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miROMeS Report Summary

Project ID: 655777
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - miROMeS (miRNA biomarkers of Osseous Metastases in Serum from breast cancer patients)

Reporting period: 2015-06-01 to 2017-05-31

Summary of the context and overall objectives of the project

Breast cancer is increasingly viewed as manageable for many years since patient survival rates have steadily increased, in which 82-88% of patients are expected to be alive after 5 years. However, bone metastasis is a common complication for breast cancer patients, the rate of bone metastasis still affecting over 70% of advanced-stage patients. This is a complicating factor for patient care, as patients may subsequently develop osteolytic fractures or spinal compression, leading to chronic pain. By the time breast cancer bone metastasis is diagnosed, the disease is considered incurable. Therefore it is a necessity to evaluate the predictive value of candidate biomarkers for bone metastasis in breast cancer patients to improve the detection of high-risk patients. Assessing biomarker prognosis value will also permit to adapt treatment for patients at risk of developing bone metastasis by given preventative treatments such as adjuvant bisphosphonates and/or new preventive therapies. This research emphasizes the importance of health and wellbeing where patient quality of life and patient relapse remains a challenge. Identification of patients at risk of bone metastasis development may lead to decreased drug burden, reducing bone-metastasis related fractures and the need for surgery, providing relief to the health care system. Further, in light of increased breast cancer survival, long-term disease management becomes an alternate health objective.
Circulating breast tumor cells that escape the primary tumor and invade the bone marrow express sets of deregulated genes and microRNAs (miRNAs) that facilitate their bone tropism and enhance engraftment of disseminated tumor cell in bone marrow which may subsequently induce osteolytic lesions. Indeed, transcriptional profiling of primary tumors has revealed gene and miRNA signatures that correlate with metastatic bone relapse in patients. Insight into the molecular mechanisms of cancer cell dissemination to bone has been obtained through associations between miRNA expression in primary tumor and metastasis with the involvement of specifically cognate genes during bone metastasis formation. MiRNAs are short and single-stranded RNAs that repress gene expression and that have been suggested as biomarkers of bone disease, driven by their remarkable stability and accessibility in blood and by the property of circulating miRNAs as a reflect of tumor miRNAs.
In this, the utility of miRNAs as biomarkers of bone disease has been studied in the present research that has combined the expertise from the ERS in miRNA biomarker analysis and the host laboratory’s expertise in bone metastasis for the following objectives:
(i)- To identify genes and miRNAs in breast primary tumors the deregulation of which is associated to bone metastasis occurrence.
(ii)- To identify serum miRNA signatures of bone metastasis in breast cancer patients with bone metastasis compared to patients without bone metastasis.
(iii)- To evaluate the predictive value of candidate miRNA biomarkers in a focused miRNA panel using serum from patients with long-term follow-up data

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

(i)- Using a number of publically available data sets, we performed high-throughput analyses on primary tumors and showed that a high lysyl oxidase (LOX) expression in primary tumors from colorectal cancer patients was associated with poor clinical outcome and that LOX was expressed by tumor cells in the bone marrow from colorectal patients with bone metastasis. Indeed, we have shown that LOX is a strong determinant of tumor cell colonization in bone: it promotes survival of tumor cells in the bone marrow and impairs bone homeostasis, leading to the formation of osteolytic lesions.
We have also shown that estrogen related receptor alpha (ERRα) expression is higher in castration-resistant prostate cancer patients with bone metastases than without, and that ERRα in prostate cancer alters molecular signaling in the stroma via the regulation of periostin and TGF expression in bone metastases and in primary tumor.
Finally, we have provided novel evidence that miR-30 family members (miR-30a, miR-30b, miR-30c, miR-30d and miR-30e) act as bone metastasis suppressor genes in breast cancer, inhibiting tumor cell invasion, osteomimicry and bone destruction. We identified a number of genes associated with osteoclastogenesis stimulation (IL-8, IL-11), osteoblastogenesis inhibition (DKK-1), tumor cell osteomimicry (RUNX2, CDH11) and invasiveness (CTGF, ITGA5, ITGB3) that were direct and/or indirect targets for repression by miR-30s. Among these genes, integrin ITGA5 was a previously unknown miR-30 target. By silencing ITGA5 in breast cancer cells, colonization of the bone marrow by tumor cells was drastically reduced in vivo. Overall, our data indicate that miR-30s employ multiple mechanisms to impede breast cancer bone metastasis. These findings may pave the way to a new field of therapeutic interventions in breast cancer patients with bone metastasis.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

(ii)- MiRNA expression that are predictive of patients response were analyzed in serum samples from triple-negative and hormone-sensitive luminal A patients who are metastasis-free (n=3), have bone metastasis (n=3) or soft tissue metastasis only (n=3). Serum samples were extracted by Exiqon Biofluids and expression profiling of the miRome was performed by RT-qPCR on 2x384 well plates for each sample (Exiqon LNA Probe Array). MiRNA level was expressed as relative quantitation calculated using the mean of an external miRNA calibrator and compared to the mean of luminal A and triple-negative serum patients (Ct). Data was analyzed on GenePattern to identify miRNAs that were significantly different between patients having no metastasis compared to patients with visceral and/or bone metastasis in serum from luminal A or triple negative. --> See Figure 1 attached

Each column represents 1 patient (3 patients per groups). Yellow box indicates miRNAs that were significantly differently expressed (t-test, p<0.05).
As regard to luminal breast cancer, statistical analyses identified 58 miRNAs that were differently expressed in serum from patients with bone and visceral metastasis compared to serum from patients with no metastasis. Bioinformatic analysis was conducted on these miRNAs to select a miRNA data set. The miRNAs will be validated by a custom-designed focus panel in the test phase and in the validation phase, using the serum of 40 women with early breast cancer from the Adjuvant Zoledronic Acid to Reduce Recurrence (AZURE) trial who subsequently relapsed at skeletal sites only, visceral sites only or did not relapse at 10+ years follow up. --> See figure 2 attached

As regard to triple negative breast cancer, statistical analysis identified 12 miRNAs that were differently expressed in serum from patients with no metastasis compared to patients with bone or lung metastasis. To establish a miRNA data set that can be used in the test phase and in the validation phase in a cohort, we analyzed samples arising from a patient-derived xenograft model (PDX) that retain the histological and genetic characteristics of the original triple-negative tumor. --> See figure 3

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