Regression of experimental NIS-expressing breast cancer brain metastases in response to radioiodide/gemcitabine dual therapy.

Treating breast cancer brain metastases (BCBMs) is challenging. Na+/I- symporter (NIS) expression in BCBMs would permit their selective targeting with radioiodide (131I-). We show impressive enhancement of tumor response by combining131I- with gemcitabine (GEM), a cytotoxic radiosensitizer. Nude mice mammary fat-pad (MFP) tumors and BCBMs were generated with braintropic MDA-MB-231Br cells transduced with bicistronically-linked NIS and firefly luciferase cDNAs. Response was monitored in vivo via bioluminescent imaging and NIS tumor expression.131I-/GEM therapy inhibited MFP tumor growth more effectively than either agent alone. BCBMs were treated with: high or low-dose GEM (58 or 14.5 mg/Kg×4); 131I- (1mCi or 2×0.5 mCi 7 days apart); and 131I-/GEM therapy. By post-injection day (PID) 25, 82-86% of controls and 78-83% of 131I--treated BCBM grew, whereas 17% low-dose and 36% high-dose GEM regressed. The latter tumors were smaller than the controls with comparable NIS expression (~20% of cells). High and low-dose 131I-/ GEM combinations caused 89% and 57% tumor regression, respectively. High-dose GEM/131I- delayed tumor growth: tumors increased 5-fold in size by PID45 (controls by PID18). Although fewer than 25% of cells expressed NIS, GEM/131I- caused dramatic tumor regression in NIS-transduced BCBMs. This effect was synergistic, and supports the hypothesis that GEM radiosensitizes cells to 131I-

Disease-modifying polymorphisms and C609Y mutation of RET associated with high penetrance of phaeochromocytoma and low rate of MTC in MEN2A.

Mutations of the rearranged during transfection (RET) proto-oncogene, located on chromosome 10q11.2, cause multiple endocrine neoplasia type 2A (MEN2A). Patients with mutations at the codon 609 usually exhibit a high penetrance of medullary thyroid cancer (MTC), but a sufficiently low penetrance of phaeochromocytoma that screening for this latter complication has been called to question. Patients with other RET mutations are at higher risk of younger age onset phaeochromocytoma if they also possess other RET polymorphisms (L769L, S836S, G691S and S904S), but there are no similar data for patients with 609 mutations. We investigated the unusual phenotypic presentation in a family with MEN2A due to a C609Y mutation in RET. Sanger sequencing of the entire RET-coding region and exon-intron boundaries was performed. Five family members were C609Y mutation positive: 3/5 initially presented with phaeochromocytoma, but only 1/5 had MTC. The index case aged 73 years had no evidence of MTC, but presented with phaeochromocytoma. Family members also possessed the G691S and S904S RET polymorphisms. We illustrate a high penetrance of phaeochromocytoma and low penetrance of MTC in patients with a RET C609Y mutation and polymorphisms G691S and S904S. These data highlight the need for life-long screening for the complications of MEN2A in these patients and support the role for the screening of RET polymorphisms for the purposes of risk stratification. LEARNING POINTS: C609Y RET mutations may be associated with a life-long risk of phaeochromocytoma indicating the importance of life-long screening for this condition in patients with MEN2A.C609Y RET mutations may be associated with a lower risk of MTC than often quoted, questioning the need for early prophylactic thyroid surgery discussion at the age of 5 years.There may be a role for the routine screening of RET polymorphisms, and this is greatly facilitated by the increasing ease of access to next-generation sequencing

PI3K activation is associated with intracellular sodium/iodide symporter protein expression in breast cancer

Background: The sodium/iodide symporter (NIS) is a membrane glycoprotein mediating active iodide uptake in the thyroid gland and is the molecular basis for radioiodide imaging and therapeutic ablation of thyroid carcinomas. NIS is expressed in the lactating mammary gland and in many human breast tumors, raising interest in similar use for diagnosis and treatment. However, few human breast tumors have clinically evident iodide uptake ability. We previously identified PI3K signaling as important in NIS upregulation in transgenic mouse models of breast cancer, and the PI3K pathway is commonly activated in human breast cancer. Methods: NIS expression, subcellular localization, and function were analyzed in MCF-7 human breast cancer cells and MCF-7 cells stably or transiently expressing PI3K p110alpha subunit using Western blot of whole cell lysate, cell surface biotinylation Western blot and immunofluorescence, and radioiodide uptake assay, respectively. NIS localization was determined in a human breast cancer tissue microarray using immunohistochemical staining (IHC) and was correlated with preexisting pAkt IHC data. Statistical analysis consisted of Student's t-test (in vitro studies) or Fisher's Exact Test (in vivo correlational studies). Results: In this study, we demonstrate that PI3K activation in MCF-7 human mammary carcinoma cells leads to expression of underglycosylated NIS lacking cell surface trafficking necessary for iodide uptake ability. PI3K activation also appears to interfere with cell surface trafficking of exogenous NIS as well as all-trans retinoic acid-induced endogenous NIS. A correlation between NIS expression and upregulation of PI3K signaling was found in a human breast cancer tissue microarray. Conclusion: Thus, the PI3K pathway likely plays a major role in the discordance between NIS expression and iodide uptake in breast cancer patients. Further study is warranted to realize the application of NIS-mediated radioiodide ablation in breast cancer

A PCR-mutagenesis strategy for rapid detection of mutations in codon 634 of the ret proto-oncogene related to MEN 2A.

BACKGROUND: Multiple endocrine neoplasias type 2A (MEN 2A) is a dominantly inherited cancer syndrome. Missence mutations in the codon encoding cysteine 634 of the ret proto-oncogene have been found in 85% of the MEN 2A families. The main tumour type always present in MEN 2A is medullar thyroid carcinoma (MTC). Only 25% of all MTC are hereditary, and generally they are identified by a careful family history. However, some familial MTCs are not easily detected by this means and underdiagnosis of MEN 2A is suspected. METHODS: DNA samples from MEN 2A patients were amplified by PCR. The products were incubated with the restriction enzyme Bst ApI or Bgl I. The samples were loaded in non-denaturing 10% Polyacrilamyde Gel and run at 120 volts for 40 min. The gels were stained with 10 μg/ml ethidium bromide, and the bands were visualized under a UV lamp. RESULTS: We developed a PCR-mutagenic method to check the integrity of the three bases of the cysteine 634 codon. CONCLUSION: The method can be used to detect inherited mutations in MTC patients without a clear family history. The method is relatively simple to use as a routine test in these patients to decrease the underdiagnosis of MEN 2A. In addition, the assay can be used to screen affected families with any mutation in cysteine 634

Induction of Sodium/Iodide Symporter (NIS) Expression and Radioiodine Uptake in Non-Thyroid Cancer Cells

Background: This study was designed to explore the therapeutic potential of suppressing MAP kinase and PI3K/Akt pathways and histone deacetylase (HDAC) to induce the expression of sodium/iodide symporter (NIS) and radioiodine uptake in non-thyroid cancer cells. Methods: We tested the effects of the MEK inhibitor RDEA119, the Akt inhibitor perifosine, and the HDAC inhibitor SAHA on NIS expression in thirteen human cancer cell lines derived from melanoma, hepatic carcinoma, gastric carcinoma, colon carcinoma, breast carcinoma, and brain cancers. We also examined radioiodine uptake and histone acetylation at the NIS promoter in selected cells. Results: Overall, the three inhibitors could induce NIS expression, to various extents, in melanoma and all the epithelial carcinoma-derived cells but not in brain cancer-derived cells. SAHA was most effective and its effect could be significantly enhanced by RDEA119 and perifosine. The expression of NIS, at both mRNA and protein levels, was most robust in the melanoma cell M14, hepatic carcinoma cell HepG2, and the gastric carcinoma cell MKN-7 cell. Radioiodine uptake was correspondingly induced, accompanied by robust increase in histone acetylation at the NIS promoter, in these cells when treated with the three inhibitors. Conclusions: This is the first demonstration that simultaneously suppressing the MAP kinase and PI3K/Akt pathways and HDAC could induce robust NIS expression and radioiodine uptake in certain non-thyroid human cancer cells, providing novel therapeutic implications for adjunct radioiodine treatment of these cancers

Combined Vorinostat and Chloroquine Inhibit Sodium Iodide Symporter Endocytosis and Enhance Radionuclide Uptake In Vivo

Purpose Patients with aggressive thyroid cancer are frequently failed by the central therapy of ablative radioiodide (RAI) uptake, due to reduced plasma membrane (PM) localization of the sodium/iodide symporter (NIS). We aimed to understand how NIS is endocytosed away from the PM of human thyroid cancer cells, and whether this was druggable in vivo.Experimental DesignInformed by analysis of endocytic gene expression in patients with aggressive thyroid cancer, we used mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET to understand the mechanisms of NIS endocytosis in transformed cell lines and patient-derived human primary thyroid cells. Systemic drug responses were monitored via 99mTc pertechnetate gamma counting and gene expression in BALB/c mice.ResultsWe identify an acidic dipeptide within the NIS C-terminus which mediates binding to the 2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor vorinostat/ SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence.ConclusionsNIS internalisation is specifically druggable in vivo. Our data therefore provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer.<br/

DNA topoisomerases participate in fragility of the oncogene RET

Fragile site breakage was previously shown to result in rearrangement of the RET oncogene, resembling the rearrangements found in thyroid cancer. Common fragile sites are specific regions of the genome with a high susceptibility to DNA breakage under conditions that partially inhibit DNA replication, and often coincide with genes deleted, amplified, or rearranged in cancer. While a substantial amount of work has been performed investigating DNA repair and cell cycle checkpoint proteins vital for maintaining stability at fragile sites, little is known about the initial events leading to DNA breakage at these sites. The purpose of this study was to investigate these initial events through the detection of aphidicolin (APH)-induced DNA breakage within the RET oncogene, in which 144 APHinduced DNA breakpoints were mapped on the nucleotide level in human thyroid cells within intron 11 of RET, the breakpoint cluster region found in patients. These breakpoints were located at or near DNA topoisomerase I and/or II predicted cleavage sites, as well as at DNA secondary structural features recognized and preferentially cleaved by DNA topoisomerases I and II. Co-treatment of thyroid cells with APH and the topoisomerase catalytic inhibitors, betulinic acid and merbarone, significantly decreased APH-induced fragile site breakage within RET intron 11 and within the common fragile site FRA3B. These data demonstrate that DNA topoisomerases I and II are involved in initiating APH-induced common fragile site breakage at RET, and may engage the recognition of DNA secondary structures formed during perturbed DNA replication

FAK acts as a suppressor of RTK-MAP kinase signalling in Drosophila melanogaster epithelia and human cancer cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours

Animal Models of Cancer in the Head and Neck Region

Animal models that resemble the cancers of the head and neck region are of paramount importance in studying the carcinogenesis of these diseases. Although several methods for modeling cancer in the head and neck are available, none are fully satisfactory. Subcutaneous xenograft models of cancer in nude mice are often used in preclinical studies. However, these models are problematic in several aspects as they lack the specific interactions that exist between the tumor cells and their native environment. Establishment of tumors at the orthotopic sites restore these distinct patterns of interactions between the tumor and the host organs that are lost or altered when the tumors are established in ectopic sites. With regard to the transgenic model of cancer in the head and neck region, it should be kept in mind that the transgene used to drive the malignant transformation may not be representative of the carcinogenic process found in human tumors. Low penetrance of tumor formation also translates into high cost and time commitment in performing studies with transgenic models. In this review, we will discuss some of the commonly used methods for modeling cancer in the head and neck region including squamous cell carcinoma of the head and neck as well as thyroid carcinoma