Breast cancer is a leading malignancy affecting the female population worldwide. Most morbidity is caused by metastases that remain incurable to date. TGF-β1 has been identified as a key driving force behind metastatic breast cancer, with promising therapeutic implications.
Methods and Findings
Employing immunohistochemistry (IHC) analysis, we report, to our knowledge for the first time, that asporin is overexpressed in the stroma of most human breast cancers and is not expressed in normal breast tissue. In vitro, asporin is secreted by breast fibroblasts upon exposure to conditioned medium from some but not all human breast cancer cells. While hormone receptor (HR) positive cells cause strong asporin expression, triple-negative breast cancer (TNBC) cells suppress it. Further, our findings show that soluble IL-1β, secreted by TNBC cells, is responsible for inhibiting asporin in normal and cancer-associated fibroblasts. Using recombinant protein, as well as a synthetic peptide fragment, we demonstrate the ability of asporin to inhibit TGF-β1-mediated SMAD2 phosphorylation, epithelial to mesenchymal transition, and stemness in breast cancer cells. In two in vivo murine models of TNBC, we observed that tumors expressing asporin exhibit significantly reduced growth (2-fold; p = 0.01) and metastatic properties (3-fold; p = 0.045). A retrospective IHC study performed on human breast carcinoma (n = 180) demonstrates that asporin expression is lowest in TNBC and HER2+ tumors, while HR+ tumors have significantly higher asporin expression (4-fold; p = 0.001). Assessment of asporin expression and patient outcome (n = 60; 10-y follow-up) shows that low protein levels in the primary breast lesion significantly delineate patients with bad outcome regardless of the tumor HR status (area under the curve = 0.87; 95% CI 0.78–0.96; p = 0.0001). Survival analysis, based on gene expression (n = 375; 25-y follow-up), confirmed that low asporin levels are associated with a reduced likelihood of survival (hazard ratio = 0.58; 95% CI 0.37–0.91; p = 0.017). Although these data highlight the potential of asporin to serve as a prognostic marker, confirmation of the clinical value would require a prospective study on a much larger patient cohort.
Our data show that asporin is a stroma-derived inhibitor of TGF-β1 and a tumor suppressor in breast cancer. High asporin expression is significantly associated with less aggressive tumors, stratifying patients according to the clinical outcome. Future pre-clinical studies should consider options for increasing asporin expression in TNBC as a promising strategy for targeted therapy.
Andrei Turtoi and colleagues describe a mechanistic role for stroma-derived asporin in breast cancer development.
Breast cancer is the most common cancer in women worldwide. Nearly 1.7 million new cases were diagnosed in 2012, and half a million women died from the disease. Breast cancer begins when cells in the breast that normally make milk (epithelial cells) acquire genetic changes that allow them to divide uncontrollably and to move around the body (metastasize). Uncontrolled cell division leads to the formation of a lump that can be detected by mammography (a breast X-ray) or by manual breast examination. Breast cancer is treated by surgical removal of the lump or, if the cancer has started to spread, by removal of the whole breast (mastectomy). After surgery, women often receive chemotherapy or radiotherapy to kill any remaining cancer cells, and women whose tumors express receptors for the female sex hormones estrogen and progesterone or for HER2, a growth factor receptor, are treated with drugs that block these receptors; estrogen, progesterone, and HER2 all control breast cell growth. Nowadays, the prognosis (outlook) for women living in high-income countries who develop breast cancer is generally good—nearly 90% of such women are still alive five years after diagnosis.
Why Was This Study Done?
The cells surrounding cancer cells—cancer-associated fibroblasts and other components of the stroma—support cancer growth and metastasis and are good targets for new cancer therapies. But, although there is mounting evidence that cancer cells actively adapt the stroma so that it produces various factors the tumor needs to grow and spread, very few molecules produced by the stroma that might serve as targets for drug development have been identified. Here, the researchers investigate whether a molecule called asporin might represent one such target. Asporin, which is highly expressed in the stroma of breast tumors, inhibits a growth factor called TGF-β1. TGF-β1 is involved in maintaining healthy joints, but is also a key molecule in the development of metastatic breast cancer. Most particularly, it modulates an important step in metastasis called the epithelial to mesenchymal transition and it regulates “stemness” in cancer cells. Stem cells are a special type of cell that can multiply indefinitely; tumor cells often look and behave very much like stem cells.
What Did the Researchers Do and Find?
Using a technique called immunohistochemistry, the researchers first showed that asporin is highly expressed in the stroma of most human breast cancers but not in normal breast tissue. Next, they showed that breast fibroblasts secrete asporin when exposed to conditioned medium from some human breast cancer cell lines (breast cancer cells adapted to grow continuously in the laboratory; conditioned medium is the solution in which cells have been grown). Specifically, conditioned medium from hormone receptor positive cells induced strong asporin expression by breast fibroblasts, whereas medium from breast cancer cells not expressing estrogen or progesterone receptors or HER2 receptors (triple-negative breast cancer cells) suppressed asporin expression. Other experiments showed that TGF-β1 secreted by breast cancer cells induces asporin expression in breast fibroblasts, and that asporin, in turn, inhibits TGF-β1-mediated induction of the epithelial to mesenchymal transition and stemness in breast cancer cells. Triple negative breast cancers appear to inhibit stromal expression of asporin at least in part via expression of the soluble signaling protein interleukin-1β. Notably, in mouse models of triple-negative breast cancer, tumors engineered to express asporin grew slower and metastasized less than tumors not expressing asporin. Finally, among women with breast cancer, asporin expression was low in triple-negative and HER2-positive tumors but significantly higher in hormone receptor positive tumors, and low asporin levels in primary breast lesions were associated with a reduced likelihood of survival independent of hormone receptor and HER2 expression.
What Do These Findings Mean?
These findings suggest that asporin is a stroma-derived inhibitor of TGF-β1 and a tumor suppressor in breast cancer. Importantly, they also provide preliminary evidence that high asporin expression is associated with less aggressive tumors (hormone receptor positive tumors), whereas low asporin expression is associated with more aggressive tumors (triple negative tumors and HER2-positive tumors). Thus, asporin expression might provide a new prognostic marker for breast cancer. However, before asporin can be used as a biomarker to predict outcomes in women with breast cancer and to identify those women in need of more aggressive treatment, these findings need to be confirmed in large prospective clinical studies. If these findings are confirmed, methods for increasing asporin expression in the stromal tissues of triple negative breast cancer could be a promising strategy for targeted therapy for this group of breast cancers, which currently have a poor prognosis.
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001871.
The US National Cancer Institute provides comprehensive information about cancer (in English and Spanish), including detailed information for patients and professionals about breast cancer and an online booklet for patients
Cancer Research UK, a not-for-profit organization, provides information about cancer; its detailed information about breast cancer includes sections on tests for hormone receptors and HER2, on treatments that target hormone receptors and treatments that target HER2, and on triple negative breast cancer
Breastcancer.org is a not-for-profit organization that provides up-to-date information about breast cancer (in English and Spanish), including information on hormone receptor status, HER2 status, and triple negative breast cancer
The UK National Health Service Choices website has information and personal stories about breast cancer; the not-for-profit organization Healthtalk.org also provides personal stories about dealing with breast cancer