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.     

Up-regulation of bone marrow stromal protein 2 (BST2) in breast cancer with bone metastasis

Background  

Bone metastases are frequent complications of breast cancer. Recent literature implicates multiple chemokines in the formation of bone metastases in breast cancer. However, the molecular mechanism of metastatic bone disease in breast cancer remains unknown. We have recently made the novel observation of the BST2 protein expression in human breast cancer cell lines. The purpose of our present study is to investigate the expression and the role of BST2 in bone metastatic breast cancer.     

Treatment of bone metastases before the onset of pain

Purpose

Bone metastases are often asymptomatic and are not diagnosed until after the onset of bone pain. However, bone structural integrity may have diminished considerably before pain onset, resulting in increased risk of skeletal-related events. Therefore, we evaluated whether bisphosphonate therapy was differentially beneficial depending on initiation before or after the onset of bone pain.

Methods

Exploratory analyses were performed in patients with bone metastases from breast cancer or lung cancer/other solid tumors enrolled in two randomized trials comparing monthly zoledronic acid versus pamidronate (breast cancer) or placebo (lung cancer/other solid tumors). Analyses included proportion of patients with one or more skeletal-related events, time to first skeletal-related event, and skeletal morbidity rate in patients with and without baseline pain.

Results

Approximately 80 % of patients reported baseline pain. Similar to overall trial results, zoledronic acid reduced the skeletal morbidity rate in all groups. Although some subsets lacked statistical power, benefits were generally greater in patients without baseline pain. For example, in breast cancer, zoledronic acid increased the 25th quartile of time to first skeletal-related event versus pamidronate by 522 days in patients with no baseline pain (median not reached for either group), but by only 10 days in patients with baseline pain. Similar trends were observed in lung cancer.

Conclusions

Benefits from zoledronic acid appeared to be greater if introduced before bone pain onset. Early diagnosis and treatment of bone metastases may delay onset of skeletal-related events.     

Primary breast cancer stem-like cells metastasise to bone,switch phenotype and acquire a bone tropism signature

Background:

Bone metastases represent a common and severe complication in breast cancer, and the involvement of cancer stem cells (CSCs) in the promotion of bone metastasis is currently under discussion. Here, we used a human-in-mice model to study bone metastasis formation due to primary breast CSCs-like colonisation.

Methods:

Primary CD44⁺CD24⁻ breast CSCs-like were transduced by a luciferase-lentiviral vector and injected through subcutaneous and intracardiac (IC) routes in non-obese/severe-combined immunodeficient (NOD/SCID) mice carrying subcutaneous human bone implants. The CSCs-like localisation was monitored by in vivo luciferase imaging. Bone metastatic CSCs-like were analysed through immunohistochemistry and flow cytometry, and gene expression analyses were performed by microarray techniques.

Results:

Breast CSCs-like colonised the human-implanted bone, resulting in bone remodelling. Bone metastatic lesions were histologically apparent by tumour cell expression of epithelial markers and vimentin. The bone-isolated CSCs-like were CD44⁻CD24⁺ and showed tumorigenic abilities after injection in secondary mice. CD44⁻CD24⁺ CSCs-like displayed a distinct bone tropism signature that was enriched in genes that discriminate bone metastases of breast cancer from metastases at other organs.

Conclusion:

Breast CSCs-like promote bone metastasis and display a CSCs-like bone tropism signature. This signature has clinical prognostic relevance, because it efficiently discriminates osteotropic breast cancers from tumour metastases at other sites.     

Post-operative breast cancer patients diagnosed with skeletal metastasis without bone pain had fewer skeletal-related events and deaths than those with bone pain

Background  

Skeletal metastases are often accompanied by bone pain. To investigate the clinical meaning of bone pain associated with skeletal metastasis in breast cancer patients after surgery, we explored whether the presence of bone pain was due to skeletal-related events (SREs) or survival (cause specific death, CSD), retrospectively.     

Atypical femoral fracture after receiving antiresorptive drugs in breast cancer patients with bone metastasis

Background

Atypical femoral fracture (AFF) occurs with minor trauma in patients receiving antiresorptive drugs such as bisphosphonate and denosumab. We hypothesized that patients with bone metastasis who receive higher doses of antiresorptive drugs tend to experience AFF more frequently. This study aimed to investigate the prevalence rate of AFF in patients receiving antiresorptive drugs for bone metastasis of breast cancer.

Methods

Based on the database from our hospital, patients with breast cancer between March and September 2014 were investigated. Thirty-two patients with bone metastasis who received higher doses of antiresorptive drugs were included for analysis and defined as the metastasis (M) group. For the control (C) group, 32 patients in the same period with breast cancer without bone metastasis who did not undergo antiresorptive drug therapy were included. We evaluated the localized periosteal thickening of the lateral cortex (beaking) and femoral neck-shaft angle in CT scout view, the periods from induction of antiresorptive drugs to the appearance of beaking, and the occurrence rate of complete fracture. The 2 groups were compared.

Results

Of the 64 limbs in 32 patients of the M group, 8 limbs in 6 patients showed beaking at the subtrochanteric area (12.5%). After the occurrence of beaking, 5 limbs in 3 patients eventually had a complete fracture with minor trauma (7.8%). On the other hand, no beaking was observed in the C group.

Conclusions

The frequency of AFF in patients with breast cancer receiving bisphosphonate and/or denosumab for bone metastasis was high. More attention should be paid to the occurrence of AFF in these patients than osteoporotic patients.

     

Pain control with zoledronic acid in patients with breast cancer and metastatic bone disease

Background

Effective management of pain associated with metastatic bone disease is paramount in the care of cancer patients. Bisphosphonates are recommended in treating skeletal complications and there is evidence that they also control pain.

Objective

To investigate the effects of zoledronic acid, a potent nitrogen-containing bisphosphonate, in reducing pain in patients with advanced breast cancer and bone metastases.

Patients and methods

Thirty-two patients (age range 37–79 years, median 56 years) with histologically proven breast cancer and bone metastases received a 15-minute infusion of zoledronic acid 4mg every 4 weeks for a mean of eight cycles (range 1–13) in a phase II pilot study. The primary efficacy endpoint was the control of pain as determined by changes in pain and analgesic scores.

Results

Zoledronic acid substantially reduced pain scores compared with baseline, with over 80% of patients reporting mild or no pain at the end of the trial (82% had moderate to very severe pain on entry). The percentage of patients requiring no analgesics increased from 9% to 35% following zoledronic acid treatment. At baseline, 47% of patients required opioids to control pain, whereas 27% required opioids at the end of the trial. Zoledronic acid appeared to be well tolerated, with no patients reporting serious adverse events.

Conclusion

These results support previous data indicating that zoledronic acid is effective in patients with breast cancer and painful bone metastases. Follow-up controlled trials are required to further investigate the role of zoledronic acid in the management of cancer patients with metastatic disease.     

Cost analysis of skeletal-related events in Spanish patients with bone metastases from solid tumours

Purpose

To estimate the cost per skeletal-related event (SRE) in patients with bone metastases secondary to solid tumours in the Spanish healthcare setting.

Methods

Patients diagnosed with bone metastases secondary to breast, prostate or lung cancer were included in this multicentre, observational study. SREs are defined as pathologic fracture (vertebral and non-vertebral fracture), radiation to bone, spinal cord compression or surgery to bone. Health resource utilisation associated with these events (inpatient stays, outpatient, emergency room and home health visits, nursing home stays and procedures) were collected retrospectively for all SREs that occurred in the 97 days prior to enrolment and prospectively during follow-up. Unit costs were obtained from the 2010 eSalud healthcare costs database.

Results

A total of 93 Spanish patients with solid tumours were included (31 had breast cancer, 21 prostate cancer and 41 lung cancer), contributing a total of 143 SREs to this cost analysis. Inpatient stays (between 9.0 and 29.9 days of mean length of stay per inpatient stay by SRE type) and outpatient visits (between 1.7 and 6.4 mean visits per SRE type) were the most frequently reported types of health resources utilised. The mean cost per SRE was between €2,377.79 (radiation to bone) and €7,902.62 (spinal cord compression).

Conclusion

SREs are associated with a significant consumption of healthcare resources that generate a substantial economic burden for the Spanish healthcare system.     

Epithelial cells expressing cytokeratins-19 and bone marrow micrometastases in patients with breast cancer at the time of primary surgery: clinical outcome during long-term follow-up

Purpose

To evaluate the detection of epithelial cells in bone marrow of breast cancer patients as an indicator of metastatic disease.

Patients and methods

Between 2001 and 2005, bone marrow biopsies were taken from 79 breast cancer patients during primary surgery. Specimens were stained immunocytochemically for epithelial cells expressing cytokeratins or epithelial membrane antigen. The long-term outcomes of these patients were analyzed.

Results

In 51 CK-positive results of 79 patients, epithelial cells were found in the bone marrow (BM) biopsies. These patients were designated CK(+). The rate of tumor recurrence or cancer-related death was significantly higher in CK(+) patients than in CK-negative patients. Multivariate analysis using the Cox regression model revealed BM status as a prognostic parameter independent of axillary lymph node status.

Conclusion

Disseminated epithelial cells in BM are associated with poor clinical outcome in breast cancer patients. However, the presence of these cells is not a sufficient parameter, suggesting that epithelial cells in the BM of breast cancer patients at the time of surgery have limited metastatic potential. The role of these cells needs to be further evaluated.     

The presence of disseminated tumour cells in the bone marrow is inversely related to circulating free DNA in plasma in breast cancer dormancy

Background:

The aim of this study was to gain insight into breast cancer dormancy by examining different measures of minimal residual disease (MRD) over time in relation to known prognostic factors.

Methods:

Sixty-four primary breast cancer patients on follow-up (a median of 8.3 years post surgery) who were disease free had sequential bone marrow aspirates and blood samples taken for the measurement of disseminated tumour cells (DTCs), circulating tumour cells (CTCs) by CellSearch and qPCR measurement of overlapping (96-bp and 291-bp) amplicons in circulating free DNA (cfDNA).

Results:

The presence of CTCs was correlated with the presence of DTCs measured by immunocytochemistry (P=0.01) but both were infrequently detected. Increasing cfDNA concentration correlated with ER, HER2 and triple-negative tumours and high tumour grade, and the 291-bp amplicon was inversely correlated with DTCs measured by CK19 qRT-PCR (P=0.047).

Conclusion:

Our results show that breast cancer patients have evidence of MRD for many years after diagnosis despite there being no overt evidence of disease. The inverse relationship between bone marrow CK19 mRNA and the 291-bp amplicon in cfDNA suggests that an inverse relationship between a measure of cell viability in the bone marrow (DTCs) and cell death in the plasma occurs during the dormancy phase of breast cancer.     

Strontium-89 (Sr-89) chloride in the treatment of various cancer patients with multiple bone metastases

Background

Although the use of Sr-89 chloride in the treatment of patients with prostate and breast cancer has been widely reported, little information is available about its use for other malignancies. Here, we retrospectively analyzed the clinical profile of Sr-89 chloride in various patients with painful bone metastases.

Methods

Entry criteria were a pathologically proven malignancy, clinically diagnosed multiple bone metastases, and adequate organ function. Sr-89 chloride (Metastron) was given by single intravenous infusion at 2 MBq/kg over 2 min. Self-reported outcome measures were used as a response index, including pain diary data on a 0–10 numeric rating scale (NRS).

Results

Fifty-four consecutive patients with painful bone metastases were treated with Sr-89 chloride at the National Cancer Center Hospital East between March 2009 and July 2011, consisting of 26 with breast/prostate cancer and 28 with other malignancies (lung 8, head and neck 6, colorectal 6, others 8). Thirteen (24 %) patients experienced a transient increase in pain, which was categorized as a flare-up response. Grade 3–4 anemia was observed in 6 patients, 3 of whom required blood transfusion. Regarding efficacy, response rates and complete response rates were 71.2 % and 34.6 %, respectively, and time to response from the initiation of treatment was 36 days (range, 13–217). No significant difference in response rates was seen between patients with breast/prostate cancer and other cancers (breast/prostate 69.2 %, other 73.1 %; p = 0.76).

Conclusions

As in patients with breast and prostate cancer, Sr-89 chloride is a promising agent for the treatment of painful bone metastases in patients with various other malignancies.     

Targeting heat shock protein 27 (HspB1) interferes with bone metastasis and tumour formation in vivo

Background:

The small stress heat shock protein 27 (Hsp27) has recently turned as a promising target for cancer treatment. Hsp27 upregulation is associated with tumour growth and resistance to chemo- and radio-therapeutic treatments, and several ongoing drugs inhibiting Hsp27 expression are under clinical trial. Hsp27 is now well described to counteract apoptosis and its elevated expression is associated with increased aggressiveness of several primary tumours. However, its role in the later stage of tumour progression and, more specifically, in the later and most deadly stage of tumour metastasis is still unclear.

Methods/results:

In the present study, we showed by qRT–PCR that Hsp27 gene is overexpressed in a large fraction of the metastatic breast cancer area in 53 patients. We further analysed the role of this protein in mice during bone metastasis invasion and establishment by using Hsp27 genetically depleted MDA-MB231/B02 human breast cancer cell line as a model. We demonstrate that Hsp27 silencing led to reduced cell migration and invasion in vitro and that in vivo it correlated with a decreased ability of breast cancer cells to metastasise and grow in the skeleton.

Conclusion:

Altogether, these data characterised Hsp27 as a potent therapeutic target in breast cancer bone metastasis and skeletal tumour growth.     

Breast Osteoblast-like Cells: A Reliable Early Marker for Bone Metastases From Breast Cancer

Background

The development of bone metastasis from breast cancer results from a functional interaction between tumor cells and osteoclasts or osteoblasts. The main aim of this study was therefore to test the hypothesis that the appearance of breast osteoblast-like cells (BOLCs) in primary mammary lesions is a precursor (and hence an early predictor) of the formation of breast cancer metastases to bone.

Diagnostic performance of <Superscript>18</Superscript>F-fluorodeoxyglucose PET/CT and bone scintigraphy in breast cancer patients with suspected bone metastasis

Background

We prospectively compared the diagnostic accuracies of PET/CT and BS in patients with suspected bone metastases from breast cancer.

Methods

This single-institution prospective study included consecutive patients with suspected bone metastases from biopsy-proven breast cancer seen at Tokai University Hospital between September 2011 and March 2014. Inclusion criteria included suspicions for bone metastases (bone pain, elevated alkaline phosphatase, elevated tumor markers, or suspected bone metastases by BS). Two nuclear medicine physicians evaluated PET/CT and BS images.

Results

Thirty patients were initially enrolled in this study. Two were excluded from the analyses because they declined to undergo imaging during follow-up. PET/CT successfully detected bone metastases in all 10 patients finally diagnosed with the condition, whereas BS identified 2. The two methods were not highly concordant in detecting osseous metastases. In 19 of 28 paired studies (68 %), 2 (10 %) were positive for metastasis, and 17 (90 %) were negative. Nine occurrences (32 %) were discordant; of these, 2 were PET/CT positive and BS negative; 5 were PET/CT positive and BS equivocal; one was PET/CT negative and BS equivocal, and one was PET/CT equivocal and BS negative.

Conclusions

Our results indicated that PET/CT was superior to BS for the diagnosis of bone metastases. On the basis of the results of previous studies as well as ours, PET/CT could replace BS as the initial modality to detect bone metastases in patients suspected for the condition.

     

BMP9 regulates cross-talk between breast cancer cells and bone marrow-derived mesenchymal stem cells

Purpose

Breast cancer cells frequently metastasize to distant organs, including bone. Interactions between breast cancer cells and the bone microenvironment are known to enhance tumor growth and osteolytic damage. Here we investigated whether BMP9 (a secretary protein) may change the bone microenvironment and, by doing so, regulate the cross-talk between breast cancer cells and bone marrow-derived mesenchymal stem cells.

Methods

After establishing a co-culture system composed of MDA-MB-231breast cancer cells and HS-5 bone marrow-derived mesenchymal stem cells, and exposure of this system to BMP9 conditioned media, we assessed putative changes in migration and invasion capacities of MDA-MB-231 cells and concomitant changes in osteogenic marker expressionin HS-5 cells and metastases-related genes in MDA-MB-231 cells.

Results

We found that BMP9 can inhibit the migration and invasion of MDA-MB-231 cells, and promote osteogenesis and proliferation of HS-5 cells, in the co-culture system. We also found that the BMP9-induced inhibition of migration and invasion of MDA-MB-231 cells may be caused by a decreased RANK ligand (RANKL) secretion by HS-5 cells, leading to a block in the AKT signaling pathway.

Conclusions

From our data we conclude that BMP9 inhibits the migration and invasion of breast cancer cells, and promotes the osteoblastic differentiation and proliferation of bone marrow-derived mesenchymal stem cells by regulating cross-talk between these two types of cells through the RANK/RANKL signaling axis.     

Menopausal symptoms and bone health in women undertaking risk reducing bilateral salpingo-oophorectomy: significant bone health issues in those not taking HRT

Background:

Women at high ovarian cancer risk, especially those with mutations in BRCA1/BRCA2, are encouraged to undergo bilateral risk-reducing salpingo-oophorectomy (BRRSPO) prior to the natural menopause. The decision to use HRT to cover the period of oestrogen deprivation up to 50 years of age is difficult because of balancing the considerations of breast cancer risk, bone and cardiovascular health.

Methods:

We reviewed by questionnaire 289 women after BRRSPO aged ⩽48 years because of high ovarian cancer risk; 212 (73%) of women responded.

Results:

Previous HRT users (n=67) had significantly worse endocrine symptom scores than 67 current users (P=0.006). A total of 123 (58%) of women had ⩾24 months of oestrogen deprivation <50 years with 78 (37%) never taking HRT. Bone density (DXA) evaluations were available on 119 (56%) women: bone loss with a T score of ⩽−1.0 was present in 5 out of 31 (16%) women with no period of oestrogen deprivation <50 years compared with 37 out of 78 (47%) of those with ⩾24 months of oestrogen deprivation (P=0.03).

Interpretation:

Women undergoing BRRSPO <50 years should be counselled concerning the risks/benefits of HRT, taking into consideration the benefits on symptoms, bone health and cardiovascular health, and that the risks of breast cancer from oestrogen-only HRT appear to be relatively small.     

Different patterns of change in bone turnover markers during treatment with bone-modifying agents for breast cancer patients with bone metastases

Background

Bone-modifying agents are effective for treatment of breast cancer patients with bone metastases. Since their action is mediated through suppression of the osteoclast function, their efficacy can be determined by monitoring bone turnover markers. However, the clinical significance of these markers is yet to be compared.

Methods

For this study, 52 breast cancer patients with bone metastases treated with zoledronic acid (n = 36) or denosumab (n = 22) were enrolled (6 patients were treated sequentially with both agents). Serum tartrate-resistant acid phosphatase-5b (TRACP-5b), pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (1CTP), N-terminal cross-linking telopeptides of type I collagen (NTX) and bone-specific alkaline phosphatase (BAP) were measured at pretreatment and 1, 3 and 6 months after treatment.

Results

Serum TRACP-5b (p < 0.0001), NTX (p = 0.0007) and BAP (p = 0.0032) decreased significantly after treatment. The baseline median value of TRACP-5b (457.5 mU/dL, range 173–1630 mU/dL) decreased to 137 mU/dL (91–795 mU/dL) 1 month after treatment. Reduction in serum NTX and BAP was greatest after 3 and 6 months, respectively. TRACP-5b, NTX and BAP were above normal levels at baseline in 62.5, 25 and 35.3 % of patients, respectively, and nearly 80 % of these patients attained normal levels during the treatment.

Conclusions

Although bone-modifying agents reduced the baseline levels of TRACP-5b, NTX and BAP significantly, the reduction patterns differed. TRACP-5b appears to affect levels most quickly and sensitively, possibly due to its direct link to the number and activity of osteoclasts. These findings suggest that the efficacy of TRACP-5b is clinically significant when considering which bone-modifying agents to use for breast cancer patients with bone metastases.

     

A high-risk 70-gene signature is not associated with the detection of tumor cell dissemination to the bone marrow

Purpose

The 70-gene signature (70-GS) is a prognostic tool, grouping patients in risk groups to assess their need for adjuvant chemotherapy. Tumor cell dissemination to the bone marrow is a marker of minimal residual disease and associated with impaired survival. In this study, we aimed to evaluate whether 70-GS is associated with the presence of disseminated tumor cells (DTCs) in the bone marrow of patients with early breast cancer.

Methods

In patients with hormone receptor-positive HER2-negative early breast cancer, the 70-GS was obtained and the presence of DTCs was immunohistochemically evaluated using cytokeratin staining with the A45-B/B3 antibody.

Results

149 patients were included into the analysis. 40 (27%) had a high-risk 70-GS and 35 (23%) had detectable DTCs in their bone marrow. 9 (22%) of the 40 patients with high-risk 70-GS and 26 (24%) of the 109 patients with a low-risk 70-GS were positive for DTCs (p = 0.863).

Conclusions

As both 70-GS and DTC detection are known prognostic factors but do not seem to correlate, a follow-up on a larger cohort is warranted to evaluate if a combination of the two is able to better stratify the relapse risk in early breast cancer patients.

     

Potential antitumor effects of nitrogen-containing bisphosphonate in hormone receptor negative breast cancer patients with bone metastases

Background  

This retrospective study evaluated, according to hormone receptor status, the antitumor effects of bisphosphonate especially on survival and disease progression in breast cancer patients with metastatic bone disease.