Chemotherapy-induced differential cell cycle arrest in B-cell lymphomas affects their sensitivity to Wee1 inhibition

Chemotherapeutic agents, e.g., cytarabine and doxorubicin, cause DNA damage. However, it remains unknown whether such agents differentially regulate cell cycle arrest in distinct types of B-cell lymphomas, and whether this phenotype can be exploited for developing new therapies. We treated various types of B cells, including primary and B lymphoma cells, with cytarabine or doxorubicin, and determined DNA damage responses, cell cycle regulation and sensitivity to a Wee1 inhibitor. We found that cyclin A2/B1 upregulation appears to be an intrinsic programmed response to DNA damage; however, different types of B cells arrest in distinct phases of the cell cycle. The Wee1 inhibitor significantly enhanced the apoptosis of G2 phase-arrested B-cell lymphomas by inducing premature entry into mitosis and mitotic catastrophe, whereas it did not affect G1/S-phase-arrested lymphomas. Cytarabine-induced G1-arrest can be converted to G2-arrest by doxorubicin treatment in certain B-cell lymphomas, which correlates with newly acquired sensitivity to the Wee1 inhibitor. Consequently, the Wee1 inhibitor together with cytarabine or doxorubicin inhibited tumor growth in vitro and in vivo more effectively, providing a potential new therapy for treating B-cell lymphomas. We propose that the differential cell cycle arrest can be exploited to enhance the chemosensitivity of B-cell lymphomas

Fulvestrant inhibits growth of triple negative breast cancer and synergizes with tamoxifen in ER alpha positive breast cancer by up-regulation of ER beta

The estrogen receptor-alpha (ER alpha) is used as a predictive marker for antiestrogen therapy in breast cancer patients. In addition to aromatase inhibitors, ER alpha can be targeted at the receptor level using the receptor modulator tamoxifen or by the pure anti-estrogen fulvestrant. The role of the second ER, ER-beta (ER beta), as a therapeutic target or prognostic marker in breast cancer is still elusive. Hitherto, it is not known if ER alpha+/ER beta+ breast cancers would benefit from a treatment strategy combining tamoxifen and fulvestrant or if fulvestrant exert any therapeutic effects in ER alpha-/ER beta+ breast cancer. Here, we report that fulvestrant up-regulated ER beta in ER alpha+/ER beta+ breast cancer and in triple negative ER beta+ breast cancers (ER alpha-/ER beta+). In ER alpha+/ER beta+ breast cancer, a combination therapy of tamoxifen and fulvestrant significantly reduced tumor growth compared to either treatment alone both in vivo and in vitro. In ER alpha-/ER beta+ breast cancer fulvestrant had potent effects on cancer growth, in vivo as well as in vitro, and this effect was dependent on intrinsically expressed levels of ER beta. The role of ER beta was further confirmed in cells where ER beta was knocked-in or knocked-down. Inhibition of DNA methyltransferase (DNMT) increased the levels of ER beta and fulvestrant exerted similar potency on DNMT activity as the DNMT inhibitor decitabine. We conclude that fulvestrant may have therapeutic potential in additional groups of breast cancer patients; i) in ER alpha+/ER beta+ breast cancer where fulvestrant synergizes with tamoxifen and ii) in triple negative/ER beta+ breast cancer patients, a subgroup of breast cancer patients with poor prognosis.Funding Agencies|Swedish Cancer Society; Swedish Research Council [2013-2457]; LiU-Cancer; Research Funds of Linkoping University Hospital</p

Leptin signals via TGFB1 to promote metastatic potential and stemness in breast cancer.

Epidemiological studies have shown obesity to be linked with poorer outcomes in breast cancer patients. The molecular mechanisms responsible for the increased risk of invasive/metastatic disease with obesity are complex, but may include elevated levels of adipokines such as leptin. Using physiological levels of leptin found in obesity in a novel chronic in vitro treatment model (≤200 ng/ml for 14 days), we confirmed the occurrence of leptin-mediated changes in growth, apoptosis and metastatic behavior, and gene expression changes representing epithelial-to-mesenchymal transition (EMT) and a cancer stem cell (CSC) like phenotype in breast epithelial and cancer cell lines (MCF10A, MCF10AT1, MCF7 and MDA-MB-231). Further, we have discovered that these effects were accompanied by increased expression of TGFB1, and could be significantly reduced by co-treatment with neutralizing antibody against TGFB1, indicating that the induction of these characteristics was mediated via TGFB1. Occurring in both MCF7 and MCF10AT1 cells, it suggests these actions of leptin to be independent of estrogen receptor status. By linking leptin signalling to the established TGFB1 pathway of metastasis / EMT, this study gives a direct mechanism by which leptin can contribute to the poorer outcomes of obese cancer patients. Inhibitors of TGFB1 are in currently in phase III clinical trials in other malignancies, thus identifying the connection between leptin and TGFB1 will open new therapeutic opportunities for improving outcomes for obese breast cancer patients

Leptin decreased cell aggregation, and increased cell migration and invasion.

<p>MCF7 or MCF10AT1 cells in low serum media were treated for 14 days with 200 ng/ml leptin (LEPT) and compared to untreated controls (CTRL). (<b>a</b>) CTRL and (<b>b</b>) LEPT-treated MCF7 cells are morphologically different. <b>(c)</b> Effect on MCF7 cells by hanging drop cell aggregation assay. ***P<0.001 (n = 5 drops, Student’s t-test. Data shown is from one representative experiment of 3 independent experiments.) <b>(d)</b> Effect on invasion of MCF7 cells into matrigel. ***P<0.001 (n = 18–20 wells, Student’s t-test, data combined from 3 independent experiments). <b>(e) and (f)</b> Effect on migration rate of <b>(e)</b> MCF7 and <b>(f)</b> MCF10AT1 cells measured by wound healing scratch assay. ***P<0.001 (n = 17–24 wells, Student’s t-test, data combined from several independent experiments).</p

Increased TGFB1 protein expression in response to leptin.

<p><b>(a and b)</b> FACS histograms showing increased intracellular TGFB1 levels in <b>(a)</b> MCF7 and <b>(b)</b> MCF10AT1 cells after chronic 200 ng/ml leptin treatment. (Red line–untreated control, Blue line–Leptin treated 200 ng/ml for 14 days.) <b>(c and d)</b> Dose responses for TGFB1 protein levels after leptin treatment in <b>(c)</b> MCF7 (n = 6) and <b>(d)</b> MCF10AT1 (n = 6) cells. (ANOVA was used to compare among groups with Tukey’s multiple comparison test.) *P<0.05, **P<0.01, ***P<0.001 compared to CTRL.</p

Leptin increased stem cell characteristics of breast cancer cells.

<p>Effect of leptin treatment (14 d in low serum conditions) on: aldefluor activity in <b>(a)</b> MCF7 (n = 7) and <b>(b)</b> MCF10AT1 (n = 6) breast cancer cells; CD44+/CD24- cell population in <b>(c)</b> MCF7 (n = 6) and <b>(d)</b> MCF10AT1 (n = 6) cultures; mammosphere formation by <b>(e)</b> MCF7 (n = 6) and <b>(f)</b> MCF10AT1 (n = 8) cells. (c-f) Leptin treatment was 200 ng/ml. (ANOVA was used to compare among groups with Tukey’s multiple comparison test (a and b). Student’s t-test was used to compare two groups (c-f).) *P<0.05, **P<0.01, ***P<0.001 compared to CTRL.</p

Neutralizing antibody against TGFB1 blocks leptin mediated actions.

<p>Effect of leptin (200 ng/ml), TGFB1 (2.5 ng/ml), Ab-TGFB1 (5 ng/ml), Ab-TGFB1+Leptin or Ab-TGFB1+TGFB1 treatment (all treatments for 14 days) compared with untreated controls on: <b>(a and b)</b> <i>CDH1</i> mRNA expression in <b>(a)</b> MCF7 & <b>(b)</b> MCF10AT1 cells (qPCR was normalised to <i>HPRT1</i> expression and is presented as fold-change compared to untreated control (CTRL)); <b>(c and d)</b> cell migration rate of <b>(c)</b> MCF7 and <b>(d)</b> MCF10AT1 cells; and <b>(e and f)</b> aldefluor activity of <b>(e)</b> MCF7 and <b>(f)</b> MCF10AT1 cells. (ANOVA was used to compare among groups with Tukey’s multiple comparison test. Data was combined from 2–3 experiments representative of at least 3 independent experiments.) *P<0.05, **P<0.01, ***P<0.001 compared to CTRL.</p

Breast cancer cells express LEPR and increase proliferation in response to leptin.

<p><b>(a)</b><i>LEPR</i> mRNA (qPCR, relative to <i>HPRT1</i>) and <b>(b)</b> LEPR protein expression (western blot) in breast epithelial cell lines. (Student’s t-test was used to compare differences in mRNA levels between pairs of cell lines—MCF10A vs MCF10AT1 and MCF7 vs MDA-MB-231. n = 9–11 wells combined from several experiments.) ***P<0.001. <b>(c)</b> MCF7 and <b>(d)</b> MCF10AT1 cells increased proliferation in response to 72 hr of acute leptin treatment, as measured by MTT assay. (n = 6–8 wells. ANOVA was used to compare among groups with Tukey’s multiple comparison test. **P<0.01, ***P<0.001 compared to control (CTRL)).</p

Sequences of primers used for quantitative RT-PCR.

<p>Sequences of primers used for quantitative RT-PCR.</p