is a non-protein-coding locus that associates with breast cancer risk in women and rats. Here, we have shown that the resistant rat Mcs5a
allele prevents cancer induced by three distinctly acting mammary carcinoma-inducing treatments, indicating that Mcs5a
does not control a specific initial step of mammary epithelial cell transformation. In addition, formation of ISCs [15
] was not affected by Mcs5a
, suggesting that Mcs5a
acts during early carcinoma progression. In the mammary gland transplantation assay, it was demonstrated that Mcs5a
acts in a non-mammary cell-autonomous fashion. This, together with the observation that Fbxo10
differential expression manifests only in T cells [14
], led to the hypothesis that Mcs5a
does not solely function in the mammary parenchyma to modulate mammary cancer susceptibility but instead acts through the immune system. To assess the involvement of the immune system, we conducted a bone marrow transplantation assay, in which we used irradiation to eliminate the recipient rat bone marrow in order to replace it with bone marrow cells from donor rats of the same or opposite Mcs5a
genotype. At the maximal dose of radiation, only partial replacement could be achieved as a higher dose of radiation resulted in ovarian and mammary organ damage that compromised mammary carcinoma development. At the maximal dose of radiation, the mammary carcinoma multiplicities of the control transplant groups S:S and R:R were approximately twofold lower compared with the average DMBA-induced mammary carcinoma multiplicity routinely obtained in non-irradiated rats of the same genotypes (Figure ) and this was due to radiation organ damage. Nevertheless, partial replacement (average of greater than 50%) of the immune cells of the Mcs5a
-resistant congenic rats with immune cells of the susceptible congenic control rats (transplant group S:R) yielded a mammary carcinoma multiplicity intermediate to the control transplant groups R:R and S:S. This intermediate phenotype of partially reconstituted rats is quantitatively comparable to the intermediate mammary carcinoma multiplicity phenotype of rats that are genetically heterozygous for the Mcs5a
-resistant allele [5
]. We concluded that Mcs5a
acts through components of the immune system to modulate mammary carcinoma multiplicity.
Specific T-cell phenotypes are also under control of the synthetically interacting Mcs5a elements that control Fbxo10 transcript levels and mammary carcinoma multiplicity. We found an overabundance of γδTCR+ T cells (important in mucosal cell surface surveillance), but not CD4+ (T helper) and CD8+ (cytotoxic) T cells, in the spleen of Mcs5a-resistant congenic rats compared with susceptible congenic rats. In addition, the CD4+, CD8+, and γδTCR+ T-cell populations harbored an increased percentage of CD62Lhigh-expressing cells in Mcs5a-resistant congenic rats. The resistant Mcs5a allele was also found to be associated with an increased proliferation rate of activated splenic T cells as well as the production of the Th1 cytokines IL-2, IFNγ, and TNF of activated T cells from spleen and, to a greater extent, from inguinal mammary LNs. The production of Th2 or Th17 cytokines was not affected by Mcs5a. T cells from Mcs5a-resistant congenic rats showed lower CD25 expression (IL2Rα) but higher cellular reduced thiol levels and mitochondrial membrane potential and increased IL-2 production. Considering these results, we propose that higher proliferative response in Mcs5a-resistant congenic T cells could be due primarily to increased cytokine signaling and lower oxidative stress.
Considering the altered T-cell homeostasis and functionality associated with the resistant Mcs5a allele, we speculate that T cells act at the mammary gland to control mammary carcinoma susceptibility. To begin to explore this hypothesis, we compared the T-cell population within the mammary epithelium between the susceptible congenic control and the Mcs5a-resistant congenic rats. We found a higher abundance of mammary epithelium-residing CD3+ T cells, mainly consisting of γδTCR+ T cells in the DMBA-treated Mcs5a-resistant congenic rats. In contrast to the splenic γδTCR+ T cells, most of the γδTCR+ T cells in the mammary gland are double-positive for CD4 and CD8 and also expressed NK-receptor P1 CD161α. This result suggests that the mammary γδTCR+ T cells may harbor increased cytotoxic characteristics that are potentially capable of killing emerging tumor cells. In contrast to normal and carcinogen-exposed mammary gland, frank mammary carcinomas have a low abundance of γδTCR+ T cells. The mammary carcinoma T-cell population consists mainly of αβTCR+ T cells. This raises an interesting possibility that immunoprevention of breast cancer versus breast cancer therapy may need to focus on different T-cell class targets, namely γδTCR+ T cells for prevention and αβTCR+ T cells for therapy.
T-cell abundance in the mammary epithelium is consistent with the observations that Mcs5a
-resistant congenic rats have an increased population of CD62Lhigh
-expressing T cells and that CD62L (L-selectin) expression was highest in γδTCR+
as compared with CD4+
splenic T cells (Figure ). L-selectin is a cell adhesion protein involved in leukocyte-endothelial 'rolling' to facilitate extravasation of leukocytes from blood and lymphatic vessels and in homing of leukocytes to secondary LNs and tumors in extralymphoid organs [17
T cells have been implicated in the protection against breast cancer in women. Decreased peripheral abundance of γδTCR+
T cells and their diminished IFNγ production were recently shown to be associated with breast cancer [20
]. Treatment of osteoporosis with bisphosphonates, compounds known as γδTCR+
T-cell agonists [21
], has been associated with reduced breast cancer risk [22
]. Additionally, the Th1 cytokine IFNγ was implicated in tumor immune surveillance in mice [23
Here, we have shown that the human-rat conserved non-protein-coding MCS5A/Mcs5a locus acts in a non-mammary cell-autonomous fashion through the immune system and modifies the homeostasis and functions of T cells. Each of these phenotypes or a combination of them has the potential to underlie the mammary carcinoma resistance phenotype mediated by the resistant Mcs5a allele. Possible mechanisms include immune surveillance or immune cell-produced Th1 cytokines altering the cellular composition/differentiation of the mammary parenchyma or both. In the future, additional mechanistic studies on the immunological aspect of Mcs5a will address the contribution of each of the observed T-cell phenotypes to Mcs5a-mediated mammary carcinoma resistance.