The role of estrogens and the microenvironment in lymphoma progression and drug sensitivity

Sammanfattning: Lymphomas are a group of cancers which originate from lymphatic cells. More than 90% of the lymphomas belong to the group of Non-Hodgkin lymphoma (NHL), which in itself is a non-homogeneous group consisting of different NHL subtypes. Traditionally, lymphomas are not thought of as hormone-related cancers. This is strange considering that epidemiological studies show a clear gender difference in incidence for most lymphomas with men being more affected and females showing a better prognosis when compared to males. The aim of this thesis was to study molecular mechanisms involved in this gender difference with a focus on sex hormones, particularly estrogens acting through estrogen receptor  (ER). Furthermore, since both survival and drug sensitivity of lymphomas can be affected by its microenvironment, we analyzed the impact of stromal cells on ibrutinib sensitivity of mantle cell lymphoma (MCL) and how stromal cell-mediated ibrutinib resistance can be overcome. Paper I. In this study, gender aspects and estrogen regulation of diffuse large B-cell lymphoma (DLBCL) was studied. When studying DLBCL in a xenograft mouse model, we observed a faster tumor growth in males than in females. Tumor growth was significantly inhibited when mice were treated with the ERβ agonist diarylpropionitrile (DPN). This was the case both for germinal center and activated B-cell like subtype DLBCL. In addition, when analyzing nuclear ERβ1 expression by immunohistochemistry in primary DLBCLs, ERβ1 expression was found in 89% of the cases. No gender difference in ER1 expression was found. However, the association between ERβ1 expression and prognosis was complex and shown to be dependent on drug treatment. These results suggest that targeting estrogen signaling via ERβ1 could be a new approach in treating DLBCL. Furthermore, ERβ1 expression might be used as a prognostic marker. Paper II. In this article, we showed that castration of C57BL6 male mice accelerated tumor growth of lymphoma in C57Bl6 mice engrafted with murine EG7 T cell lymphoma cells. By analyzing the tumor tissue, we observed stronger Ki67 and weaker TUNEL staining in castrated mice, indicating increased proliferation and decreased apoptosis. Treatment of intact male mice with the androgen receptor antagonist Bicalutamide had no effect on lymphoma growth, suggesting that signaling via the androgen receptor is not involved in the enhanced EG7 lymphoma growth following castration. In contrast, accelerated lymphoma growth was observed when mice were treated with aromatase inhibitors (AIs), which inhibit androgen-to- estrogen conversion. Similar effects where seen in mice grafted with human Granta-519 MCL cells or when treated with different AIs. Moreover, dihydrotestosterone, which can’t be converted to estrogens by aromatase, did not have any impact of lymphoma growth in male mice. This further supported an indirect effect of androgens on lymphoma growth, which involves the conversion of endogenous testosterone to estrogens. We conclude that blocking estrogen synthesis may accelerate lymphomas growth, which highlights the protective role estrogens may have on lymphoma progression. Furthermore, our results may raise concern for increased lymphoma risk (at least progression) in breast cancer patients treated with AIs. Paper III. In this report we showed that MCL tumor growth derived from engrafted human MCL and Burkitt lymphoma (BL) cells was inhibited by treatment with selective agonists of ER. Using the selective ER agonist DPN, we in tumors derived from Granta-519 MCL cells also demonstrated reduced expression of BAFF and GRB7, two markers of B-cell proliferation and survival. In addition, DPN treatment inhibited the expression of VEGF-C, LYVE-1, CD34 and angiogenin, markers of angiogenesis and lymphangiogenesis. Moreover, by investigating Raji BL cells engrafted to mice, we found that DPN treatment reduced tumor dissemination. Importantly, ER protein expression was demonstrated in primary MCL specimens. These results support that lymphomas are under estrogen control and suggest that treatment with ER agonists may be a new approach in the treatment of at least some lymphoma subtypes. Paper IV. By using a MCL-stromal cell co-culture system, we demonstrate that stromal cells protect MCL from ibrutinib-induced apoptosis and support MCL cell regrowth after drug removal by impairing ibrutinib-mediated repression of phosphoinositide-3-kinase (PI3K)/AKT signaling. Importantly, the stromal cell-mediated ibrutinib resistance was overcome by inhibiting AKT activity using the PI3Kα specific inhibitor BYL719. Enhanced pro-apoptotic effect by combined ibrutinib plus BYL719 treatment was also observed in primary MCL cells when co-cultured with stromal cells. Moreover, the combined treatment with ibrutinib and BYL719 enhanced inhibition of MCL tumor growth in vivo in xenograft experiments. We found that a direct interaction between MCL cells and stromal cell conferred the ibrutinib resistance and that this involved the adhesion molecule VLA-4. Blocking VLA-4 abrogated the ibrutinib resistance as assayed in a cell apoptosis and a cell regrowth assay. Our results suggest that combined treatment with ibrutinib and a PI3Kα inhibitor, alternatively blocking VLA-4, may be a promising therapeutic strategy to overcome stromal cell-mediated ibrutinib resistance in MCL.