Microcalcifications in breast cancer: Novel insights into the molecular mechanism and functional consequences

Shane O’Grady, PhD student, RCSI

Cancer is a disease that will, unfortunately, touch all of our lives at some point, directly or indirectly. Despite decades of gradual, hard-won incremental improvements to available treatment options, survival is still strongly associated with the stage at which a tumour is first detected: patients diagnosed with stage 4 breast cancer have a 5 year survival rate approximately 4 times lower than those presenting in the clinic with a stage 1 tumour [1]. The ability to detect breast cancer at an early, more easily treated stage has been a significant contributor to improved survival rates observed in recent decades. The adoption of mammographic screening programmes in many countries has been linked to a marked increase in early detection and improved prognosis. In 2015 (latest available statistics) over 145,000 women were screened in Ireland under the government funded BreastCheck programme, resulting in the detection of 986 cancers.

Breast tumours can be detected by several features in mammogram images but one of the most common are the presence of small deposits of calcified material known as microcalcifications. Whilst these calcifications do not constitute a definitive cancer diagnosis (indeed, most will be subsequently confirmed as benign findings by biopsy) the detection of microcalcifications is cause for concern and, in many cases, are the only detectable sign to the presence of a tumour. Calcifications can be characterised by their chemical composition (benign conditions usually display calcifications consisting of calcium oxalate whilst cancer associated calcifications are composed of hydroxyapatite) and morphology (calcifications of a linear ”branching” pattern, due to formation along the interior lumen of the breast ductal system are highly suggestive of cancer).

In addition to their efficacy in the detection of breast cancer, the presence of microcalcifications within a breast tumour may confer useful prognostic information. Breast tumours with associated calcifications display a 3-fold increased rate of HER2 overexpression [2]. Microcalcifications have also been associated with decreased survival [3-5], increased risk of recurrence [6-8], high tumour grade [9, 10] and increased likelihood of spread to the lymph nodes [4, 5, 9]. In vitro experiments have also shown a potent pro-inflammatory effect for microcalcifications. Stimulation of breast cancer cells with hydroxyapatite nanoparticles leads to significant upregulations in IL-1β and COX2 expression as well as increased matrix metalloproteinase activity [11-13], suggesting a direct pro-tumourigenic effect of microcalcifications.

Clearly, the presence of microcalcifications in a tumour is a clinically important attribute and a detailed understanding of their formation may inform our understanding of the early stages of breast tumourigenesis. Previous research from our lab developed the first in vitro model of microcalcification formation, demonstrating the role of an active, cell-regulated process with many similarities to physiological, osteoblast-mediated mineralisation [14, 15]. Culturing the 4T1 murine breast cancer cell line with the same reagents used in in-vitro studies to induce mineralisation in osteoblasts results in formation of hydroxyapatite calcifications. Successful mineralisation was highly dependent on expression of the alkaline phosphatase enzyme, which was increased during the mineralisation process.

My PhD research has aimed to expand on earlier findings from our lab by significantly increasing the number of cell lines tested for the ability to mineralise under in vitro conditions and asses the role of several mineralisation-associated genes known to be expressed in breast tumours. Breast cancers display high expression of many genes most typically associated with cells of the skeletal system, and this “osteomimicry” has been shown to significantly alter tumour behaviour. For example, the osteogenic transcription factor RUNX2 has been linked to an increased ability to invade and migrate through nearby tissue, resulting in metastasis [16]. Thus far, the number of these mineralisation-associated genes that have been investigated in relation to breast microcalcifications is low.

We have identified several novel regulators of breast calcification including calcium transport proteins, magnesium ions and inflammatory cytokines. Cells cultured under mineralisation promoting conditions demonstrate upregulation of pro-mineralisation factors including BMP2 and RUNX2 with a parallel loss of anti-mineralisation factors MGP and ENPP1. These results provide direct in vitro evidence for a shift towards an osteogenic phenotype previously hypothesised based on evidence from patient studies. Raman spectroscopy analysis confirmed the chemical composition of in vitro mineralisation samples as hydroxyapatite, a form of calcium-phosphate commonly associated with malignancy. In addition, we have shown that stimulation of breast cancer cells with synthetic hydroxyapatite particles representative of malignant calcifications upregulated a range of inflammatory mediators in a process dependent on calcium signalling. The presence of an inflammatory microenvironment is a significant contributor to cancer progression [17].

The development of an effective in vitro model of mammary calcification has allowed for a significant enhancement in our understanding in the formation of mammary calcifications. This work builds on our previous findings and further demonstrates the role of a cell-mediated process involving an alteration in calcium transport pathways, expression of osteoblastic markers and a loss of endogenous mineralisation inhibitors.

References

1. NCRI. Cancer in Ireland 1994-2015 with estimates for 2015-2017: Annual Report of the National Cancer Registry. 2017;

2. Elias, S.G., et al., Imaging features of HER2 overexpression in breast cancer: a systematic review and meta-analysis. Cancer Epidemiol Biomarkers Prev, 2014. 23(8): p. 1464-83.

3. Tabár, L., et al., A novel method for prediction of long-term outcome of women with T1a, T1b, and 10–14 mm invasive breast cancers: a prospective study. The Lancet, 2000. 355(9202): p. 429-433.

4. Tabar, L., et al., Mammographic tumor features can predict long-term outcomes reliably in women with 1–14-mm invasive breast carcinoma. Cancer, 2004. 101(8): p. 1745-1759.

5. Ling, H., et al., Malignant calcification is an important unfavorable prognostic factor in primary invasive breast cancer. Asia-Pacific Journal of Clinical Oncology, 2013. 9(2): p. 139-145.

6. Qi, X., et al., Mammographic calcification can predict outcome in women with breast cancer treated with breast-conserving surgery. Oncol Lett, 2017. 14(1): p. 79-88.

7. Rauch, G.M., et al., Microcalcifications in 1657 Patients with Pure Ductal Carcinoma in Situ of the Breast: Correlation with Clinical, Histopathologic, Biologic Features, and Local Recurrence. Ann Surg Oncol, 2016. 23(2): p. 482-9.

8. Woodard, G.A., et al., Qualitative Radiogenomics: Association between Oncotype DX Test Recurrence Score and BI-RADS Mammographic and Breast MR Imaging Features. Radiology, 2017: p. 162333.

9. Zheng, K., et al., Relationship between mammographic calcifications and the clinicopathologic characteristics of breast cancer in Western China: a retrospective multi-center study of 7317 female patients. Breast Cancer Res Treat, 2017.

10. Nyante, S.J., et al., The association between mammographic calcifications and breast cancer prognostic factors in a population-based registry cohort. Cancer, 2017. 123(2): p. 219-227.

11. Cooke, M., et al., Phosphocitrate Inhibits Calcium Hydroxyapatite Induced Mitogenesis and Upregulation of Matrix Metalloproteinase-1, Interleukin-1β and Cyclooxygenase-2 mRNA in Human Breast Cancer Cell Lines. Breast Cancer Research and Treatment, 2003. 79(2): p. 253-263.

12. Morgan, M.P., et al., Calcium hydroxyapatite promotes mitogenesis and matrix metalloproteinase expression in human breast cancer cell lines. Molecular Carcinogenesis, 2001. 32(3): p. 111-117.

13. Morgan, M.P., et al., Basic calcium phosphate crystal–induced prostaglandin E2 production in human fibroblasts: Role of cyclooxygenase 1, cyclooxygenase 2, and interleukin-1β. Arthritis & Rheumatism, 2004. 50(5): p. 1642-1649.

14. Cox, R.F., et al., Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation. Br J Cancer, 2012. 106(3): p. 525-537.

15. Cox, R.F., et al., Osteomimicry of Mammary Adenocarcinoma Cells In Vitro; Increased Expression of Bone Matrix Proteins and Proliferation within a 3D Collagen Environment. PLoS ONE, 2012. 7(7): p. e41679.

16. Leong, D.T., et al., Cancer-related ectopic expression of the bone-related transcription factor RUNX2 in non-osseous metastatic tumor cells is linked to cell proliferation and motility. Breast Cancer Res, 2010. 12.

17. Mantovani, A., et al., Cancer-related inflammation. Nature, 2008. 454: p. 436.