STAT3 is a genetic modifier of TGF-beta induced EMT in KRAS mutant pancreatic cancer.

Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here, we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of the STAT3-responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.

Ablation of Long Noncoding RNA Hoxb3os Exacerbates Cystogenesis in Mouse Polycystic Kidney Disease.

SIGNIFICANCE STATEMENT: Long noncoding RNAs (lncRNAs) are a class of nonprotein coding RNAs with pivotal functions in development and disease. They have emerged as an exciting new drug target category for many common conditions. However, the role of lncRNAs in autosomal dominant polycystic kidney disease (ADPKD) has been understudied. This study provides evidence implicating a lncRNA in the pathogenesis of ADPKD. We report that Hoxb3os is downregulated in ADPKD and regulates mammalian target of rapamycin (mTOR)/Akt pathway in the in vivo mouse kidney. Ablating the expression of Hoxb3os in mouse polycystic kidney disease (PKD) activated mTOR complex 2 (mTORC2) signaling and exacerbated the cystic phenotype. The results from our study provide genetic proof of concept for future studies that focus on targeting lncRNAs as a treatment option in PKD. BACKGROUND: ADPKD is a monogenic disorder characterized by the formation of kidney cysts and is primarily caused by mutations in two genes, PKD1 and PKD2 . METHODS: In this study, we investigated the role of lncRNA Hoxb3os in ADPKD by ablating its expression in the mouse. RESULTS: Hoxb3os -null mice were viable and had grossly normal kidney morphology but displayed activation of mTOR/Akt signaling and subsequent increase in kidney cell proliferation. To determine the role of Hoxb3os in cystogenesis, we crossed the Hoxb3os -null mouse to two orthologous Pkd1 mouse models: Pkhd1/Cre; Pkd1F/F (rapid cyst progression) and Pkd1RC/RC (slow cyst progression). Ablation of Hoxb3os exacerbated cyst growth in both models. To gain insight into the mechanism whereby Hoxb3os inhibition promotes cystogenesis, we performed western blot analysis of mTOR/Akt pathway between Pkd1 single-knockout and Pkd1 - Hoxb3os double-knockout (DKO) mice. Compared with single-knockout, DKO mice presented with enhanced levels of total and phosphorylated Rictor. This was accompanied by increased phosphorylation of Akt at Ser 473 , a known mTORC2 effector site. Physiologically, kidneys from DKO mice displayed between 50% and 60% increase in cell proliferation and cyst number. CONCLUSIONS: The results from this study indicate that ablation of Hoxb3os in mouse PKD exacerbates cystogenesis and dysregulates mTORC2.

Oncogenic dependency plays a dominant role in the immune response to cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human malignancies. Advanced PDAC is considered incurable. Nearly 90% of pancreatic cancers are caused by oncogenic KRAS mutations. The mechanisms of primary or acquired resistance to KRAS inhibition are currently unknown. Here, we propose that oncogenic dependency, rather than KRAS mutation per se, plays a dominant role in the immune response to cancer, including late-stage PDAC. Classifying tumor samples according to KRAS activity scores allows accurate prediction of tumor immune composition and therapy response. Dual RAS/MAPK pathway blockade combining KRAS and MEK inhibitors is more effective than the selective KRAS inhibitor alone in attenuating MAPK activation and unblocking the influx of T cells into the tumor. Lowering KRAS activity in established tumors promotes immune infiltration, but with a limited antitumor effect, whereas combining KRAS/MEK inhibition with immune checkpoint blockade achieves durable regression in preclinical models. The results are directly applicable to stratifying human PDAC based on KRAS dependency values and immune cell composition to improve therapeutic design.

STAT3 is a genetic modifier of TGF-beta induced EMT in KRAS mutant pancreatic cancer.

Oncogenic mutations in KRAS are among the most common in cancer. Classical models suggest that loss of epithelial characteristics and the acquisition of mesenchymal traits are associated with cancer aggressiveness and therapy resistance. However, the mechanistic link between these phenotypes and mutant KRAS biology remains to be established. Here we identify STAT3 as a genetic modifier of TGF-beta-induced epithelial to mesenchymal transition. Gene expression profiling of pancreatic cancer cells identifies more than 200 genes commonly regulated by STAT3 and oncogenic KRAS. Functional classification of STAT3 responsive program reveals its major role in tumor maintenance and epithelial homeostasis. The signatures of STAT3-activated cell states can be projected onto human KRAS mutant tumors, suggesting that they faithfully reflect characteristics of human disease. These observations have implications for therapeutic intervention and tumor aggressiveness.

KRAS drives immune evasion in a genetic model of pancreatic cancer.

Immune evasion is a hallmark of KRAS-driven cancers, but the underlying causes remain unresolved. Here, we use a mouse model of pancreatic ductal adenocarcinoma to inactivate KRAS by CRISPR-mediated genome editing. We demonstrate that at an advanced tumor stage, dependence on KRAS for tumor growth is reduced and is manifested in the suppression of antitumor immunity. KRAS-deficient cells retain the ability to form tumors in immunodeficient mice. However, they fail to evade the host immune system in syngeneic wild-type mice, triggering strong antitumor response. We uncover changes both in tumor cells and host immune cells attributable to oncogenic KRAS expression. We identify BRAF and MYC as key mediators of KRAS-driven tumor immune suppression and show that loss of BRAF effectively blocks tumor growth in mice. Applying our results to human PDAC we show that lowering KRAS activity is likewise associated with a more vigorous immune environment.

IL-6 promotes MYC-induced B cell lymphomagenesis independent of STAT3.

The inflammatory cytokine IL-6 is known to play a causal role in the promotion of cancer, although the underlying mechanisms remain to be completely understood. Interplay between endogenous and environmental cues determines the fate of cancer development. The Eµ-myc transgenic mouse expresses elevated levels of c-Myc in the B cell lineage and develops B cell lymphomas with associated mutations in p53 or other genes linked to apoptosis. We generated Eµ-myc mice that either lacked the IL-6 gene, or lacked the STAT3 gene specifically in B cells to determine the role of the IL-6/JAK/STAT3 pathway in tumor development. Using the Eµ-myc lymphoma mouse model, we demonstrate that IL-6 is a critical tumor promoter during early stages of B cell lymphomagenesis. IL-6 is shown to inhibit the expression of tumor suppressors, notably BIM and PTEN, and this may contribute to advancing MYC-driven B cell tumorigenesis. Several miRNAs known to target BIM and PTEN are upregulated by IL-6 and likely lead to the stable suppression of pro-apoptotic pathways early during the tumorigenic process. STAT3, a classical downstream effector of IL-6, appears dispensable for Eµ-myc driven lymphomagenesis. We conclude that the growth-promoting and anti-apoptotic mechanisms activated by IL-6 are critically involved in Eµ-myc driven tumor initiation and progression, but the B cell intrinsic expression of STAT3 is not required.

STAT3 is a master regulator of epithelial identity and KRAS-driven tumorigenesis.

A dichotomy exists regarding the role of signal transducer and activator of transcription 3 (STAT3) in cancer. Functional and genetic studies demonstrate either an intrinsic requirement for STAT3 or a suppressive effect on common types of cancer. These contrasting actions of STAT3 imply context dependency. To examine mechanisms that underlie STAT3 function in cancer, we evaluated the impact of STAT3 activity in KRAS-driven lung and pancreatic cancer. Our study defines a fundamental and previously unrecognized function of STAT3 in the maintenance of epithelial cell identity and differentiation. Loss of STAT3 preferentially associates with the acquisition of mesenchymal-like phenotypes and more aggressive tumor behavior. In contrast, persistent STAT3 activation through Tyr705 phosphorylation confers a differentiated epithelial morphology that impacts tumorigenic potential. Our results imply a mechanism in which quantitative differences of STAT3 Tyr705 phosphorylation, as compared with other activation modes, direct discrete outcomes in tumor progression.

PRISM II: an open-label study to assess effectiveness of dextromethorphan/quinidine for pseudobulbar affect in patients with dementia, stroke or traumatic brain injury.

BACKGROUND: Phase 3 trials supporting dextromethorphan/quinidine (DM/Q) use as a treatment for pseudobulbar affect (PBA) were conducted in patients with amyotrophic lateral sclerosis (ALS) or multiple sclerosis (MS). The PRISM II study provides additional DM/Q experience with PBA secondary to dementia, stroke, or traumatic brain injury (TBI). METHODS: Participants in this open-label, multicenter, 90-day trial received DM/Q 20/10 mg twice daily. The primary outcome was the Center for Neurologic Study-Lability Scale (CNS-LS), assessing change in PBA episode frequency and severity. The CNS-LS final visit score was compared to baseline (primary analysis) and to the response in a previously conducted placebo-controlled trial with DM/Q in patients with ALS or MS. Secondary outcomes included change in PBA episode count and Clinical Global Impression of Change with respect to PBA as rated by a clinician (CGI-C) and by the patient or caregiver (PGI-C). RESULTS: The study enrolled 367 participants with PBA secondary to dementia, stroke, or TBI. Mean (standard deviation [SD]) CNS-LS score improved significantly from 20.4 (4.4) at baseline to 12.8 (5.0) at Day 90/Final Visit (change, -7.7 [6.1]; P < .001, 95 % CI: -8.4, -7.0). This magnitude of improvement was consistent with DM/Q improvement in the earlier phase-3, placebo-controlled trial (mean [95 % CI] change from baseline, -8.2 [-9.4, -7.0]) and numerically exceeds the improvement seen with placebo in that study (-5.7 [-6.8, -4.7]). Reduction in PBA episode count was 72.3 % at Day 90/Final Visit compared with baseline (P < .001). Scores on CGI-C and PGI-C showed that 76.6 and 72.4 % of participants, respectively, were "much" or "very much" improved with respect to PBA. The most frequently occurring adverse events (AEs) were diarrhea (5.4 %), headache (4.1 %), urinary tract infection (2.7 %), and dizziness (2.5 %); 9.8 % had AEs that led to discontinuation. Serious AEs were reported in 6.3 %; however, none were considered treatment related. CONCLUSIONS: DM/Q was shown to be an effective and well-tolerated treatment for PBA secondary to dementia, stroke, or TBI. The magnitude of PBA improvement was similar to that reported in patients with PBA secondary to ALS or MS, and the adverse event profile was consistent with the known safety profile of DM/Q. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01799941, registered on 25 February 2013.

An open-label study to assess safety, tolerability, and effectiveness of dextromethorphan/quinidine for pseudobulbar affect in dementia: PRISM II results.

BACKGROUND: Dextromethorphan (DM)/quinidine (Q) is an approved treatment for pseudobulbar affect (PBA) based on trials in amyotrophic lateral sclerosis or multiple sclerosis. PRISM II evaluated DM/Q effectiveness and tolerability for PBA secondary to dementia, stroke, or traumatic brain injury; dementia cohort results are reported. METHODS: This was an open-label, multicenter, 90 day trial; patients received DM/Q 20/10 mg twice daily. Primary outcome was change in Center for Neurologic Study-Lability Scale (CNS-LS) score. Secondary outcomes included PBA episode count and Clinical and Patient/Caregiver Global Impression of Change scores with respect to PBA (CGI-C/PGI-C). RESULTS: 134 patients were treated. CNS-LS improved by a mean (SD) of 7.2 (6.0) points at Day 90/Endpoint (P<.001) vs. baseline. PBA episodes were reduced 67.7% (P<.001) vs. baseline; global measures showed 77.5% CGI-C and 76.5% PGI-C "much"/"very much" improved. Adverse events included headache (7.5%), urinary tract infection (4.5%), and diarrhea (3.7%); few patients dropped out for adverse events (10.4%). CONCLUSIONS: DM/Q significantly reduced PBA symptoms in patients with dementia; reported adverse events were consistent with the known safety profile of DM/Q. Trial Registration clinicaltrials.gov identifier: NCT01799941.

External urethral sphincter motor unit recruitment patterns during micturition in the spinally intact and transected adult rat.

In the rat, external urethral sphincter (EUS) activation during micturition consists of three sequential phases: 1) an increase in tonic EUS activity during passive filling and active contraction of the bladder (guarding reflex), 2) synchronized phasic activity (EUS bursting) associated with voiding, and 3) sustained tonic EUS activity that persists after bladder contraction. These phases are perturbed following spinal cord injury. The purpose of the present study was to characterize individual EUS motor unit (MU) patterns during micturition in the spinally intact and transected adult rat. EUS MU activity was recorded from either the L5 or L6 ventral root (intact) or EUS muscle (transected) during continuous flow cystometry in urethane-anesthetized adult female Sprague-Dawley rats. With the use of bladder pressure threshold and timing of activation, four distinct patterns of EUS MU activity were identified in the intact rat: low threshold sustained, medium/high threshold sustained, medium/high threshold not sustained, and burst only. In general, these MUs displayed little frequency modulation during active contraction, generated high-frequency bursts of action potentials during EUS bursting, and varied in terms of the duration of sustained tonic activity. In contrast, three general patterns of EUS MU activity were identified in the transected rat: low threshold, medium threshold, and high threshold. These MUs exhibited considerable frequency modulation during active contraction of the bladder, no bursting behavior and little to no sustained firing. The prominent frequency modulation of EUS MUs is likely due to the enhanced guarding reflex seen in EUS whole muscle electromyogram recordings in transected rats (D'Amico SC, Schuster IP, Collins WF 3rd. Exp Neurol 228: 59-68, 2011). In addition, EUS MU recruitment in transected rats more closely followed predictions by the size principle than in intact rats. This may reflect the influence of local synaptic circuits or intrinsic properties of EUS motoneurons that are active in intact rats but attenuated or absent in transected rats.

Partial agonist, but not pure antiestrogens stimulate doming, a marker of normal differentiation, in the MCF-7 breast cancer cell line.

UNLABELLED: Abstract Background: The mechanisms by which tamoxifen inhibits breast tumor growth are not completely understood. Partial agonist antiestrogens such as tamoxifen may cause the estrogen receptor (ER) to interact with genes different from those activated by ER bound to estradiol. Doming is a property often associated with, and considered a marker of, differentiation in mammary epithelial cells in culture. This study compared the ability of pure and partial agonist antiestrogens to stimulate doming. MATERIALS AND METHODS: MCF-7 cells grown in medium with 10% calf serum were treated with antiestrogens. Domes were counted in three rows (width of the 4× field) across the flask. RESULTS: Three partial agonist antiestrogens [4-hydroxytamoxifen (OHT), H1285 and RU 39,411] caused dome formation. None of the pure antiestrogens tested (ICI 164,384, ICI 182,780 and RU 58,668) caused doming. Doming was stimulated in a dose-dependent manner starting at 1 nM OHT with maximum stimulation at 10-100 nM. Estradiol did not stimulate doming, but blocked doming at 1%-10% of the OHT concentration. Trichostatin A (TSA) reduced the level of estrogen receptor alpha (ERα) and adding it 24 h before adding OHT prevented dome formation. CONCLUSIONS: OHT and the other partial agonist antiestrogens appear to act through the ER to stimulate doming. The ability of tamoxifen to induce a marker of differentiation may play a role in its inhibition of breast tumors. If so, then the fact that other partial agonist antiestrogens share this ability, but that pure antiestrogens lack it, may be an important consideration in developing new antiestrogens for breast cancer therapy.

Quantification of external urethral sphincter and bladder activity during micturition in the intact and spinally transected adult rat.

The goal of this study was to determine the effect of chronic mid-thoracic spinal cord transection on the time course of external urethral sphincter (EUS) and bladder activity associated with micturition events in the rat. Adult female Sprague-Dawley rats, either spinally intact or transected (T(9)-T(10)), were anesthetized with urethane and set up for continuous flow urodynamic recording of bladder intravesical pressure (BP) and EUS electromyography (EMG). Spinal transections were performed under isoflurane anesthesia 1-8 weeks prior to the terminal experiment. Four major differences between intact and transected rats were observed: 1) While the frequency of micturition events in the intact rat was dependent upon the rate of bladder filling, the bladder contraction and associated EUS activation in transected rats exhibited an intrinsic rhythm that was independent of the rate of bladder filling and post-transection survival time. 2) EUS activation was augmented at the beginning of active bladder contraction in the transected rat, indicating an amplified guarding reflex. 3) Phasic EUS activity at the peak of bladder contraction (EUS bursting) in the intact rat was markedly reduced or absent in the transected rat. 4) The sustained tonic EUS activity following bladder relaxation in the intact rat was absent in the transected rat. These data are discussed in the context of understanding the pathophysiology of spinal cord injury (SCI) induced destrusor-sphincter dyssynergia (DSD).

The small G protein Rac1 activates phospholipase Cdelta1 through phospholipase Cbeta2.

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cbeta2 (PLCbeta2) to increase intracellular Ca(2+) levels. This increased Ca(2+) can in turn activate the very robust PLCdelta1 to synergize Ca(2+) signals. We have previously found that PLCbeta2 will bind to and inhibit PLCdelta1 in solution by an unknown mechanism and that PLCbeta2.PLCdelta1 complexes can be disrupted by Gbetagamma subunits. However, because the major populations of PLCbeta2 and PLCdelta1 are cytosolic, their regulation by Gbetagamma subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCbeta2 and PLCbeta3 are the regions that result in PLCdelta1 binding and inhibition. In cells, PLCbeta2.PLCdelta1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHbeta2 dissociates the complex. Using PHbeta2 as a tool to assess the contribution of PLCbeta inhibition of PLCdelta1 to Ca(2+) release, we found that, although PHbeta2 only results in a 25% inhibition of PLCdelta1 in solution, in cells the presence of PHbeta2 appears to eliminates Ca(2+) release suggesting a large threshold effect. We found that the small plasma membrane population of PLCbeta2.PLCdelta1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCdelta1 is controlled by the amount of bound PLCbeta2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCbeta2, PLCdelta1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.