Notch3 as a novel therapeutic target in metastatic medullary thyroid cancer.

BACKGROUND: Medullary thyroid cancer portends poor survival once liver metastasis occurs. We hypothesize that Notch3 overexpression in medullary thyroid cancer liver metastasis will decrease proliferation and growth of the tumor. METHODS: TT cells were modified genetically to overexpress Notch3 in the presence of doxycycline, creating the TT-Notch3 cell line. Mice were injected intrasplenically with either TT-Notch3 or control vector TT-TRE cells. Each cell line had 3 treatment groups: control with 12 weeks of standard chow, early DOX with doxycycline chow at day 0 and for 70 days thereafter, and late DOX with doxycycline chow at 8 weeks. Each animal underwent micro-computed tomography to evaluate for tumor formation and tumor quantification was performed. Animals were killed at 12 weeks, and the harvested liver was stained with Ki-67, hematoxylin and eosin, and Notch3. RESULTS: Induction of Notch3 did not prevent formation of medullary thyroid cancer liver metastases as all mice in the early DOX group developed tumors. However, induction of Notch after medullary thyroid cancer liver tumor formation decreased tumor size, as seen on micro-computed tomography scans (late DOX group). This translated to a 37-fold decrease in tumor volume (P = .001). Notch3 overexpression also resulted in decreased Ki-67 index (P = .038). Moreover, Notch3 induction led to increased areas of neutrophil infiltration and necrosis on hematoxylin and eosin staining of the tumors CONCLUSION: Notch3 overexpression demonstrates an antiproliferative effect on established metastatic medullary thyroid cancer liver tumors and is a potential therapeutic target in treatment.

Neuroendocrine Tumor-Targeted Upconversion Nanoparticle-Based Micelles for Simultaneous NIR-Controlled Combination Chemotherapy and Photodynamic Therapy, and Fluorescence Imaging.

Although neuroendocrine tumors (NETs) are slow growing, they are frequently metastatic at the time of discovery and no longer amenable to curative surgery, emphasizing the need for the development of other treatments. In this study, multifunctional upconversion nanoparticle (UCNP)-based theranostic micelles are developed for NET-targeted and near-infrared (NIR)-controlled combination chemotherapy and photodynamic therapy (PDT), and bioimaging. The theranostic micelle is formed by individual UCNP functionalized with light-sensitive amphiphilic block copolymers poly(4,5-dimethoxy-2-nitrobenzyl methacrylate)-polyethylene glycol (PNBMA-PEG) and Rose Bengal (RB) photosensitizers. A hydrophobic anticancer drug, AB3, is loaded into the micelles. The NIR-activated UCNPs emit multiple luminescence bands, including UV, 540 nm, and 650 nm. The UV peaks overlap with the absorption peak of photocleavable hydrophobic PNBMA segments, triggering a rapid drug release due to the NIR-induced hydrophobic-to-hydrophilic transition of the micelle core and thus enabling NIR-controlled chemotherapy. RB molecules are activated via luminescence resonance energy transfer to generate 1O2 for NIR-induced PDT. Meanwhile, the 650 nm emission allows for efficient fluorescence imaging. KE108, a true pansomatostatin nonapeptide, as an NET-targeting ligand, drastically increases the tumoral uptake of the micelles. Intravenously injected AB3-loaded UCNP-based micelles conjugated with RB and KE108-enabling NET-targeted combination chemotherapy and PDT-induce the best antitumor efficacy.

Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo.

Novel therapies for neuroendocrine (NE) cancers are desperately needed as they frequently present as metastatic disease and cause debilitating symptoms by secreting excessive hormones. Induction of Notch isoforms has a tumor suppressive effect in NE cancer cell lines, and we have observed that histone deacetylase inhibitors (HDACi) potently activate Notch. In this study, we describe the potential for Burkholderia thailandensis-derived class I HDACi thailandepsin A (TDP-A) as a Notch activator and therapeutic agent against NE cancer. IC50 for TDP-A was determined to be 4-6 nM in NE cancer cell lines (BON, MZ-CRC-1, and TT) without cytotoxicity to lung fibroblasts. The binding characteristics of TDP-A to its target HDAC1 was examined using bioluminescence resonance energy transfer (BRET). Western blot and flow cytometry analysis showed that TDP-A induces cell cycle arrest and apoptosis in a dose-dependent manner. TDP-A dose-dependently activated the Notch pathway as measured by increasing functional CBF1-luciferase reporter signal and mRNA and protein expressions of Notch isoforms, which were attenuated by pretreatment with γ-secretase inhibitor DAPT. Furthermore, TDP-A lead to changes in expression level of downstream targets of Notch pathway and reduced expression of NE cancer markers. An in vivo study demonstrated that TDP-A suppressed NE cancer progression. These results show that TDP-A, as a Notch activator, is a promising agent against NE cancers.

AB3-Loaded and Tumor-Targeted Unimolecular Micelles for Medullary Thyroid Cancer Treatment.

Medullary thyroid cancer (MTC) is often resistant to standard therapies, emphasizing the need for the development of other treatments. A new histone deacetylase inhibitor, AB3, can effectively inhibit MTC cell proliferation in vitro. However, its poor aqueous solubility and stability, fast clearance, and lack of tumor targeting ability limit its in vivo application. Therefore, multifunctional unimolecular micelles were developed for targeted delivery of AB3 for MTC therapy. The unimolecular micelles exhibited a spherical core-shell structure, uniform size distribution, and excellent stability. AB3 was encapsulated into the hydrophobic core of the unimolecular micelles, thus significantly enhancing its aqueous solubility and stability. KE108, a somatostatin analog possessing high affinity to all five subtypes of SSTR, was used as an MTC-targeting ligand. In vitro cellular uptake analyses demonstrated that the KE108 exhibited superior targeting ability in MTC cells compared to octreotide, the first clinically used somatostatin analog. Moreover, the AB3-loaded and KE108-conjugated unimolecular micelles exhibited the best efficacy in suppressing MTC cell growth and tumor marker expression in vitro. Furthermore, AB3-loaded, KE108-conjugated micelles demonstrated the best anticancer efficacy in vivo without any apparent systemic toxicity, thereby offering a promising approach for targeted MTC therapy.

KE108-conjugated unimolecular micelles loaded with a novel HDAC inhibitor thailandepsin-A for targeted neuroendocrine cancer therapy.

Neuroendocrine (NE) cancers can cause significant patient morbidity. Besides surgery, there are no curative treatments for NE cancers and their metastases, emphasizing the need for the development of other forms of therapy. In this study, multifunctional unimolecular micelles were developed for targeted NE cancer therapy. The unimolecular micelles were formed by multi-arm star amphiphilic block copolymer poly(amidoamine)-poly(valerolactone)-poly(ethylene glycol) conjugated with KE108 peptide and Cy5 dye (abbreviated as PAMAM-PVL-PEG-KE108/Cy5). The unimolecular micelles with a spherical core-shell structure exhibited a uniform size distribution and excellent stability. The hydrophobic drug thailandepsin-A (TDP-A), a recently discovered HDAC inhibitor, was physically encapsulated into the hydrophobic core of the micelles. KE108 peptide, a somatostatin analog possessing high affinity for all five subtypes of somatostatin receptors (SSTR 1-5), commonly overexpressed in NE cancer cells, was used for the first time as an NE cancer targeting ligand. KE108 exhibited superior targeting abilities compared to other common somatostatin analogs, such as octreotide, in NE cancer cell lines. The in vitro assays demonstrated that the TDP-A-loaded, KE108-targeted micelles exhibited the best capabilities in suppressing NE cancer cell growth. Moreover, the in vivo near-infrared fluorescence imaging on NE-tumor-bearing nude mice showed that KE108-conjugated micelles exhibited the greatest tumor accumulation due to their passive targeting and active targeting capabilities. Finally, TDP-A-loaded and KE108-conjugated micelles possessed the best anticancer efficacy without detectable systemic toxicity. Thus, these novel TDP-A-loaded and KE108-conjugated unimolecular micelles offer a promising approach for targeted NE cancer therapy.

Generation of Novel Thyroid Cancer Stem-Like Cell Clones: Effects of Resveratrol and Valproic Acid.

Anaplastic thyroid cancer is an aggressive and highly lethal cancer for which conventional therapies have proved ineffective. Cancer stem-like cells (CSCs) represent a small fraction of cells in the cancer that are resistant to chemotherapy and radiation therapy and are responsible for tumor reoccurrence and metastasis. We characterized CSCs in thyroid carcinomas and generated clones of CSC lines. Our study showed that anaplastic thyroid cancers had significantly more CSCs than well-differentiated thyroid cancers. We also showed that Aldefluor-positive cells revealed significantly higher expression of stem cell markers, self-renewal properties, thyrosphere formation, and enhanced tumorigenicity. In vivo passaging of Aldefluor-positive cells resulted in the growth of larger, more aggressive tumors. We isolated and generated two clonal spheroid CSC lines derived from anaplastic thyroid cancer that were even more enriched with stem cell markers and more tumorigenic than the freshly isolated Aldefluor-positive cells. Resveratrol and valproic acid treatment of one of the CSC lines resulted in a significant decrease in stem cell markers, Aldefluor expression, proliferation, and invasiveness, with an increase in apoptosis and thyroid differentiation markers, suggesting that these cell lines may be useful for discovering new adjuvant therapies for aggressive thyroid cancers. For the first time, we have two thyroid CSC lines that will be useful tools for the study of thyroid CSC targeted therapies.

Thailandepsin A-loaded and octreotide-functionalized unimolecular micelles for targeted neuroendocrine cancer therapy.

Due to the overexpression of somatostatin receptors in neuroendocrine (NE) cancers, drug nanocarriers conjugated with somatostatin analogs, such as octreotide (OCT), for targeted NE cancer therapy may offer increased therapeutic efficacies and decreased adverse effects. In this study, OCT-functionalized unimolecular micelles were prepared using individual hyperbranched polymer molecules consisting of a hyperbranched polymer core (Boltorn(®) H40) and approximately 25 amphiphilic polylactide-poly(ethlyene glycol) (PLA-PEG) block copolymer arms (H40-PLA-PEG-OCH3/OCT). The resulting micelles, exhibiting a uniform core-shell shape and an average hydrodynamic diameter size of 66 nm, were loaded with thailandepsin-A (TDP-A), a relatively new naturally produced histone deacetylase (HDAC) inhibitor. In vitro studies using flow cytometry and confocal laser scanning microscopy (CLSM) demonstrated that OCT conjugation enhanced the cellular uptake of the unimolecular micelles. Consequently, TDP-A-loaded and OCT-conjugated micelles exhibited the highest cytotoxicity and caused the highest reduction of NE tumor markers. Finally, the in vivo studies on NE cancer bearing nude mice demonstrated that TDP-A-loaded and OCT-conjugated micelles possessed superior anticancer activity in comparison with other TDP-A formulations or drug alone, while showing no detectable systemic toxicity. Thus, these TDP-A-loaded and OCT-conjugated micelles offer a promising approach for targeted NE cancer therapy.

Tumor-suppressor role of Notch3 in medullary thyroid carcinoma revealed by genetic and pharmacological induction.

Notch1-3 are transmembrane receptors that appear to be absent in medullary thyroid cancer (MTC). Previous research has shown that induction of Notch1 has a tumor-suppressor effect in MTC cell lines, but little is known about the biologic consequences of Notch3 activation for the progression of the disease. We elucidate the role of Notch3 in MTC by genetic (doxycycline-inducible Notch3 intracellular domain) and pharmacologic [AB3, novel histone deacetylase (HDAC) inhibitor] approaches. We find that overexpression of Notch3 leads to the dose-dependent reduction of neuroendocrine tumor markers. In addition, Notch3 activity is required to suppress MTC cell proliferation, and the extent of growth repression depends on the amount of Notch3 protein expressed. Moreover, activation of Notch3 induces apoptosis. The translational significance of this finding is highlighted by our observation that MTC tumors lack active Notch3 protein and reinstitution of this isoform could be a therapeutic strategy to treat patients with MTC. We demonstrate, for the first time, that overexpression of Notch3 in MTC cells can alter malignant neuroendocrine phenotype in both in vitro and in vivo models. In addition, our study provides a strong rationale for using Notch3 as a therapeutic target to provide novel pharmacologic treatment options for MTC.

The roles of the epithelial-mesenchymal transition marker PRRX1 and miR-146b-5p in papillary thyroid carcinoma progression.

Thyroid carcinoma is the most common endocrine malignancy, and papillary thyroid carcinoma represents the most common thyroid cancer. Papillary thyroid carcinomas that invade locally or metastasize are associated with a poor prognosis. We found that, during epithelial-mesenchymal transition (EMT) induced by transforming growth factor-ß1 (TGF-ß1), papillary thyroid carcinoma cells acquired increased cancer stem cell-like features and the transcription factor paired-related homeobox protein 1 (PRRX1; alias PRX-1), a newly identified EMT inducer, was markedly up-regulated. miR-146b-5p was also transiently up-regulated during EMT, and in siRNA experiments miR-146b-5p had an inhibitory role on cell proliferation and invasion during TGF-ß1-induced EMT. We conclude that papillary thyroid carcinoma tumor cells exhibit increased cancer stem cell-like features during TGF-ß1-induced EMT, that miR-146b-5p has a role in cell proliferation and invasion, and that PRRX1 plays an important role in papillary thyroid carcinoma EMT and disease progression.

The novel histone deacetylase inhibitor thailandepsin A inhibits anaplastic thyroid cancer growth.

BACKGROUND: Anaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with antiproliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. METHODS: Human-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay, and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. RESULTS: TDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly adenosine diphosphate ribose polymerase were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently, and the antiapoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase after TDP-A treatment. CONCLUSIONS: TDP-A induces a notable dose- and time-dependent antiproliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.

MK-2206 causes growth suppression and reduces neuroendocrine tumor marker production in medullary thyroid cancer through Akt inhibition.

BACKGROUND: Development of targeted therapies for medullary thyroid cancer (MTC) has focused on inhibition of the rearranged during transfection (RET) proto-oncogene. Akt has been demonstrated to be a downstream target of RET via the key mediator phosphoinositide-3-kinase. MK-2206 is an orally administered allosteric Akt inhibitor that has exhibited minimal toxicity in phase I trials. We explored the antitumor effects of this compound in MTC. METHODS: Human MTC-TT cells were treated with MK-2206 (0-20 µM) for 8 days. Assays for cell viability were performed at multiple time points with MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide). The mechanism of action, mechanism of growth inhibition, and production of neuroendocrine tumor markers were assessed with Western blot analysis. RESULTS: MK-2206 suppressed MTC cell proliferation in a dose-dependent manner (p ≤ 0.001). Levels of Akt phosphorylated at serine 473 declined with increasing doses of MK-2206, indicating successful Akt inhibition. The apoptotic proteins cleaved poly (ADP-ribose) polymerase and cleaved caspase-3 increased in a dose-dependent manner with MK-2206, while the apoptosis inhibitor survivin was markedly reduced. Importantly, the antitumor effects of MK-2206 were independent of RET inhibition, as the levels of RET protein were not blocked. CONCLUSIONS: MK-2206 significantly suppresses MTC proliferation without RET inhibition. Given its high oral bioavailability and low toxicity profile, phase II studies with this drug alone or in combination with RET inhibitors are warranted.

Resveratrol induces differentiation markers expression in anaplastic thyroid carcinoma via activation of Notch1 signaling and suppresses cell growth.

Anaplastic thyroid carcinoma (ATC) is an extremely aggressive malignancy with undifferentiated features, for which conventional treatments, including radioactive iodine ablation, are usually not effective. Recent evidence suggests that the Notch1 pathway is important in the regulation of thyroid cancer cell growth and expression of thyrocyte differentiation markers. However, drug development targeting Notch1 signaling in ATC remains largely underexplored. Previously, we have identified resveratrol out of over 7,000 compounds as the most potent Notch pathway activator using a high-throughput screening method. In this study, we showed that resveratrol treatment (10-50 µmol/L) suppressed ATC cell growth in a dose-dependent manner for both HTh7 and 8505C cell lines via S-phase cell-cycle arrest and apoptosis. Resveratrol induced functional Notch1 protein expression and activated the pathway by transcriptional regulation. In addition, the expression of thyroid-specific genes including TTF1, TTF2, Pax8, and sodium iodide symporter (NIS) was upregulated in both ATC cell lines with resveratrol treatment. Notch1 siRNA interference totally abrogated the induction of TTF1 and Pax8 but not of TTF2. Moreover, Notch1 silencing by siRNA decreased resveratrol-induced NIS expression. In summary, our data indicate that resveratrol inhibits cell growth and enhances redifferentiation in ATC cells dependent upon the activation of Notch1 signaling. These findings provide the first documentation for the role of resveratrol in ATC redifferentiation, suggesting that activation of Notch1 signaling could be a potential therapeutic strategy for patients with ATC and thus warrants further clinical investigation.

Neuroendocrine phenotype alteration and growth suppression through apoptosis by MK-2206, an allosteric inhibitor of AKT, in carcinoid cell lines in vitro.

Carcinoids are neuroendocrine malignancies characterized by their overproduction of various bioactive hormones that lead to the carcinoid syndrome. We have shown previously that AKT serves as a key regulator of growth and phenotypic expression of tumor markers in carcinoids by the genetic depletion of AKT expression. However, no small-molecule inhibitor of AKT kinase activity has been developed until recently. MK-2206, a novel allosteric inhibitor of AKT, is currently undergoing clinical trials for the treatment of solid tumors. In this study, we explored the effect of MK-2206 on carcinoid cell proliferation and bioactive hormone production in vitro in two carcinoid cell lines - pancreatic carcinoid BON and bronchopulmonary H727. Treatment with MK-2206 effectively suppressed AKT phosphorylation at serine 473 and significantly reduced cell proliferation in a dose-dependent manner. Most importantly, MK-2206 treatment resulted in a significant reduction in ASCL1, CgA, and NSE expression, collectively recognized as markers of neuroendocrine tumor malignancy. Furthermore, MK-2206-treated cells showed an increase in levels of cleaved PARP and cleaved caspase-3, with a concomitant reduction in levels of Mcl-1 and XIAP, indicating that the antiproliferative effect of MK-2206 occurs through the induction of apoptosis. In conclusion, MK-2206 suppresses carcinoid tumor growth, and alters its neuroendocrine phenotype, indicating that this drug may be beneficial for patients with carcinoid syndrome. These studies merit further clinical investigation.

Treatment of macular seborrheic keratoses using a novel 1927-nm fractional thulium fiber laser.

OBJECTIVES: Macular seborrheic keratoses (SK) are common, benign growths. A novel fractionated 1927-nm thulium fiber laser was selected to investigate efficacy and safety in the clearance of macular SK in nonfacial areas at a private dermatologic laser center. STUDY DESIGN: Six subjects (average age 56.8 ± 9.5) with at least four 3-mm or larger macular SK on one extremity, neck, or chest were enrolled and provided informed consent. Subjects received three treatments at 3- to 4-week intervals. Subjects were treated at settings of 10 to 20 mJ with 30% to 55% surface area coverage and four to eight passes at each session. Investigators performed a global extremity SK count, an SK count within a 5- by 5-cm (25 cm(2)) square on transparency mapping, and lesion thickness grading. Side effects were assessed. Patients rated pain using a visual analog scale. RESULTS: At baseline, average global SK count was 43.2 ± 27.3. At 1-month follow-up, 20.7 ± 16.6 lesions remained. At 3- and 6-month follow-up, 17.7 ± 23.8 and 14.7 ± 18.6 lesions remained, respectively. A decrease in thickness and SK counts within the 5- by 5-cm square was also observed. Moderate erythema and mild edema were post-treatment responses. CONCLUSION: Nonablative fractional resurfacing using the 1927-nm thulium fiber laser is a safe and effective treatment modality to decrease nonfacial macular SK.

1,927-nm fractional thulium fiber laser for the treatment of nonfacial photodamage: a pilot study.

BACKGROUND: A 1,927-nm wavelength was recently added to the 1,550-nm erbium-doped fiber laser. This wavelength possesses a higher absorption coefficient for water than the 1,550-nm, conferring greater ability to target epidermal processes. OBJECTIVE: To evaluate the efficacy and safety of a novel 1,927-nm fractional thulium fiber laser in the treatment of nonfacial photodamage. METHODS: Nine participants with nonfacial photodamage underwent three laser treatments (at 3- to 4-week intervals) at pulse energies of 10 to 20 mJ and 30% to 55% surface area coverage (4-8 passes) with a 1,927-nm thulium fiber laser. A blinded assessor and participants evaluated clinical improvement of treatment areas at 1-month follow-up using a quartile grading scale (0-4). RESULTS: Nine participants with a total of 12 treatment areas completed three treatments and the 1-month follow-up visit. At follow-up, a blinded assessor rated mean improvement in photodamage as 3.25 ± 1.0 and lentigines as 3.33 ± 0.9 on a scale of 0 to 4 (0=none, 1=1-25%, 2=26-50%, 3=51-75%, and 4=76-100% improvement). Skin responses observed after treatment were moderate erythema, mild edema, itching, and desquamation. No scarring or postinflammatory hyper- or hypopigmentation was observed. CONCLUSION: The 1,927-nm fractional thulium fiber laser is a safe, effective treatment for nonfacial photodamage. Dr. Polder is a principal investigator and Dr. Suzanne Bruce is a consultant for Solta Medical.