Saif Al-Sobhi

Fatty Acid Synthase and AKT Pathway Signaling in a Subset of Papillary Thyroid Cancers

CONTEXT Fatty acid synthase (FASN) is an enzyme that plays a critical role in de novo synthesis of fatty acids. FASN is overexpressed in variety of human cancers, but its role has not been elucidated in papillary thyroid carcinoma (PTC). OBJECTIVE Our objective was to investigate the role of FASN and its relationship with phosphatidylinositol 3-kinase/AKT activation in a large series of PTC in a tissue microarray format followed by studies using PTC cell lines and Nude mice. DESIGN Analysis of apoptosis and cell cycle were evaluated by flow cytometry and DNA fragmentation assays. FASN and phospho-AKT protein expression was determined by immunohistochemistry and Western blotting. RESULTS Our data show that expression of FASN is associated with activated AKT (phospho-AKT) in a subset of PTC. Treatment of PTC cell lines (NPA-187, ONCO-DG-1, and B-CPAP) with C-75, an inhibitor of FASN, suppresses growth and induces apoptosis in all cell lines. Treatment of PTC cells with C-75 or expression of FASN small interfering RNA causes down-regulation of FASN and inactivation of AKT activity. Furthermore, treatment of PTC cell lines with C-75 results in apoptosis via the mitochondrial pathway involving the proapoptotic factor Bad, activation of Bax, activation of caspases, and down-regulation of antiapoptotic proteins. Finally, treatment of NPA-187 xenografts with C-75 results in growth inhibition of tumors in Nude mice via down-regulation of FASN expression and inactivation of AKT. CONCLUSIONS Our results suggest that FASN and activated AKT pathway may be a potential target for therapeutic intervention for the treatment of PTC.

Decreased axillary lymph node retrieval in patients after neoadjuvant chemotherapy

BACKGROUND The purpose of this study was to assess our clinical impression that fewer lymph nodes are retrieved after level I and II axillary dissection after neoadjuvant chemotherapy and whether there is a positive correlation between the total number of lymph nodes retrieved and the number of diseased lymph nodes. METHODS Patients included those with stage IIB, IIIA, and IIIB breast cancer of whom 77 had neoadjuvant chemotherapy and 58 had initial surgery only. All had modified radical mastectomy with in continuity level I and II axillary dissection. RESULTS Patients after neoadjuvant chemotherapy had 14.3 +/- 6.7 lymph nodes detected versus 16.9 +/- 8.8 (mean +/- SD; P <0.057) for those with initial surgery only. The number of positive nodes were 3.7 +/- 4.7 versus 6.6 +/- 8.7 (mean +/- SD; P <0.033) respectively and the number of negative nodes were 10.6 +/- 7.5 versus 10.4 +/- 8 (mean +/- SD; P <0.9). The correlation between the number of positive lymph nodes and the total number of lymph nodes was r = 0.58; P <0.001. CONCLUSIONS It appears that fewer lymph nodes are retrieved after level I and II axillary dissection after neoadjuvant chemotherapy. The total number of lymph nodes retrieved increases directly with the number of positive lymph nodes in patients not treated with chemotherapy.

FoxM1 and its association with matrix metalloproteinases (MMP) signaling pathway in papillary thyroid carcinoma.

CONTEXT Forkhead boxM1 (FoxM1) transcription factor has been shown to promote pathogenesis of several malignancies. FoxM1 has also been shown to be associated with matrix metalloproteinases (MMP) in various cancers. However, little is known about its function in papillary thyroid carcinoma (PTC). OBJECTIVE In this study, we investigated the role of FoxM1 in pathogenesis in a large series of PTC in a tissue microarray format followed by in vitro and in vivo studies using PTC cell lines and nude mice. DESIGN Expression of FoxM1 and its associated proteins were investigated in Middle Eastern PTC samples by immunohistochemistry. Apoptosis was measured by flow cytometry and immunoblotting. Invasion and migration studies were performed using 8-μm Transwell plates. RESULTS FoxM1 was overexpressed in 28.4% of PTC and significantly associated with activated matrix metalloproteinase-9 (MMP-9) (P = 0.0004), X-linked inhibitor of apoptosis protein (XIAP) (P = 0.0024), and B-cell lymphoma-extra large (Bcl-XL) (P = 0.0014) expression. Treatment of PTC cell lines with thiostrepton, an inhibitor of FoxM1, resulted in inhibition of cell viability via induction of apoptosis. In addition, thiostrepton treatment of PTC cells or expression of FoxM1-specific small interfering RNA down-regulated expression of FoxM1 accompanied with decreased MMP-2 and MMP-9 expression. Furthermore, inhibition of FoxM1 attenuated migration and invasion of PTC cells. Interestingly, overexpression of FoxM1 rescued the effects of thiostrepton in PTC cell lines. Finally, treatment of PTC cell line xenografts with thiostrepton resulted in growth inhibition of tumors in nude mice via down-regulation of FoxM1 and MMP-9 and MMP-2. CONCLUSION Altogether, this is the first study showing that FoxM1 and its associated signaling pathway play a critical role in the pathogenesis of PTC and may be a potential target for therapeutic intervention for treatment of these cancers.

Detection of metastatic parathyroid carcinoma with Tc-99m sestamibi imaging

PURPOSE To assess the use of Tc-99m sestamibi to localize recurrent and metastatic parathyroid carcinoma. METHODS A patient with a history of parathyroid carcinoma that was resected 6 months before had high serum calcium and high serum parathyroid hormone levels. Tc-99m sestamibi imaging was performed to localize the recurrence. RESULTS Tc-99 sestamibi imaging detected the recurrence and the metastatic lymph nodes. These findings were confirmed surgically and pathologically. CONCLUSION Tc-99m sestamibi can be useful in diagnosing and localizing metastatic parathyroid carcinoma.

Whole-body positron emission tomographic scanning in patients with adrenal cortical carcinoma: comparison with conventional imaging procedures.

Two patients with histologically documented adrenal cortical carcinoma (ACC) underwent whole-body fluorodeoxyglucose (FDG) positron emission tomographic (PET) imaging. Results were compared with those of computed tomography (CT), magnetic resonance imaging (MRI), ultrasound, bone scanning, and octreotide imaging to evaluate the role of PET and to determine any additional advantage PET may provide over conventional imaging in the management of ACC. Both patients were 26-year-old men. One patient was found to have Cushings syndrome, and the other had a clinically silent recurrent ACC. These findings indicate that PET can accurately localize ACC before operation and reliably detect its recurrence after operation. It can serve as an adjuvant imaging method in the detection of metastases and monitor the clinical course of ACC with findings that complement those of conventional imaging. It can yield additional information in defining tumor metabolic activity, necrosis, and in the earlier detection of metastases compared with CT.

High prevalence of mTOR complex activity can be targeted using Torin2 in papillary thyroid carcinoma

The mammalian target of rapamycin (mTOR) signaling cascade is a key regulatory pathway controlling initiation of messenger RNA in mammalian cells. Although dysregulation of mTOR signaling has been reported earlier in cancers, there is paucity of data about mTOR expression in papillary thyroid carcinoma (PTC). Therefore, in this study, we investigated the presence of mTORC2 and mTORC1 complexes in a large cohort of >500 PTC samples. Our clinical data showed the presence of active mTORC1 and mTORC2 in 81 and 39% of PTC samples, respectively. Interestingly, coexpression of mTORC1 and mTORC2 activity was seen in a 32.5% (164/504) of the PTC studied and this association was statistically significant (P = 0.0244). mTOR signaling complex was also found to be associated with activated AKT and 4E-BP1. In vitro, using Torin2, a second-generation mTOR inhibitor or gene silencing of mTOR expression prevented mTORC1 and mTORC2 activity leading to inactivation of P70S6, 4E-BP1, AKT and Bad. Inhibition of mTOR activity led to downregulation of cyclin D1, a gene regulated by messenger RNA translation via phosphorylation of 4E-BP1. Torin2 treatment also inhibited cell viability and induced caspase-dependent apoptosis via activation of mitochondrial apoptotic pathway in PTC cells. Finally, Torin2 treatment induces anticancer effect on PTC xenograft tumor growth in nude mice via inhibition of mTORC1 and mTORC2 and its associated pathways. Our results suggest that coexpression of mTORC1 and mTORC2 is seen frequently in the clinical PTC samples and dual targeting of mTORC1 and mTORC2 activity may be an attractive therapeutic target for treatment of PTC.

Role of X-Linked Inhibitor of Apoptosis as a Prognostic Marker and Therapeutic Target in Papillary Thyroid Carcinoma

CONTEXT Papillary thyroid cancer (PTC) is the second most common cancer in females in Saudi Arabia. However, the pathogenesis of PTC is still not fully elucidated. OBJECTIVE To identify potential genes that play important role in progression of PTC, we studied the role of X-linked inhibitor of apoptosis protein (XIAP) as a potential prognostic marker and therapeutic target in a large cohort of PTC samples and cell lines. DESIGN A DNA microarray chip was used to screen for gene copy number. XIAP expression was assessed by immunohistochemistry in a tissue microarray format on a cohort of 1022 clinical samples. In vitro and in vivo studies were performed using Embelin and/or LY294002 on PTC cell lines. RESULTS XIAP was found to be amplified in 14 of 29 and overexpressed in 48.8% of PTC cases. XIAP overexpression was significantly associated with old age, extrathyroidal extension, tumor size, nodal involvement, tall-cell variant, advanced stage disease, and significantly poor disease-free survival (P = .0341). XIAP was also significantly associated with phosphorylated AKT (P < .0001), Bcl-Xl (P < .0001), and Ki67 (P = .0006) proteins. Embelin treatment caused growth inhibition and apoptosis in PTC cell lines and induced tumor regression in PTC xenograft in nude mice. Finally, the combination of suboptimal doses of Embelin and LY294002 induced a synergistic apoptotic response in PTC cells. CONCLUSION XIAP dysregulation in PTC confers an aggressive phenotype with poor outcome. In vitro and in vivo studies using an XIAP inhibitor suggest that this subgroup of PTC with overexpression of XIAP can be therapeutically targeted, either alone or in combination, to induce efficient apoptosis in these cancers.

Genomic Profiling of Thyroid Cancer Reveals a Role for Thyroglobulin in Metastasis.

Papillary thyroid carcinoma (PTC) has a wide geographic variation in incidence; it is most common in Saudi Arabia, where it is only second to breast cancer as the most common cancer among females. Genomic profiling of PTC from Saudi Arabia has not been attempted previously. We performed whole-exome sequencing of 101 PTC samples and the corresponding genomic DNA to identify genes with recurrent somatic mutations, then sequenced these genes by using a next-generation gene-panel approach in an additional 785 samples. In addition to BRAF, N-RAS, and H-RAS, which have previously been shown to be recurrently mutated in PTC, our analysis highlights additional genes, including thyroglobulin (TG), which harbored somatic mutations in 3% of the entire cohort. Surprisingly, although TG mutations were not exclusive to mutations in the RAS-MAP kinase pathway, their presence was associated with a significantly worse clinical outcome, which suggests a pathogenic role beyond driving initial oncogenesis. Analysis of metastatic PTC tissue revealed significant enrichment for TG mutations (p < 0.001), including events of apparent clonal expansion. Our results suggest a previously unknown role of TG somatic mutations in the pathogenesis of PTC and its malignant evolution.

c-Met inhibitor synergizes with tumor necrosis factor-related apoptosis-induced ligand to induce papillary thyroid carcinoma cell death.

The Met receptor tyrosine kinase is overexpressed and/or activated in variety of human malignancies. Previously we have shown that c-Met is overexpressed in Middle Eastern papillary thyroid carcinoma (PTC) and significantly associated with an aggressive phenotype, but its role has not been fully elucidated in PTC. The aim of this study was to determine the functional link between the c-Met/AKT signaling pathway and death receptor 5 (DR5) in a large cohort of PTC in a tissue microarray format followed by functional studies using PTC cell lines and nude mice. Our data showed that high expressions of p-Met and DR5 were significantly associated with an aggressive phenotype of PTC and correlated with BRAF mutation. Treatment of PTC cell lines with PHA665752, an inhibitor of c-Met tyrosine kinase, inhibited cell proliferation and induced apoptosis via the mitochondrial pathway in PTC cell lines. PHA665752 treatment or expression of c-Met small interfering (si)RNA resulted in dephosphorylation of c-Met, AKT and its downstream effector molecules. Furthermore, PHA665752 treatment upregulated DR5 expression via generation of reactive oxygen species in PTC cell lines, and synergistically potentiated death receptor-induced apoptosis with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Finally, cotreatment with PHA665752 and TRAIL caused more pronounced effects on PTC xenograft tumor growth in nude mice. Our data suggest that the c-Met/AKT pathway may be a potential target for therapeutic intervention for treatment of PTC refractory to conventionally therapeutic modalities.

Obstructive endotracheal lesions of thyroid cancer

Airway invasion is a life-threatening complication of thyroid cancer. An important issue that deserves better attention is the differentiation between the clinical features of tracheal wall invasion versus those of an obstructive endotracheal lesion. We present information on the clinical course, diagnostic modalities utilized, management instituted, along with the prognosis, and follow-up data on a group of patients presenting with obstructive endotracheal lesions of thyroid cancer. Two thousand four hundred and eighty-nine thyroid cancer patients were seen at our institution from December 1975 to May 2000. Thirteen patients presented with symptoms of respiratory distress related to obstructive endotracheal lesions. At presentation, 11 patients underwent endoscopic examination. Imaging studies consisting of I123 whole body scan (WBS), computed tomography/magnetic resonance imaging (CT/MRI) of neck and chest, whole body positron emission tomography using 18-fluoro-2-deoxy-D-glucose ((FDG)PET) were done, as also was determination of the tumour markers, serum thyroglobulin (TG) and calcitonin. Patients were followed for one to 108 months after the initial presentation. Intraluminal tracheal obstruction was severe in eight patients; five had near-total-occlusion. Paralysis of the vocal folds was present in five. Evidence of metastatic disease was present in most patients. Dissociation between iodine uptake and TG synthesis was evident in five patients during follow-up. Four patients died of cancer. Of the nine living patients; cancer persisted in six, recurred in two patients, and remitted in one. This study has identified obstructive endotracheal lesion of thyroid cancer as a distinct entity apart from tracheal wall disease. These data provide evidence that intraluminal tracheal invasion of thyroid cancer is an ominous sign and a frequent cause of morbidity.