Data presented in this work show, for the first time, that SERCA3 protein is expressed in normal breast tissue, with the highest expression levels found in normal lobular epithelial cells. SERCA3 expression was also observed, although at a lower level, in luminal ductal cells as well, whereas myoepithelial cells were consistently negative. This indicates that SERCA3 constitutes a new phenotypic marker that displays a cell-type specific expression pattern in the normal mammary gland.
Of particular interest, we show that SERCA3 expression in acini markedly decreases already in very early and common lobular lesions such as adenosis and lobular hyperplasia without atypia which are non-obligatory low risk precancerous lesions. This observation indicates that fully differentiated normal breast acinar epithelium possesses a specialized endoplasmic reticulum calcium uptake system, which, as reflected by the loss of SERCA3 expression, undergoes significant remodeling at a very early step of hyperplasia/dysplasia.
Calcium-dependent signal transduction constitutes a key component of intracellular signaling networks that control cell growth, differentiation and survival. Calcium-dependent cell activation is initiated by the release of calcium from the endoplasmic reticulum by second messengers such as inositol-1,4,5-tris
Calcium accumulation in this organelle depends entirely on the activity of SERCA enzymes. Therefore, the extent, the spatiotemporal characteristics, frequency and amplitude of calcium release from the endoplasmic reticulum are critically dependent upon, and modulated by, SERCA-dependent calcium transport.28–33
SERCA activity therefore constitutes a major negative feedback mechanism on calcium mobilization from the organelle.
It has been shown earlier that SERCA3 expression undergoes major changes during colon and gastric carcinogenesis.19,20
Whereas SERCA3 is abundantly expressed in gastric and colonic epithelium,20
SERCA3 expression is decreased in colon adenomas, and lost in adenocarcinomas.19
In addition, it has been shown, that the induction of cell differentiation by histone deacetylase inhibitors, as well as the inhibition of the APC/β-catenin-TCF-4 oncogenic pathway by dominant negative TCF-4 lead to the induction of the expression of SERCA3 in colon cancer cells in vitro
SERCA3 expression is correlated also with the state of differentiation of various lymphoid34,35
leukemia cells, and the direct pharmacological modulation of SERCA activity can induce growth factor independence, growth arrest or differentiation in a cell-type dependent manner.19,21,40
These observations, when taken together indicate that SERCA-dependent calcium sequestration into the endoplasmic reticulum can modulate cell growth and differentiation in various cell types.
Epidemiologic, as well as experimental studies indicate that calcium plays an important role in breast cancer prevention and in the control of breast cancer cell growth.38,39
The observation that endoplasmic reticulum calcium pump expression becomes anomalous already in very early non-obligatory precancerous lesions indicates that proper calcium uptake into the organelle is involved in the establishment and maintenance of the fully differentiated breast acinar phenotype, and suggests that a previously unknown defect of calcium accumulation into the endoplasmic reticulum, related to the loss of SERCA3 expression, may be involved in the formation of early/premalignant steps of breast tumorigenesis.
When investigated in invasive ductal or lobular breast carcinoma, SERCA3 expression, although variable, was always decreased when compared to normal acini, and in several cases was below detection limits. While tubular carcinoma, a histologically well differentiated lesion, displayed SERCA3 expression almost similar to normal acini, and stronger than normal ducts, SERCA3 expression in invasive ductal carcinoma was lower than in normal controls (lymphocytes, endothelial cells and normal acini). SERCA3 expression in invasive ductal carcinoma was inversely proportional to Elston-Ellis (EE) grade, as well as to individual components of the EE grading system, with the strongest correlation being observed with nuclear grade. Moreover, when tumors were stratified simultaneously for nuclear grade (NG) and proliferative activity (S), a highly significant correlation could be observed between grade and SERCA3 expression among the NG1/S1, NG2/S2 and NG3/S3 groups. In benign ductal lesions (columnar changes, apocrine metaplasia, ductal hyperplasia with or without atypia), the expression of SERCA3 was decreased when compared to normal controls and remained heterogeneous in the range of weak to moderate expression levels.
When investigated in the context of estrogen and progesterone receptor expression, the loss of SERCA3 expression was found to be correlated with hormone receptor negative status in invasive ductal carcinomas, and an even stronger correlation could be seen when lesions were stratified simultaneously for hormone receptor status and nuclear grade. In addition, SERCA3 expression was also significantly decreased in triple negative (HR0, HER-2 0) carcinomas.
Taken together, data presented in this work show that normal acinar differentiation in breast is associated with strong SERCA3 expression. This is drastically decreased already at the earliest stages of morphologically detectable anomalies of normal acinar architecture, and remains low at further stages of lobular carcinogenesis. Regarding ductal pathology, we show that although normal breast ducts express SERCA3 at significant levels, its expression is comparable or decreased in early benign ductal lesions. In invasive ductal carcinoma SERCA3 expression is inversely correlated with nuclear grade and proliferative activity, as well as with hormone receptor expression. SERCA3 loss is the most marked in high nuclear grade, highly proliferating, hormone receptor negative tumors. The correlation of the degree of SERCA3 expression with several established markers of breast cancer biology connects, for the first time, abnormal endoplasmic reticulum calcium pump expression to breast tumorigenesis.
Cellular calcium homeostasis is maintained by a functionally interconnected network of calcium pumps, calcium channels and calcium binding proteins. The activity of many components of this network is regulated by calcium itself in a concentration-dependent manner, and interconnectedness and partial functional redundancies of the components are typical of the cellular calcium homeostatic “toolkit”.14,33
Decreased SERCA3 expression may thus have complex consequences on global cellular calcium homeostasis, cell survival and responsiveness to external stimuli. SERCA3 has been shown earlier to be associated with the inositol-1,4,5-tris
-phosphate-mobilisable sub-compartment of the endoplasmic reticulum in platelets.41
It is tempting to speculate that SERCA3 loss in breast epithelial cells may also lead to decreased calcium accumulation into the inositol-1,4,5-tris
-phosphate-mobilisable calcium pool, leading to the acquisition by the cell of a more autonomous signaling configuration42
with a decreased or lost capacity to respond to extracellular stimuli that signal via
-phosphate-induced calcium mobilization. These effects may be explored in further investigations using, for example, genetically engineered calcium indicators (GECIs43
) expressed in mammary epithelium in model systems that recapitulate the formation and progression of benign and malignant breast tumors.
In conclusion, observations presented in this work indicate that endoplasmic reticulum calcium pump function may be involved in the process of breast tumorigenesis already at a very early stage. Detection of SERCA3 expression may prove useful for the immunophenotypic characterization of benign and malignant lesions. A better understanding of the molecular implications of the endoplasmic reticulum calcium homeostatic defect reflected by the loss of SERCA3 expression may open new avenues in the understanding of early steps of breast tumorigenesis.