mTOR inhibitors counteract tamoxifen-induced activation of breast cancer stem cells.

Breast cancer cells with stem cell characteristics (CSC) are a distinct cell population with phenotypic similarities to mammary stem cells. CSCs are important drivers of tumorigenesis and the metastatic process. Tamoxifen is the most widely used hormonal therapy for estrogen receptor (ER) positive cancers. In our study, tamoxifen was effective in reducing proliferation of ER + adherent cancer cells, but not their CSC population. We isolated, expanded and incubated CSC from seven breast cancers with or without tamoxifen. By genome-wide transcriptional analysis we identified tamoxifen-induced transcriptional pathways associated with ribosomal biogenesis and mRNA translation, both regulated by the mTOR-pathway. We observed induction of the key mTOR downstream targets S6K1, S6RP and 4E-BP1 in-patient derived CSCs by tamoxifen on protein level. Using the mTOR inhibitors rapamycin, everolimus and PF-04691502 (a dual PI3K/mTOR inhibitor) and in combination with tamoxifen, significant reduction in mammosphere formation was observed. Hence, we suggest that the CSC population play a significant role during endocrine resistance through activity of the mTOR pathway. In addition, tamoxifen further stimulates the mTOR-pathway but can be antagonized using mTOR-inhibitors.

Hodgkin-lymphoma-derived cells show high sensitivity to dactinomycin and paclitaxel.

Depending on stage and risk factors, up to 30% of patients with advanced Hodgkin lymphoma (HL) progress or relapse. Patients with pleural effusions have a particularly poor prognosis and this stage of HL is regularly resistant to chemotherapy. All currently available HL cell lines are derived from late stage HL patients. In the present study we measured the sensitivity of these HL lines against the 26 most frequently used cytostatic drugs. We used a novel fluorescent short-term survival assay where the cell was incubated with the drugs for 4 days. The precise number of differentially stained live and dead cells was determined using a custom-built automated laser confocal fluorescent microscope. We found that HL cells, independently of their origin, showed very similar sensitivity patterns for several of the drugs. All HL cell lines were highly sensitive to dactinomycin, paclitaxel and etoposide. Our data suggest that the inclusion of dactinomycin and paclitaxel into chemotherapy protocols against late stage Hodgkin lymphoma with pleural effusion may be justified.

Differential expression of oestrogen receptors in human secondary lymphoid tissues.

Many autoimmune diseases including rheumatoid arthritis (RA), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) occur much more frequently in women than in men. There is much evidence that oestrogen is the major cause of this gender difference. Interestingly, oestrogen relieves the symptoms of RA and SS but it exacerbates SLE. This contradictory effect of oestrogen on autoimmune diseases is not well understood. Most of the effects of oestrogen are mediated by two receptors: oestrogen receptor alpha and beta (ERalpha and ERbeta). To determine whether these contradictory effects of oestrogen relate to the involvement of distinct effects of the two ERs, we investigated expression of ERalpha and ERbeta in human secondary lymphoid tissues. We observed that, in tonsils, ERbeta is expressed in lymphocytes of germinal centres (GC) and the follicular mantle zone as well as in granulocytes, while ERalpha is expressed only in activated germinal centres but not in the follicular zone. ERbeta is the predominant ER in human leucocytes from peripheral blood, spleen and in leucocytes infiltrating cancers in both males and females. In addition, in different human lymphoma cell lines including Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, ERbeta is abundant while ERalpha is not detectable. Our results indicate that ERbeta is the predominant type of ER in mature lymphocytes. We suggest that ERalpha and ERbeta have distinct roles in secondary lymphoid tissues and that further studies with ERbeta-specific agonists will help to elucidate the role of ERbeta in these tissues.

Autoimmune glomerulonephritis with spontaneous formation of splenic germinal centers in mice lacking the estrogen receptor alpha gene.

In mice, ovariectomy accelerates the progression of the end-stage renal disease glomerulosclerosis. In women, the incidence of this disease increases after menopause, and estrogen alters its progression. Polymorphisms in the human estrogen receptor alpha (ERalpha) gene have been suggested to constitute a genetic predisposition for lupus nephritis. Here we show that by 1 year of age, mice lacking ERalpha (ERalpha(-/-)) but not those lacking ERbeta (ERbeta(-/-)) exhibit immune complex-type glomerulonephritis, proteinuria, and destruction of tubular cells with severe infiltration of B lymphocytes in the kidney and the presence of anti-DNA antibodies in serum. No gender difference occurred in the incidence or severity of these symptoms. However, in female but not in male ERalpha(-/-) mice there were elevated serum levels of IgG3. Other prominent features of these mice were (i) spontaneous formation of germinal centers in the spleen in the absence of antigen challenge and (ii) infiltration of plasma cells in the kidney and plasmacytosis in the spleen. Immunohistochemistry indicated a selective expression of ERalpha protein in the germinal centers but not in the follicular mantle zone of murine spleens and human tonsils. Our results indicate that ERalpha has indispensable functions in the kidney and in germinal centers, and that defective ERalpha signaling results in glomerulonephritis.

Disruption of the estrogen receptor beta gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis.

Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor beta knockout (ERbeta-/-) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15-30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERbeta-/- mice than in WT littermates. Some of the ERbeta-/- mice also had a severe lymphoproliferative phenotype. Thus the absence of ERbeta results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERbeta in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERbeta-/- mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERbeta agonists might have clinical value in the treatment of leukemia.