Functional membrane androgen receptors in colon tumors trigger pro-apoptotic responses in vitro and reduce drastically tumor incidence in vivo

<p>Abstract</p> <p>Background</p> <p>Membrane androgen receptors (mAR) have been implicated in the regulation of cell growth, motility and apoptosis in prostate and breast cancer. Here we analyzed mAR expression and function in colon cancer.</p> <p>Results</p> <p>Using fluorescent mAR ligands we showed specific membrane staining in colon cell lines and mouse xenograft tumor tissues, while membrane staining was undetectable in healthy mouse colon tissues and non-transformed intestinal cells. Saturation/displacement assays revealed time- and concentration-dependent specific binding for testosterone with a K_Dof 2.9 nM. Stimulation of colon mAR by testosterone albumin conjugates induced rapid cytoskeleton reorganization and apoptotic responses, even in the presence of anti-androgens. The actin cytoskeleton drug cytochalasin B effectively inhibited the pro-apoptotic responses and caspase-3 activation. Interestingly, <it>in vivo </it>studies revealed that mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors.</p> <p>Conclusion</p> <p>Our results demonstrate for the first time that functional mARs are predominantly expressed in colon tumors and that their activation results in induction of anti-tumor responses <it>in vitro </it>and extensive reduction of tumor incidence <it>in vivo</it>.</p

Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-β signaling

TGF-β and related growth factors critically regulate cell potency and functions. Smad7 is induced by TGF-βs and inhibits the physiological functions of TGF-β signaling. This study describes an unexpected finding that Smad7 promotes self-renewal of embryonic stem cells (ESCs) in a manner independent of its inhibition on TGF-β signaling. Instead, Smad7 acts to induce activation of transcription factor signal transducers and activators of transcription 3 (STAT3) in ESCs. Smad7 activates STAT3 through its direct binding to the cytokine receptor upstream of STAT3 activation. In agreement with the role of STAT3 in maintaining ESC pluripotency, Smad7 promotes ESC self-renewal and induced pluripotent stem cell reprogramming. This finding illustrates a regulatory mechanism for Smad7 in maintaining pluripotency, and likely in cancer and inflammation

Membrane Androgen-Rezeptor-Aktivierung auslöst pro-apoptotische Reaktionen in vitro und in vivo und blockiert die Migration bei Darmkrebs

The classical intracellular androgen receptors (iAR) mediate genomic androgen signals, which take at least more than half an hour. However, the rapid or non-genomic action of androgens takes only seconds to few minutes and involves the activation of androgen membrane binding sites. Although the molecular identity of those membrane binding sites remains still unknown, their expression has been reported in many cell types, including various tumor cells. Activation of membrane androgen receptors (mAR) in prostate and breast cancer cells has been implicated in the regulation of cell growth, motility and apoptosis. Here we analyzed mAR expression and function in colon cancer. Using fluorescent mAR ligands we showed specific membrane staining in mouse colon tumor tissues and in iAR silenced Caco2 cell lines. Stimulation of colon-mAR by testosterone-albumin-conjugates induced rapid actin and tubulin cytoskeleton reorganization and generated apoptotic responses, even in the presence of anti-androgens. We showed that long-term activation of mAR in Caco2 cell lines down-regulated the activity of PI-3K and Akt and induced de-phosphorylation/activation of the pro-apoptotic Bad. Treatment of APCmin/+ mice significantly decreased the expression of p-AKT and p-Bad levels in tumor tissue. Moreover, mAR activation resulted in a 65% reduction of tumor incidence in chemically induced Balb/c mice colon tumors and an 80% reduction of tumor incidence in APCmin/+ mice colon tumors. Furthermore, mAR activation strongly inhibited Caco2 cell migration. In accordance with this, vinculin, a protein controlling cell adhesion and actin reorganization, was effectively phosphorylated upon mAR activation. Phosphorylation inhibitors genistein and PP2 inhibited actin reorganization and restored motility. Moreover, blocking actin reorganization by cytochalasin B and silencing vinculin by appropriate siRNA’s restored the migration potential. From these results we conclude that mAR activation inhibits the pro-survival signals Akt/Bad in vitro and in vivo, induces potent proapoptoric responses and blocks migration of colon cancer cells via regulation of vinculin signaling and actin reorganization. Our results point to a central role of mAR in the induction of anti-tumor responses in colon cancer.Die klassischen intrazellulären Androgenrezeptoren (iAR) vermitteln die genomische Androgenwirkung, die wenigstens 30 Minuten Zeit erfordert. Im Gegensatz hierzu benötigen die schnellen, nichtgenomischen Androgeneffekte nur einige Sekunden bis wenige Minuten durch die Aktivierung von Androgenbindungsstellen in der Zellmembran. Derartige membranständige Androgenbindungsstellen wurden schon in vielen Zelltypen inklusive Tumorzellen nachgewiesen, obwohl die molekulare Identität dieser Bindungsstellen noch immer unbekannt ist. Die Aktivierung dieser membranständigen Androgenrezeptoren (mAR) steht im Zusammenhang mit der Regulation von Zellwachstum, Motilität und Apoptose. Mit der vorliegenden Arbeit wurde die Expression und Funktion von mAR bei Kolonkarzinomen untersucht. Mithilfe fluoreszierender mAR-Liganden konnten membranständige Rezeptoren spezifisch angefärbt werden in Kolonkarzinomgewebe der Maus und in Caco2-Zellen, die nicht über iAR verfügen. Die Stimulierung von mAR in Dickdarmgewebe durch Testosteron-Albumin-Konjugate führte zu einer raschen Reorganisation des Aktin- und Tubulinnetzwerkes und löste Apoptose selbst in Anwesenheit von Antiandrogenen aus. Die längeranhaltende Aktivierung von mAR in Caco2-Zellen führte zu verminderter PI-3- und Akt-Kinaseaktivität und zur Dephosphorylierung und mithin Aktivierung von proapoptotischem Bad. Eine entsprechende Behandlung von APCmin/+-Mäusen verringerte die Expression von p-Akt und von p-Bad signifikant in Tumorgewebe. Darüber hinaus führte die Aktivierung von mAR zu einer Verminderung der Tumorinzidenz um 65% bei chemisch induzierten Dickdarmtumoren von Balb/c-Mäusen und um 80% bei APCmin/+-Mäusen. Weiterhin hemmte die mAR-Aktivierung die Zellmigration von Caco2-Zellen stark. In Übereinstimmung mit diesem Befund war Vinculin, ein Protein, das Zelladhäsion und Aktinreorganisation reguliert, nach mAR-Aktivierung deutlich phosphoryliert. Die Phosphorylierungsinhibitoren Genistein und PP2 hemmten die Aktinreorganisation und reaktivierten die Zellmotilität. Zusätzlich konnte die Blockade der Aktinreorganisation durch Cytochalasin B oder die auf siRNA basierende Herunterregulation von Vinculin das Migrationspotential der Zellen wiederherstellen. Aus diesen Daten kanngeschlossen werden, dass die Aktivierung von mAR die überlebensfördernden Akt/Bad-Signale in vitro und in vivo hemmt, wirkungsvolle proapoptotische Zellantworten induziert und die Migration von Kolontumorzellen über die Regulierung des Vinculin-Signalweges und der Aktinreorganisation blockiert. Diese Erkenntnisse deuten auf eine zentrale Rolle von mAR für die Induktion von Antitumorantworten bei Kolonkarzinom

Pre-expanded Muscle-sparing Latissimus Dorsi Flaps for Reconstruction of Severe Scar Contractures on the Anterior Chest

ABSTRACT: Objective: To investigate the utility of pre-expanded muscle-sparing latissimus dorsi flaps in the reconstruction of deformities secondary to severe scar contractures on the anterior chest. Methods: The function of the latissimus dorsi was preserved with blood supply from the main or lateral branch of the thoracodorsal artery. The entire treatment period was divided into two stages, during which segmental latissimus dorsi flaps were pre-expanded in stage I and anterior chest scar deformities were reconstructed in stage II.During stage I, the musculocutaneous perforators arising from the lateral branch of the thoracodorsal artery were determined by ultrasound preoperatively; the flap design included the anterior segment of the latissimus dorsi supplied by the musculocutaneous perforators from the lateral branch; and a tissue expander was placed following flap dissection and then infused with saline intermittently for 4–6 months.In stage II, the chest scars were excised, and breast tissues were repositioned; the continuity of the medial branch of the thoracodorsal nerve to the muscle was preserved when reconstruction was performed using the segmental latissimus dorsi flaps supplied by the main or lateral branch of the thoracodorsal artery. Results: From October 2010 to October 2019, 21 patients (on 24 sides) underwent reconstructive procedures for extensive scar contractures on the anterior chest. All flaps survived, and their donor sites were sutured directly. During a follow-up of 3 months to 8 years, the flaps became soft and exhibited color similar to that of the adjacent tissues. The limited neck and shoulder movements improved, and postoperatively, all female patients were satisfied with the shape of their breasts. Additionally, neither apparent weakening on the adduction, internal rotation, or extension strength of the shoulder joint on the affected side nor marked depression deformity in the back was observed. Conclusion: Pre-expanded muscle-sparing latissimus dorsi flaps with blood supply from the main or lateral branch of the thoracodorsal artery proved to be a desirable option for the reconstruction of extensive scar contractures on the anterior chest

Stimulation of the amino acid transport SLC6A19 by JAK2

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutationV617FJAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na+ coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2,V617FJAK2 or inactiveK882EJAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2mM) to the bath generated a current (Ile), which was significantly increased following coexpression of JAK2 orV617FJAK2, but not by coexpression ofK882EJAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40μM) resulted in a gradual decline of Ile. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of Ile following inhibition of carrier insertion by brefeldin A (5μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells

Fine-needle aspiration for periprosthetic fluid removal after implantation of a remote internal-port tissue expander

Background: Use of internal filling ports in tissue expander–based reconstructions are advantageous because of easier self-care, lower infection rates, and fewer instances of capsule formation. The appearance of periprosthetic fluid accumulation after internal-port tissue expander implantation is a common complication that warrants treatment. In this study, we introduced a noninvasive method using fine-needle aspiration (FNA) to remove fluids accumulated after implantation of a remote internal-port tissue expander. Methods: In this study, 245 patients who underwent implantation of remote internal-port tissue expanders in our hospital from July 1, 2012, to July 1, 2019, were included and divided into two groups. In the control group, patients underwent tissue expander implantation before July 1, 2016, and large quantities of fluids were removed with surgical aspiration procedures in most cases. In the FNA group, the patients underwent implantation after July 1, 2016, and large quantities of fluids were removed first with the FNA procedure. Patients’ demographic data, indications for FNA application, and related complications were collected and analyzed. Results: Overall, 395 expanders were placed in 245 patients. Postoperative management was similar in both groups. Fluids were managed with 23 expanders in the control group and with 31 expanders in the FNA group. There was no difference in the fluid aspiration rate between the two groups. The surgical aspiration rate was 11.1% (23/208) in the control group. The success rate of FNA was 90.3% (28/31). In the FNA group, the surgical aspiration rate was 1.6% (3/187), which was significantly lower than that in the control group. There were no significant differences in complications between the two groups. Conclusion: FNA can be used for periprosthetic fluid removal after the implantation of a remote internal-port tissue expander in most cases. This method is more convenient and safer than surgical aspiration for the postoperative management of internal-port tissue expander implantation