the expression of EGFR, ERBB2, ERBB3, and ERBB4 on radical prostatectomy specimens (n = 285) arrayed on six tissue microarrays. By combining EGFR, ERBB2, and ERBB3 protein expression in a decision tree model, we identified an association with biochemical recurrence (log rank = 25.295, p < 0.001), development of bone metastases (log rank = 23.228, p < 0.001), and cancer-specific mortality (log rank = 24.586, p < 0.001). CONCLUSIONS: Our study revealed that specific protein expression patterns of ERBB family members are associated with an increased risk of PC progression and mortality.