The present study aims to define the relationship between HSulf-2 and ductal carcinoma
in situ progression to invasive ductal carcinoma using the MCF10DCIS progression model. Although HSulf-2 has been reported to be up-regulated in breast cancer [
23], its role in breast cancer progression has not been clearly defined. Here we utilized a unique cell line which expresses HSulf-2 and has the ability to form ductal lesions similar to those found in DCIS pathology in the human breast. By utilizing mouse mammary fat pad injections to evaluate the impact of HSulf-2 depleted MCF10DCIS cells on tumor growth, we found that HSulf-2 knockdown significantly attenuated tumor size, promoted apoptosis and retained comedo lesions for a longer period of time. It is notable that apoptosis was predominantly limited to the inner center or luminal area of comedo structures in HSulf-2 depleted xenografts. This indicates that loss of HSulf-2 selectively renders inner luminal cells of comedo lesions to undergo apoptosis presumably due to the tumor microenvironment resulting in culmination of the apoptotic program, which triggers spontaneous apoptosis in comedo lesions [
33]. HSulf-2 loss up-regulated both the number and size of comedo structures with intact basement membrane. A striking feature of HSulf-2 depleted xenografts is the maintenance of the integrity of basement membrane even at later stages (Week 7) of DCIS to IDC progression, which suggests that HSulf-2 presence is essential for basement membrane disintegration. Basement membrane is a physical barrier between epithelial cells and stromal cells. Many MMPs (proteases) have been shown to play important roles in the remodeling of basement membrane and invasion of surrounding tissues [
34]. Importantly, HSulf-2 silencing attenuated transition from DCIS to IDC by limiting MMP-9 expression and activities required for basement membrane degradation. Several members of the MMP family have been shown to be up-regulated prior to progression from DCIS to IDC in MCF10DCIS model [
33]. Proteolysis of extracellular matrix proteins and basement membrane by these proteases results in the disruption of this barrier to promote invasion into surrounding stroma. The effect of HSulf-2 loss was specific to MMP-9, whereas no effect on MMP-2 was observed. MMP-9 has previously been shown to be a predominant matrix protease expressed in ductal lesions [
7]. Our
in vivo data show that HSulf-2 depletion markedly attenuates tumor growth. Supporting this notion, previous studies have identified HSulf-2 as one of the top 50 genes up-regulated in DCIS to IDC [
25]. Similarly, in two different mouse models of mammary carcinoma, HSulf-2 up-regulation was associated with pro-angiogenic activity [
23]. Our data provide a novel insight by raising the possibility that HSulf-2 may play an important role in the disintegration of basement membrane and promoting invasion of surrounding tissue. In addition to retention of comedo lesions even at Week 7 of tumor growth, HSulf-2 deficient xenografts were predominantly apoptotic. Massive apoptosis was evident in the center of comedo lesions and not near the basement membrane. This could be explained in several ways: a) it can be postulated that cells in the center of comedo lesions are often highly hypoxic and have a decreased supply of nutrients and b) these cells are separated from extracellular matrix protein of basement membrane and, hence, lack adhesion, and that HSulf-2 knockdown further sensitizes these cells to apoptosis due to lack of survival signals (growth factor and adhesion mediated). In other words, HSulf-2 depletion might pave way for luminal clearance in these comedo lesions as a result of apoptosis. Previous reports have also documented that HSulf-2 promotes cellular resistance to apoptosis in HCC cell lines [
35]. Our study suggests that progression of DCIS to IDC might depend on HSulf-2 activities. Therefore, therapeutically targeting this enzyme either by shRNA or by a small molecule inhibitor may serve to improve our chances of controlling the progression of DCIS to IDC. Our data do not concur with a more recent study highlighting a tumor suppressor role of HSulf-2 in MDA231 cell line [
24]. This study adequately addressed the role of HSulf-2 in the context of metastatic propensity of highly aggressive MDA231 cell line. However, caution should be exercised, as enhanced expression of HSulf-2 might promote nontargeted effects on tumor growth. Secondly, the specificity of substrates of HSulf-2 -HSPGs located at the cell surface could contribute to the differential response to the presence of HSulf-2 based on the binding affinity of specific HSPGS towards different growth factors. Thus, it is plausible that observed differences could partly depend on the nature of specific substrates (HSPGs) expressed in the different cell lines with HSulf-2 expression. Mechanistically, HSulf-2 has been shown to attenuate bFGF2 signaling but promotes Wnt signaling [
36‐
38]. Activated Wnt signaling is common in mammary tumors despite lack of mutations in Wnt pathway genes [
39]. Therefore, HSulf-2 presence may promote autocrine induction of Wnt signaling during breast tumorigenesis as previously reported [
22].
In all, this is the first report which highlights the critical role of HSulf-2 in the progression of DCIS to IDC in MCF10DCIS cell line xenograft model. Validation of this finding in human tumors could lead to HSulf-2 as a biomarker of breast cancer progression. Additionally, we propose that therapeutic targeting of HSulf-2 could lead to improved clinical outcome in patients with breast cancer