STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa

CDK4/CDK6 Inhibitors Synergize with Midostaurin, Avapritinib, and Nintedanib in Inducing Growth Inhibition in <i>KIT</i> D816V⁺ Neoplastic Mast Cells

In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V+ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38− stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials

STAT5 is Expressed in CD34+/CD38− Stem Cells and Serves as a Potential Molecular Target in Ph-Negative Myeloproliferative Neoplasms

International audienc

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis

<div><p>A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after <i>toll-like</i> receptor (TLR)-activation and glucose-deprivation or co-treatment with 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an <i>in-vivo</i> mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our <i>in vitro</i> observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone <i>Listeria monocytogenes</i> mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.</p></div

Impact of AMPK activators on IL-12p40/IL-10 induction.

<p>Human monocytes were preincubated for 90 minutes with different concentrations of A-769662 (a, b) or AICAR (c, d) or medium and then stimulated with LPS. Secretion of IL-10 (a, c) and IL-12p40 (b, d) was determined from 20 hr culture supernatants by ELISA. Data are representative of 3–5 independent experiments and presented as % response ± SD. A-769662 treatment alone induced no significant cytokine production: IL-10: 4 times below detection level, IL-12p40: 3 times below detection level 1x 1.8 pg/mL, Similarly, AICAR treatment alone induced no significant cytokine production: IL-10: 3x ≤ 3.1 pg/mL, IL-12p40: 3 times below detection level. *p ≤ 0.05, ***p ≤ 0.01.</p

2-DG protects mice from Listeria monocytogenes <i>(L</i>.<i>m</i>.<i>)</i> infection.

<p>BALB/c mice (n = 5) were pretreated i.p. with 2-DG or PBS for 3 days and then challenged with 5 x 10⁴ <i>L</i>.<i>m</i>. On day 3 after infection the number of bacteria in the spleen and the liver were determined (a). Liver histomorphology was analyzed by H&E staining on day 3 after infection (b). Granulomatous lesions are indicated (by arrows). **p ≤ 0.005. (c) Livers (n = 3 per group) were analyzed by immunohistochemical staining of F4/80 on day 3 post infection. Representative images from each group of mice are shown (brown color, F4/80 staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: 50 μm. Inserts: the high-power views. (d) Quantitative analysis of F4/80 staining in the liver specimens using HistoQuest software. The box-plot analyses represent the number of F4/80-positive cells (designated as “Count”) and also the total and mean areas of F4/80-positive cells (designated as “Total Area” and “Mean Area”, respectively); the total area values reflect both the magnitude and morphology/size of positive cells, while the mean area values reflect predominantly the morphology/size of positive cells. ***p ≤ 0.001; only significant p values are shown.</p

Modulation of cytokine production by 2-DG in murine BMDMs.

<p>BMDMs from BALB/c mice were cultured in 6 well plates and treated with 1, 3, and 5 mM 2-DG and <i>L</i>.<i>m</i>. as indicated. IL-10 (a) and IL-12p40 (b) were determined from 20 hour culture supernatants by ELISA. *p ≤ 0.05, **p ≤ 0.01 <i>L</i>.<i>m</i>. <i>Listeria monocytogenes</i>. 2-DG treatment alone induced the following cytokine levels: IL-10: 4 times ≤ 84.5 pg/mL, IL12p40: 4 times ≤ 5.1 pg/mL.</p

2-DG differentially modulates pro- and anti-inflammatory cytokine secretion in human monocytes.

<p>Human monocytes were preincubated for 90 minutes with different concentrations of 2-DG or medium and then stimulated with LPS. Secretion of IL-10 (a) and IL-12p40 (b) IL-23p19 (c), IL-1β (d), IL-6 (e) TNF-α (f) was determined from 18–24 hr culture supernatants by ELISA. Pretreatment of monocytes with rapamycin (100 nM) served as control. Data are representative of 5 independent experiments and presented as % response ± SD. In unstimulated cultures cytokines were undetectable. 2-DG treatment alone induced no significant cytokine production: IL-10: 2 times below detection level, 3 times ≤ 1.4 pg/mL, IL-12p40: 4 times below detection level, 1x 3.8 pg/mL, IL-23p19: 5 times below detection level, IL-1β: 2 times below detection level, 3 times ≤ 22.2 pg/mL, IL-6: 5 times below 11.6 pg/mL, TNF-α 1x below detection level 4 times ≤ 2.8 pg/mL; Mean cytokine levels after LPS stimulation in the absence of 2-DG were: IL-10, 904 ± 1096 pg/mL; IL-12p40, 535 ± 509 pg/mL; IL-23p19, 136 ± 152 pg/mL; IL-1β, 9471 ± 10692 pg/mL; IL-6, 1608 ± 414 pg/mL; TNF-α, 1596 ± 642 pg/mL. *p ≤ 0.05, ***p ≤ 0.005.</p

Modulation of inflammatory mediators by 2-DG at the transcriptional level and its impact of 2-DG on mTOR signaling in human monocytes.

<p>mRNA levels of IL-10, IL-12B(p40), IL-23A(p19) in human monocytes were assessed by real-time PCR analysis. Monocytes were pre-treated with 2-DG for 90 minutes or medium and then stimulated with LPS for 4 hr. Expression levels were normalized to house keeping genes and shown relative to LPS-stimulated cells. Data are displayed as means ± SD of two independent experiments. Significance was assessed by two-tailed t test; ** p ≤ 0.01 (a). Monocytes were preincubated for 15 minutes with respective mM doses of 2-DG, rapamycin (100 nM) or medium as indicated and then stimulated with LPS. Whole cell lysates were analyzed by immunoblotting using specific antibodies against phospho-S6RP (b)<b>.</b> Data are representative of 2 independent experiments.</p

Impact of 2-DG on macrophage polarization.

<p>Human macrophages were differentiated for 7 d with M-CSF and then activated for 1 d with the indicated stimuli. (A) Secretion of IL-10, IL-12p40, IL-12p70, IL-23p19 and CCL-13 was determined from 20 hr culture supernatants by Luminex analysis. Data are representative of 3 independent experiments and presented as mean ± SD. (B) Surface expression of macrophage markers was measured by flow cytometry. Data are representative of 3 independent experiments and presented as mean ± SD. n.d. not detectable; *p≤0.05, **p≤0.01, ***p≤0.001.</p