ResearchPad - tumor-biology:-general-tumorigenesis-progression-and-metastasis https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SAT-141 Glycoprotein NMB (GPNMB) Is Pro-Tumorigenic in TSC2-Null Cancer Cells and Is a Potential Drug Target and Biomarker for Lymphangioleiomyomatosis (LAM)]]> https://www.researchpad.co/article/elastic_article_9551 Lymphangioleiomyomatosis (LAM) is an estrogen-sensitive lung disease found almost exclusively in women that is characterized by hyperproliferation of smooth muscle cells forming small tumors, or LAM lesions throughout the lungs of patients. Growth of these tumors leads to progressive loss of pulmonary function, and sometimes subsequent lung transplantation. LAM tumor cells contain mutations in either the TSC1 or TSC2 genes, leading to activation of the mTORC1 pathway. In fact, mTOR inhibitors such as sirolimus are commonly used to treat LAM; however, these drugs are not always effective and have significant side effects, suggesting the need for new therapeutic targets. Interestingly, another important feature of LAM cells is that they express melanocytic markers that are normally found in melanocytes or melanoma cells. From RNASeq analysis of a mouse model for LAM that we designed, we discovered significant upregulation of the melanocytic marker Glycoprotein Non-Metastatic Melanoma Protein B (GPNMB), a type I transmembrane protein. GPNMB was not only highly expressed in our mouse model (a uterine specific TSC2-null mouse), it was also expressed in TSC2-null cell lines, and human LAM patient lung samples. In our hands, knocking down GPNMB expression by siRNA directed against GPNMB mRNA decreased migration and proliferation in TSC2-null cells. Additionally, we found that GPNMB’s large ectodomain is shed by TSC2-null cells and can be detected in the blood of human patients with LAM. Finally, MMP 2 and 9 can be secreted as a result of ectodomain shedding and its interaction with integrins. Accordingly, we did indeed see a decrease in MMP 2/9 expression in TSC2-null cells with reduced GPNMB expression from treatment with siRNA directed against GPNMB mRNA. Overall, our results demonstrate the potential importance of GPNMB in LAM tumor progression, and suggest that GPNMB may be a possible LAM biomarker and target for its treatment.

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<![CDATA[SAT-147 Endogenous Expression of TCF21 by CRISPR/dCas9 System Results in Different Biological Responses in Adrenocortical Carcinoma and Hepatocarcinoma]]> https://www.researchpad.co/article/elastic_article_8780 Transcription factor 21 (TCF21/POD-1/Epicardin) inhibits the expression of SF-1 (NR5A1) by binding to the promoter E-box site in adrenocortical carcinoma (ACC). In contrast, TCF21 promotes increased expression of LRH-1 in hepatocarcinoma cell line, HepG2 cells, by binding to the Small Heterodimer Partner (SHP/NR0B2) promoter region, an LRH-1 negative regulator. Epigenetic alteration induced TCF21 loss of function and has been associated with increased of cellular migration and invasion. In ACC, TCF21 promoter is hypermethylated and less expressed. Our aim was to evaluate the effect of TCF21 by expressing or silencing TCF21 in adrenocortical pediatric adenoma, ACA-T7 cells, in ACC cell lines SW-13 and H295R cell line, and in HepG2 cell line. Were used CRISPR/dCas9/TCF21 and pCMVMycPOD1 or siRNATCF21 to express and silence TCF21, respectively. Increased expression of TCF21 in H295RpCMVMycPOD1 and SW-13CRISPR/dCas9/TCF21 cells resulted in significantly decreased cell migration and invasion (53.2±36.1%/70.8±26.5% and 82.6±4.2%/100%, respectively). In ACA-T7/siRNATCF21 cells, the inhibition of TCF21 resulted in significant increase migration and invasion capacity (45.0±12.7%/33.1±17.4%) compared with ACA-T7 cells. Higher TCF21 expression in HepG2CRISPR/dCas9/TCF21 increased invasion [147.08±16.54% (p<0.0001)]. Analysis of metalloproteinase genes expression showed that TCF21 significantly (p<0.01) increased MMP8 expression in SW-13CRISPR/dCas9/TCF21 and H295R/pCMVMycPod-1 whereas decreased MMP9 and MMP2 (p< 0.0001). The opposite effect was observed in ACA-T7/siRNATCF21. Moreover, in HepG2CRISPR/dCas9/TCF21 cells was observed an increase of MMP2 and MMP9 expression (p<0.001). These results suggest that TCF21 regulate epithelial mesenchymal transition and vice versa (EMT/MET) in tumors depending on cellular context. Supported by Fapesp and Capes.

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<![CDATA[SAT-LB26 A Dual Role for IGF-1R in Mammary Tumorigenesis]]> https://www.researchpad.co/article/elastic_article_8586 The insulin-like growth factor type 1 receptor (IGF-1R) is now thought to have a dual function in breast cancer. Several studies have shown overexpression of the IGF-1R pathway results in increased tumor cell proliferation and survival. Recent loss-of-function models have shown decreased mammary tumorigenesis latency and increased metastasis. These recent studies correlate with analyses of human patient datasets identifying worse overall survival with low IGF-1R expression. Similarly, inhibition of IGF-1R in the clinic has had no affect or has led to worse outcomes supporting the hypothesis that the IGF-1R may have tumor suppressive properties. Our prior published studies revealed loss of IGF-1R function results in heightened tumor epithelial stress, which alters the tumor microenvironment to be permissive for metastasis. Therefore, we asked does the loss of IGF-1R inherently change the tumor epithelium or is it simply the alterations of the microenvironment that lead to a metastatic primary tumor? We first analyzed cell invasion of primary tumor epithelial cells from a mouse tumor model driven by the Wnt1 oncogene (MMTV-Wnt1) and with reduced IGF-1R signaling by expression of a dominant-negative transgene (MMTV-dnIGF-1R). Epithelial cells from the MMTV-Wnt1/dnIGF-1R (bigenic) tumors invaded at the same rate as MMTV-Wnt1 tumor epithelial cells by tail vein injection suggesting invasive capacity is unchanged with reduced IGF-1R signaling. Interestingly, size of lung micrometastases from tail vein injected bigenic tumor epithelial cells was significantly reduced and the number also decreased over time in part due to a proliferative defect determined by immunostaining for pH3. Similarly, bigenic primary tumor epithelial cells injected into the mammary gland fat pad failed to form tumors suggesting alterations in cell adhesion. Consistent with this observation, E-cadherin gene and protein expression were decreased in the bigenic tumor epithelium compared to MMTV-Wnt1 tumors. In vitro analysis of cell adhesion in MMTV-Wnt1 primary epithelial cells resulted in both K8+ (luminal) and K14+ (basal) tumor epithelial cells adherence, while only K14+ cells from bigenic tumors adhered to collagen. Similarly, the lung micrometastases from tail vein injections exhibited predominantly K14+ cells. Analysis of MMTV-Wnt1 and bigenic primary tumors using single cell RNAseq revealed alterations in both stromal and epithelial populations that may contribute to bigenic primary tumor growth and metastasis. These data support the conclusion that inhibiting IGF-1R signaling results in both alterations to the tumor epithelium (partial EMT) and microenvironment that result in metastasis, but also, that the IGF-1R deficient metastatic cells need a niche or paracine signaling to proliferate.

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<![CDATA[SAT-148 Ectopic ACTH Secretion Has Varied Presentation and Requires Individualized Treatment - One Size Does Not Fit All]]> https://www.researchpad.co/article/elastic_article_8565 Ectopic ACTH secretion (EAS) presents in myriad ways. We present five cases of EAS to highlight similarities and differences in presentation and treatment. The first woman with known metastatic lung neuroendocrine tumour (NET) for two years presented with facial fullness, proximal weakness, worsening hypertension and hypokalaemia. Random cortisol of 2742nmol/L (99.39mcg/dL), with adrenocorticotrophic hormone (ACTH) of 201ng/L (5-50), was in keeping with EAS. She received medical treatment followed by bilateral adrenalectomy with EAS resolution and development of adrenal insufficiency. She is doing well. The second woman with proximal weakness was evaluated by neurologists. All neurological tests were normal but facial fullness and easy bruising was noted. Random cortisol was 875nmol/L (31.71mcg/dL) and ACTH was 90 ng/L. Imaging revealed metastatic liver disease with unknown primary and biopsy confirmed NET. Cortisol rose despite medical treatment and she died within fifteen months. The third woman with significant smoking history presented with haemoptysis and breathlessness. A right lung mass was suspected on chest X-ray and confirmed with CT. Endobronchial ultrasound-guided biopsy revealed small cell lung cancer (SCLC). She developed generalised weakness and severe hypokalaemia. Random cortisol of 1645nmol/L (59.63mcg/dL) with ACTH of 282ng/L suggested EAS. Despite medical treatment, she died within two weeks. The fourth woman presented with confusion, hypertension and severe hypokalaemia. Morning cortisol of 8557nmol/L (310.19mcg/dL) and random ACTH of 73ng/L were suggestive of EAS. CT demonstrated left lung mass with widespread metastases. She deteriorated and died within 2 weeks. Our only man had incidentally discovered metastatic liver lesions on ultrasound. Further imaging revealed prostatic mass and biopsy showed small cell neuroendocrine cancer. He presented with severe hypokalaemia. Random cortisol was 1065nmol/L (38.61mcg/dL) and ACTH was 188ng/L. He was commenced on medical treatment but declined rapidly and died.

All our patients had profound hypokalaemia and metastatic disease at presentation. Many patients do not exhibit classical cushingoid features as EAS tends to develop acutely and underlying malignancy drives weight loss. A high index of suspicion is required to make a diagnosis. EAS should be considered in patients with proximal myopathy, pigmentation, resistant or severe hypokalaemia or hypertension and known or suspected malignancy. Early and quick control of cortisol excess is essential to minimise cardiometabolic abnormalities, severe infections and thromboembolic complications. Prognosis depends upon age, frailty, comorbidity, nature of neoplasm and extent of hypercortisolaemia. Adrenolytics with or without bilateral adrenalectomy, reduction in tumour burden and management of complications are the mainstay of treatment.

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<![CDATA[SAT-144 Cooperative Mechanism of SREBP-Dependent Cholesterol Synthesis Pathway and P53 on Malignant Formation in Breast Cancer]]> https://www.researchpad.co/article/elastic_article_7149 p53 is mutated more than half of human cancers, and mutant p53, a gain of function, can actively have functional consequences with tumorigenesis. However, its action of molecular mechanisms, particularly in vivo conditions, has not been fully are clarified. Here, we generated KO and KI (R280K) breast cancer cell lines for p53 using CRISPR/Cas9 system, and then performed a three-dimensional culture model. We found that the introduction of mutant p53 was solely able to mediate the transformation to poor architectural structure. Interestingly, our findings in statin-effect along with cholesterol synthesis pathway, especially isoprenoid dependency, revealed that this pathway is necessary and sufficient for the regulation of malignant architecture in SREBP2-dependent manner with cooperatively being controlled by mutant p53 on 3D-cultured breast cancer. Furthermore, in accordance with the malignancy progresses, SREBP2 was accumulated in nuclear and nuclear membrane portion with enhancement in malignant formation. In addition, we found that mutant p53 interacts with SREBP2, and consistently mutant p53 was associated with DHCR7 promoter in parallel with binding pattern of SREBP2. Thus, our results provide the novel insight into the mutant p53, a gain of function, and its linkage to poor architectural structure in 3D-cultured breast cancer cells via SREBP2-dependent isoprenoids regulation as potential therapeutic targets.

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<![CDATA[SAT-119 Targeting Glutamate Metabolism and Signaling in ER+, Endocrine Therapy-Resistant Breast Cancer]]> https://www.researchpad.co/article/elastic_article_7107 Estrogen receptor-positive (ER+) breast cancer is the most commonly diagnosed form of this malignancy. Aromatase inhibitors and selective estrogen receptor modulators or degraders (SERMS, SERDs) can be highly effective in treating ER+ breast cancer, but de novo and acquired resistance to these interventions is a persistent clinical problem. Endocrine therapy resistant breast cancer cells rewire their metabolism to support cellular demands associated with rapid proliferation and/or increased invasion and metastasis. An important feature of this metabolic flexibility is conversion of glutamine to glutamate, an amino acid integral to protection of cells from oxidative stress. Consistent with this, we show multiple cellular models of ER+, endocrine resistant breast cancer cells markedly increase glutamate release and upregulate expression of essential glutamine/glutamate metabolic enzymes and transporters, including the glutamate/cystine antiporter xCT, glutamate dehydrogenase (GLUD1/2), and/or the glutamine importer SLC1A5. Riluzole (RIL) is FDA-approved for the treatment of amyotrophic lateral sclerosis (ALS), and has several proposed mechanisms of action, including suppression of glutamate release and increased glutamate uptake. We show ER+, endocrine responsive and resistant breast cancer cells are growth-inhibited by RIL. This is due to an increase in cell death, particularly in endocrine resistant breast cancer cells, and cell cycle arrest. Interestingly, histologic subtype confers a different cell cycle arrest profile, with invasive ductal cancer (IDC) models arresting in G1 but invasive lobular cancer (ILC) models arresting in G2/M. Isobologram analysis of RIL plus SERMs or SERDs shows additive-to-synergistic activity in a subset of ER+ cell line models, and preliminary studies show combination activity in patient-derived explants (PDEs). Mechanistically, we tested whether signaling through metabotropic glutamate receptors (mGluRs, GRMs) and/or cystine import contribute to RIL’s growth-inhibitory phenotype. Antagonists of mGluRs/GRMs don’t phenocopy the effects of RIL, suggesting extracellular glutamate signaling through these receptors is not a key mechanism. Rescue experiments with β-mercaptoethanol to promote cystine uptake through transporters other than xCT show partial reversal of RIL-mediated cell cycle arrest in some cells, suggesting xCT may contribute to RIL-induced growth inhibition. In summary, we show RIL may be a viable addition to endocrine therapy in ER+ breast cancer. Ongoing studies will test additional mechanism(s) by which RIL may attenuate the growth of ER+ breast cancer models in vitro, including inhibition of protein kinase C and casein kinase 1 delta. We are further testing RIL efficacy alone and in combination with a SERD in primary tumors and lung metastases in a ER+ patient-derived xenograft (PDX) model.

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<![CDATA[SAT-130 The Intermediate Prolactin Receptor Is a Breast Cancer Oncogene]]> https://www.researchpad.co/article/elastic_article_6830 Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed with canonical long mPRLr (mPRLrL). mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr isoform, the intermediate hPRLr (hPRLrI). As determined by tissue microarray (TMA), hPRLrI is expressed in >85% of breast cancer, with expression increasing as a function of both tumor grade and Ki67 status. To confirm the oncogenic potential of hPRLrI, isoform-specific hPRLrI knock-down (KD) was performed in breast cancer cell line MCF7. hPRLrI KD resulted in a significant decrease in proliferation, migration, and anchorage-independent growth. Given the homology between mPRLrT and hPRLrI, we hypothesized hPRLrI may similarly induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). hPRLrL/I co-expression in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL/I co-expression is necessary to induce transformation of normal mammary epithelia. To test our hypothesis in vivo, we established MCF10A xenografts using female NSG mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I cohort, significantly over that of expressing either isoform alone. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover is impaired in breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. Regarding differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the oncogenicity of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue.

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<![CDATA[SAT-118 Rapid Decompensation from Complications of Severe Hypercortisolism in an Unusual Presentation of an Ectopic ACTH-Secreting Neuroendocrine Tumor]]> https://www.researchpad.co/article/elastic_article_6750 Background: Ectopic Cushing’s syndrome from an ACTH-secreting neuroendocrine tumor (NET) is a rare condition whose onset and disease progression is often more aggressive than other forms of Cushing’s syndrome due to complications from severe hypercortisolism.

Clinical Case: A 75-year old woman presented with profound proximal muscle weakness, severe hypokalemia, Cushingoid features, and biopsy-proven Candida esophagitis. Initial testing was consistent with ACTH-dependent Cushing syndrome: elevated 24 hour urinary cortisol excretion (1,310.54 mcg/24h; n <50 mcg/24h), abnormal 1 mg dexamethasone suppression test (68.3 ug/dL), and elevated ACTH level (200 pg/mL; n: 7.2–63.3 pg/mL). MRI was negative for a pituitary lesion but abdominal CT revealed an 8.8 cm liver mass with biopsy consistent with a well-differentiated neuroendocrine tumor, WHO Grade 2. Subsequent 68Ga-DOTATATE-PET/CT noted DOTATATE uptake in the liver lesion, a 0.9 cm right pulmonary nodule, and the pancreatic tail without CT correlate. Initially, the patient was prescribed mifepristone and spironolactone for hypokalemia. Given her NET of unknown primary, metastatic disease, and immunocompromised state due to hypercortisolism, the patient was not a candidate for surgical resection of her NET but was instead referred for bilateral adrenalectomy. However, she rapidly decompensated from complications of her hypercortisolism prior to surgery. Her weakness progressed to immobility, and she developed acute psychosis manifested as agitation and mutism. The patient was immediately admitted to the hospital where she developed new-onset atrial flutter and myelosuppression requiring multiple transfusions. She underwent urgent bilateral adrenalectomy, but despite surgery, her post-operative course was complicated by hypoxemic respiratory failure and shock. The patient shortly thereafter expired from pulseless electrical activity arrest.

Conclusion: This atypical case of an ectopic ACTH-secreting NET highlights the life-threatening complications associated with severe hypercortisolism, including: opportunistic infection, severe metabolic abnormalities, psychosis, myopathy, and critical illness that can incite myelosuppression and unstable arrhythmias. These patients can quickly deteriorate and are at high risk for mortality. Early diagnosis and swift reversal of their hypercortisolism with bilateral adrenalectomy are oftentimes needed to prevent these potentially fatal complications.

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<![CDATA[SAT-137 The Effect of Hypertriglyceridemia on Triple Negative Breast Cancer Progression]]> https://www.researchpad.co/article/elastic_article_6709 Obesity is associated with increased cancer risk and cancer-associated mortality1,2. Hypertriglyceridemia (HTG), a component of the metabolic syndrome which frequently co-exists with obesity, has been associated with increased breast cancer risk and mortality in triple negative breast cancer (TNBC)3,4. To determine if HTG is causally related to enhanced TNBC progression in the absence of other obesity-associated characteristics, TNBC growth and metastasis in a mouse model of HTG was examined. Mice overexpressing human apolipoprotein C3 (AC3) were backcrossed onto FVB/N background and crossed with recombination-activating gene 1 (Rag1) knockout mice to generate immunodeficient HTG mice. AC3 mice relative to wild-type (WT) littermates showed a 20-fold higher circulating triglycerides (p < 0.0001) and elevated very low density lipoprotein (VLDL) cholesterol (p = 0.001). No differences in body weight, body composition, blood glucose or plasma insulin levels were observed between the two groups, allowing for investigation on the influence of HTG on TNBC without confounders such as hyperinsulinemia or hyperglycemia. AC3 mice orthotopically implanted with the mouse mammary tumor cell line, Mvt1, showed both increased tumor growth (AC3 vs WT: 1157.0 ± 84.2 vs 707.2 ± 58.6 mm3, p = 0.0009) and lung metastasis (AC3 vs WT: 57.3 ± 3.0 vs 32.9 ± 5.3 mm3, p = 0.001) relative to WT mice. Immunodeficient Rag1/AC3 mice likewise, showed increased tumor growth compared to WT controls when implanted with human TNBC MDA-MB-231 cells (AC3 vs WT: 363.2 ± 113.9 vs 92.95 ± 16.2 mm3, p = 0.038). To investigate how HTG affects tumor lipid metabolism, serum and tumors from both groups were analyzed by liquid chromatography/mass spectrometry. Total alkyl-acyl, di-acyl-phosphatidylcholines and sphingomyelin concentrations were higher in the serum of AC3 mice relative to WT. In contrast, no overall difference in tumor phospholipid or acylcarnitine content was noted between AC3 and WT mice, suggesting no difference in fatty acid oxidation in the setting of HTG. Mvt1 tumors from AC3 and WT mice were analyzed by RNA sequencing. Decreased expression of genes associated with cholesterol synthesis (Fdft1, Pvmk, Acss2) were found in tumors from AC3 mice. Tumors from AC3 mice also showed decreased protein expression of LDLR, which is associated with LDL cholesterol uptake. Overall, these findings suggest that HTG, independently of other obesity-associated characteristics such as hyperinsulinemia and hyperglycemia, leads to changes in intracellular lipid metabolism and promotes TNBC progression.

References: 1Chan, D. S. M. et al. Ann. Oncol. Off. J. Eur. Soc. Med. Oncol.25, 1901-1914 (2014). 2Pierobon, M. & Frankenfeld, C. L. Breast Cancer Res. Treat.137, 307-314 (2013). 3Lofterød, T. et al. BMC Cancer18, 654 (2018).4Goodwin, P. J. et al. Nutr. Cancer27, 284-292 (1997).

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<![CDATA[SAT-131 Asymptomatic Severe Hypoglycemia with Lactic Acidosis in a Case of Non-Hodgkin’s Lymphoma - an Unusual Phenomenon of Hyper-Warburgism]]> https://www.researchpad.co/article/elastic_article_6559 INTRODUCTION

Hypoglycemia in malignancy is well known with several etiologies; impaired liver function, insulin receptor autoantibodies, production of insulin-like substance by malignant cells or large tumor burden. Another possible mechanism is the Warburg effect where metabolism shifts towards glycolytic pathways over oxidative phosphorylation even under aerobic conditions leading to excess lactate production. This leads to glucose consumption due to shunting of glucose away from normal cells to cancer cells. Very few cases of lactic acidosis and severe hypoglycemia in Non-Hodgkin’s Lymphoma have been described in the literature. We report such an unusual case here.

CASE DISCUSSION

A 52-year-old, African-American male was admitted for severe malnutrition with 80 lb. weight loss, severe hypoglycemia and progressively increasing right chest wall mass. On arrival, he had no classic symptoms of hypoglycemia. Physical exam revealed persistent tachycardia, white patches on oral mucosa and posterior tongue, and an indurated mass on right chest wall with extensive swelling of right upper limb. Lab work was significant for blood glucose (BG) of 41 mg/dL (60–100), lactate 16 mmol/L (0.5 to 2), anion gap 26 mEq/L (3–10), albumin 2.3 g/dL (3.4 to 5.4) and normal renal and liver function tests. Fingerstick sugar readings were persistently in the 20s with no response to multiple boluses of dextrose and glucagon. He was started on dextrose 5% (D5) drip and intravenous solumedrol. Solumedrol was weaned off and D5 titrated down to investigate causes of hypoglycemia. BG dropped to 39 and corresponding labs showed insulin level of < 2 mcUnit/mL (2–20), C-peptide of 0.2 ng/mL (0.8–6.0) and ketone level of 0.2 mmol/L (<0.4). IGF-1 and IGF-2 were both low at 26 ng/mL (61–200) and 113 ng/mL (333–967) respectively. A CT torso with contrast showed bilateral pleural effusions, moderate pericardial effusions and a large ill-defined heterogeneous mass along anterior chest wall. He underwent ultrasound guided biopsy of the chest wall mass and diagnosed with diffuse large B-cell lymphoma. He also tested positive for HIV/AIDS and Hepatitis C. PET scan showed diffuse FDG (fluorodeoxyglucose) uptake consistent with advanced disease. He was started on chemotherapy and lactate and BG normalized soon after 1st cycle.

CONCLUSION

In our case; suppressed insulin, low C-peptide and IGF-2 levels indicate non-insulin mediated hypoglycemia due to rapid glucose utilization by cancer cells. Severe hypoglycemia with lack of neuroglycopenic symptoms suggests use of lactate (rather than glucose) as an alternative metabolic fuel for brain, thus preserving its function. Our patient presented with an exaggerated Warburg effect (hyper-Warburgism) as evident by extreme glucose consumption, severe lactic acidosis and large tumor burden on FDG/PET. Chemotherapy must be instituted timely to correct these abnormalities.

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<![CDATA[SAT-145 Cholesterol Uptake as a Critical Vulnerability in Triple Negative Breast Cancer]]> https://www.researchpad.co/article/elastic_article_6415 Triple Negative Breast Cancer (TNBC) is an aggressive subtype of cancer with poor prognosis due to high metastatic potential and lack of targeted therapies. Normal epithelial cells express the microRNA-200c (miR-200c), a potent suppressor of epithelial-to-mesenchymal transition (EMT). However, miR-200c is silenced or lost in TNBC, allowing a de-differentiated, non-epithelial phenotype and aberrant expression of genes conferring invasive and chemoresistant characteristics. Recent literature has demonstrated that EMT promotes altered tumor cell metabolism, creating novel vulnerabilities that can be exploited therapeutically. In addition to driving global metabolic changes, miR-200c-induced reversal of EMT alters key cholesterol metabolism genes that support the uptake of dietary cholesterol from the bloodstream. Intracellular cholesterol homeostasis is critical for cell survival and is carefully regulated, but how these homeostatic mechanisms adapt during tumor progression is poorly understood. Based on preliminary data, I hypothesize that TNBCs depend on exogenous cholesterol uptake and availability to maintain cell viability and an invasive phenotype. This work aims to identify novel cholesterol-related targets in breast cancer and delineate mechanisms regulating cholesterol homeostasis in normal and cancer physiology.Restoration of miR-200c in TNBC leads to alteration of the cholesterol uptake components low- and very-low-density-lipoprotein receptors LDLR and VLDLR, through direct and indirect mechanisms previously unexplored in cancer. miR-200c further inhibits Niemann-Pick Type C (NPC1), a lysosomal protein necessary for utilization of exogenous cholesterol. Interestingly, expression of NPC1 in TNBC correlates with a unique inability of cells to proliferate in the absence of exogenous LDL supply, suggesting defects in de novo cholesterol biosynthesis. Further, NPC1 inhibition leads to cell death in TNBC but not more epithelial-like breast cancers. Whether this cell death is due to disruption in critical cholesterol supply or due to defective lysosome dysfunction is currently being investigated. Overall, this work suggests a role of NPC1 in cancer cell metastasis that has not been previously explored, and identifies cholesterol uptake as a targetable dependency in TNBC.

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<![CDATA[SAT-126 Breast Adipose Tissue Extracellular Vesicles from Obese Women Increase Breast Cancer Aggressiveness - a Novel Mechanism for the Obesity-Breast Cancer Link]]> https://www.researchpad.co/article/elastic_article_6367 Background and Objectives: Breast cancer is among the most common cancer in women with 2.1 million new cases detected each year. Numerous studies have demonstrated a connection between body mass index (BMI) and cancer incidence, with obesity (BMI ≥ 30) being responsible for the development of at least 13 types of cancer, and 15% to 20% of total cancer-related mortality. The effects of extracellular vesicles (EVs) derived from the obese adipose tissue microenvironment on breast cancer have not yet been clearly elucidated.

Methods: EVs were obtained from media conditioned with human breast adipose tissue from reduction mammoplasty (n=31). Women were healthy at the time of surgery and had no history of breast cancer. Patient samples were stratified based on their body mass index (BMI), with a BMI < 25 considered healthy and a BMI ≥ 25 considered overweight/obese. Breast adipose tissue-derived EVs (AT-EVs) were characterized (Quantitative Mass Spectrometry) and used to treat human breast cancer cell lines, including the ER+ MCF7 and triple negative breast cancer (TNBC) MDA-MB-231. Effects on cell proliferation and migration in vitro, and on tumor growth in a mouse xenograft model, were examined after long-term education with EVs. RNA sequencing was performed to investigate potential reprogramming induced by AT-EVs.

Results: We found a positive correlation between protein amount per AT-EV and BMI. Quantitative proteomics of AT-EVs revealed 46 proteins that were significantly higher and 54 proteins that were significantly lower in specimens from women with a BMI ≥ 25 compared to women with a BMI < 25. AT-EVs from patients with a BMI ≥ 25 induced proliferation of MCF7 cells compared to AT-EVs from patients with a BMI < 25. Obese EVs induced a more aggressive phenotype in MDA-MB-231 cells, increasing their invasiveness in vitro. Obese EVs also increased the growth of MCF7 and MDA-MB-231 cells in vivo. Ingenuity pathway analysis of RNA-Seq data identified significant differences in mTOR signaling and canonical pathways associated with altered mitochondrial function.

Conclusion: Our studies identify a novel mechanism to explain the obesity-breast cancer link in older women. Namely, that in obesity, the breast microenvironment produces EVs capable of reprogramming breast cancer cells to grow faster and be more aggressive. Identifying which cargo in breast AT-EV mediates these effects may provide new targets for intervention.

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<![CDATA[SAT-115 A Rare Case of Metastatic Insulinoma in a Patient with Huntington’s Disease]]> https://www.researchpad.co/article/elastic_article_6313 A 49-year-old female with past medical history of Huntington’s disease presents to the ED after being found unresponsive at her assisted-living facility. She was noted to be hypoglycemic with a glucose of 21 and responded to dextrose. For the past several months, patient had symptoms of dizziness, tremors. She has no history of diabetes, and only home medication is Zyprexa. Patient required D10 drip to maintain glucose levels. Given patient’s psychiatric history, there was concern for surreptitious use of diabetic medications. Labwork showed an insulin level of 163 and an elevated C-peptide of 7.0 consistent with endogenous insulin secretion. Patient underwent a 72-hour fasting protocol which revealed a glucose of 23, C-peptide of 4.1 and insulin of 101, undetectable beta-hydroxybutyrate levels and pro-insulin level of 782 which is consistent with a diagnosis of insulinoma. She was started on diazoxide, which diminishes insulin secretion, to treat the hypoglycemia. Patient underwent a pancreas CT protocol which showed multiple hypervascular hepatic masses up to 9 cm and one 4 cm mass in pancreatic tail concerning for metastasis. Pathology revealed metastatic well-differentiated neuroendocrine tumor grade 3. She was not a candidate for surgical resection of tumors and she was started on Octreotide 200 mcg TID. Patient underwent two Y90 radioembolization treatments, 2 months apart with improvement of hypoglycemia and discontinuation of diazoxide. Patient remains on Octreotide LAR injection every 4 weeks. Insulinoma, although rare, occurs in 1-4% of the population and is the most common type of functional neuroendocrine tumor of the pancreas. It secretes insulin which results in hyperinsulinemic hypoglycemia. Diagnosis involves persistent hypoglycemia with glucose <50 mg/dL, neuroglycopenic symptoms and prompt relief after glucose administration. Gold standard diagnosis is a 72-hour fast and measurement of plasma insulin, C-peptide and pro-insulin, which detects 99% of insulinomas. After lab confirmation, imaging is necessary with either CT, MRI or EUS. Surgical resection is curative in most patients. Other treatment modalities include injection of octreotide, EUS-aided alcohol ablation, radio-frequency ablation or embolization of insulinoma, as well as targeted therapy with everolimus or sunitinib. Malignant insulinomas are extremely rare and often invade into surrounding soft tissue or have lymph node or liver metastasis. They are usually unresectable and require targeted therapy. We present the case of a 49 YO patient with Huntington’s disease who presented with severe persistent hypoglycemia secondary to metastatic insulinoma. Diagnosis of insulinoma is often delayed or missed as symptoms may be attributed to psychiatric, cardiac or neurological disorders or medication misuse. Clinicians should be aware of insulinoma as a cause of life-threatening hypoglycemia.

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<![CDATA[SAT-133 Breast Tumor Kinase (Brk/PTK6) Mediates Triple Negative Breast Cancer Cell Migration and Taxol Resistance via SH2 Domain-Dependent Activation of RhoA and AhR]]> https://www.researchpad.co/article/elastic_article_6285 Triple negative breast cancer (TNBC) patients have higher recurrence rates and a worse prognosis relative to patients diagnosed with other breast cancer subtypes. Protein tyrosine kinase 6 (PTK6; also called Brk), a soluble tyrosine kinase, is overexpressed in 86% of breast cancer patients, however its precise function in the context of TNBC is poorly defined. PTK6 expression is elevated in TNBC models in response to both cellular and endocrine stress, coordinated transcriptionally by the Hypoxia-Inducible Factors (HIFs) and glucocorticoid receptor (GR). We showed previously that PTK6 expression, but not its intrinsic kinase activity, is required for breast cancer cell motility. To further delineate the mechanisms of PTK6 signaling, we created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domains. MDA-MB-231 cells expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were less responsive to growth factor-stimulated cell motility relative to wild type or kinase dead (KM) controls. To identify signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants, we used a reverse phase protein array (RPPA), which revealed that the SH2 domain of PTK6 mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Moreover, in TNBC cells, including a taxane-refractory TNBC model, addition of AhR or Rho inhibitors to paclitaxel (Taxol) enhanced cytotoxicity. Together, these studies reveal that the SH2-domain of PTK6 is an effector of advanced cancer phenotypes in GR+ TNBC cells and identify RhoA and AhR as novel therapeutic targets in PTK6+ tumors.

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<![CDATA[SAT-138 Neutrophil Elastase Promotes Proliferative Signals in Prostate Cells Through EGFR and DDR1]]> https://www.researchpad.co/article/elastic_article_6167 Studies examining many different cancers have demonstrated that inflammation plays a critical role in tumor progression, in part through the release of proteases from stromal cells that function to either remodel the tumor microenvironment or to directly stimulate cancer cells to grow. One specific protease, neutrophil elastase (NE), has been shown to be a critical regulator of cancer growth in several mouse models. Accordingly, our laboratory demonstrated that NE, most likely from granulocytic myeloid-derived suppressor cells, potentially promotes prostate cancer progression in several different in-vivo and in-vitro models. To date, however, little is known regarding the mechanisms utilized by NE to promote tumor growth. It has been suggested that NE might cleave epidermal growth factor (EGF) or transforming growth factor-α from the cell surface to induce activation of EGFR/ERK signal transduction in an autocrine fashion. Alternatively, NE has been shown to enter into early endosomes to degrade insulin receptor substrate-I, ultimately resulting in phosphoinositol 3-kinase hyperactivity and subsequent tumor cell proliferation. Here we demonstrate that NE triggered proliferative signals in six prostate cell lines representing the spectrum of prostate cell differentiation, including normal prostatic epithelium, benign prostatic hypertrophy, and metastatic prostate cancer. Focusing on ERK signaling, we found that the stimulatory effect of NE on ERK phosphorylation was dose dependent and was abrogated by small interfering RNA induced EGFR knockdown, as well as by pretreatment of cells with irreversible EGFR inhibitor AG1478. Unlike EGF, however, NE-initiated EGFR phosphorylation was minimal. Thus, while EGFR appears to be critical for NE-induced ERK activation, perhaps it is not extensively activated directly by NE. Notably, discoidin domain receptor-1 (DDR1) was strongly expressed in normal prostate epithelium cells, but gradually decreased and had little expression in benign and metastatic prostate cancer cells sequentially. Nevertheless, similar to EGFR knockdown, silencing of DDR1 in all cell types inhibited NE mediated pERK upregulation, suggesting that DDR1 may also be important for NE-induced action. Together, our data suggest that NE, in concert with low level signals from the EGFR and DDR1, play an important role in promoting prostate cell proliferation both in normal and cancerous prostate epithelial cells.

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<![CDATA[SAT-153 PTHrP Mediated Hypercalcemia]]> https://www.researchpad.co/article/elastic_article_6157 PTHrp mediated hypercalcemia

Introduction:

PTHrp is a normal gene product, which helps tooth eruption and mammary gland development. However, PTHrp production in an adult is mostly related to solid tumor malignancy. The homology of PTHrp to PTH from the 1st to 13th amino acids decided the similar mechanism of acting at PTH-1 receptor on increasing bone resorption and calcium reabsorption at the distal renal tubule. Lung cancer is known to secrete PTHrp and PTHrp secreting hepatic cellular carcinoma (HCC) is exceedingly rare. It’s only been documented in case reports. Two cases of PTHrp secreting atypical HCC are included in this case report.

Case presentation:

67 year old with history of alcohol abuse with 17.8 x 8.8 cm mass in the left hepatic lobe presented with acute encephalopathy and calcium of 14.4 mg/dl. Patient was treated with zolendronic acid 4mg, IV fluid and calcitonin. Patient has a PTH level of 7 pg/ml and PTHrp level of 335 pg/ml. Bone turnover marker Beta crosslaps of 1595 pg/ml. 25-hydroxy vitD of 25 ng/ml and 1,25 dihydroxy vitD of 42 pg/ml. Liver biopsy demonstrated moderately differentiated hepatocellular carcinoma. The undifferentiated portion was positive with CD56, CAM 5.2 suggestive of neuroendocrine differentiation and epithelial lineage. Patient had negative metastatic bone scan and no pathologic fractures. Second case is a 63 year old male with NASH cirrhosis was admitted for hypercalcemia of 13.2 mg/dl. Patient was found to have metastatic atypical hepatocellular carcinoma and L4 vertebral lesion. PTHrp was 30 pg/ml and PTH 14 pg/ml. Bone turnover marker Beta crosslaps of 405 pg/ml. 25-hydroxy vitD of 48 ng/ml and 1,25 dihydroxy vitD of 9 pg/ml. Patient expired prior to biopsy.

Discussion:

4 cases of similar HCC were found in the literature. The treatment approach was resection of tumor, chemo or ablation. Hypercalcemia is controlled with reduction of the tumor burden and rarely by zolendronic acid alone. Both of presented cases were too advanced for surgery or chemo treatment. When tumor was not amendable for treatment, the hypercalcemia was not controllable long term. A small size study in japan administering 10mg alendronate via hepatic artery rather than IV route for patients with HCC showed enhancement of the apoptosis of HCC in addition to controlling hypercalcemia. An animal study on anti-PTHrp monoclonal murine antibody showed improved mortality of PTHrp producing pancreatic carcinoma (FA-6) in transplanted nude mice.

Conclusion:

Most of the patients with PTHrp mediated hypercalcemia have advance cancer that is not amendable for surgery or chemotherapy. A non-invasive treatment approach other than alendronate should be investigated to control PTHrp mediated hypercalcemia.

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<![CDATA[SAT-132 The Secretory Vesicle Membrane Protein, CYB561, Promotes the Growth and Metastatic Potential of Castration-Resistant Neuroendocrine Prostate Cancer]]> https://www.researchpad.co/article/elastic_article_6147 An increase in the population of neuroendocrine (NE) differentiated (NED) cells and their secretory products are closely correlated with prostate cancer (PCa) resistance to existing therapies and eventual progression to castration-resistant PCa (CRPC). It is hypothesized that NED cells secrete neuropeptides that support tumor growth and induce aggressiveness of adjacent proliferating tumor cells through a paracrine mechanism. A gene that is constitutively expressed in secretory vesicles of NE cells, and has been previously found to be highly expressed in CRPC and cancer of several tissues is Cytochrome b561 (CYB561). The CYB561 gene encodes a secretory vesicle transmembrane protein that primarily functions in the regeneration of ascorbic acid, a necessary step in the α-amidation activation process in the biosynthesis of most neuropeptides. The CYB561 protein also exhibits ferrireductase activity and may contribute in regulating iron transport and metabolism, which are two other pathways often dysregulated in cancer. These findings led us to hypothesize that CYB561 may be a key player in the NE differentiation process that drives the progression of prostate cancers into the more aggressive NE subtype. In our study, we found that CYB561 expression is higher in metastatic and NE PCa (NEPC) models compared to normal prostate epithelia, and that its expression is not affected by androgen treatment or steroid deprivation. Lentiviral-mediated knockdown of CYB561 in the NEPC cell line, PC-3, decreased the expression of genes involved in NE differentiation and labile iron pool storage, decreased cell proliferation, reduced cell survival in a colony formation assay, and slowed down cell migration in a wound-healing assay. Treatment of normal prostate epithelial cells, PNT1A, with conditioned media from CYB561 knockdown PC-3 cells led to a decrease in proliferation rate when compared to treatment of PNT1A cells with media from CYB561 expressing (control) PC-3 cells. Taken together, our findings demonstrate the role of CYB561 in supporting the growth and metastatic potential of NEPC cells, and highlights the potential use of CYB561 as a therapeutic target and biomarker that can be used to identify more aggressive disease.

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<![CDATA[SAT-124 Optimization of Experimental Conditions for Mimicking Hypoxia in Cultured Breast Cancer Cells by Using Cobalt(II) Chloride (CoCl<sub>2</sub>)]]> https://www.researchpad.co/article/elastic_article_6010 Hypoxia is a common phenomenon in solid tumor development caused by a decrease in either oxygen concentration or oxygen pressure as a result of rapid tumor cell growth. Hypoxia is characterized by stabilization of the alpha subunit of the hypoxia-inducible factor (HIF-1α) and its nuclear translocation and heterodimerization with HIF-1β. Activation of this signaling pathway involves multiple downstream effectors including carbonic anhydrase 9 (CA9, s. CAIX). A reliable method to mimic hypoxia utilizes cobalt(II) chloride (CoCl2), which directly induces the expression of HIF-1α. The aim of this study was to optimize the experimental conditions for CoCl2 treatment of breast cancer cells in vitro using three human breast cancer cell lines (MDA-MB-231, T-47D, and MCF-7 cells). We performed time- and concentration-response experiments, using various concentrations of CoCl2 (50, 100, 200, and 300 μM) for 24 and 48 hours, and measured the expression of HIF-1α and CA9 by qRT-PCR and Western blot analyses. Results demonstrated that CoCl2 downregulated HIF-1α mRNA levels but upregulated CA9 mRNA levels in a concentration- and time-dependent manner. Concomitantly, CoCl2 treatment resulted in a significant induction of HIF-1α protein levels. We further investigated the effect of the CoCl2 concentrations listed above on cell apoptosis using an in situ apoptosis detection kit. The results demonstrated that concentrations of CoCl2 up to 100 μM had no significant effect on cell apoptosis.

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<![CDATA[SAT-116 An Affair of the Heart - First Case Report of Immune Checkpoint Inhibitor Associated Cardiac Sarcoidosis Presenting with Non-PTH Medicated Hypercalcemia]]> https://www.researchpad.co/article/elastic_article_5951 Background:

Immune checkpoint inhibitors (ICI) are an effective new tool in the treatment of malignancy by rescuing exhausted T-cells and enhancing anti-tumor immunity. The offset of immune self-tolerance can result in autoimmune adverse effects involving gastrointestinal, pulmonary and endocrine systems. Lung and skin sarcoidosis have been described in association with ICI use. We present the first case of non-PTH medicated hypercalcemia due to cardiac sarcoidosis in the setting of immunotherapy.

Case Presentation:

A 71-year-old man was referred to endocrinology for hypercalcemia. He had a fourteen-year history of scalp melanoma in remission until February 2019, when routine surveillance scans suggested metastatic disease. Computer tomography of the chest showed mediastinal and hilar lymphadenopathy (largest node 5.2 cm) and numerous pulmonary nodules (largest 1.7 cm). Biopsy of the largest pulmonary nodule and mediastinal lymph node (LN) confirmed BRAF wild-type metastatic melanoma. Ipilimumab/nivolumab (antiCTLA4/antiPD-1) combination therapy was started. After two cycles, hypercalcemia was noted on routine laboratory surveillance. He was asymptomatic and physical exam was unremarkable. Initial workup revealed: calcium 10.6 mg/dL (8.6-10.2), albumin 4 g/dL (3.5 - 5.2), phosphorus 3.8 mg/dL (2.7 - 4.5), PTH <0.6 pg/mL (15.0 - 65.0), PTHrP <2.0 pmol/L (0.0 - 2.3), 25 hydroxyvitamin D 22. 9 ng/mL (30 - 60), vitamin A 0.59 mg/L (0.30 - 1.20). He denied taking calcium-containing supplements. He was treated with hydration and immunotherapy was continued for two cycles, followed by single agent nivolumab. After three months on ICI, the metastatic lesions were reduced in size by 30%. His calcium peaked at 12.5 mg/dL and was treated with 4mg of intravenous Zoledronic acid without resolution. He developed worsening functional status, symptomatic hypotension, and elevated troponins. Cardiac MRI demonstrated myocarditis and nivolumab-induced myocarditis was suspected. Surprisingly, endomyocardial biopsy revealed multiple granulomas suggestive of sarcoidosis. AFB, PAS and Congo red stains were negative. He was treated with supportive care and glucocorticoids with resolution of hypercalcemia and improved cardiac function. Unfortunately, serum 1,25 dihydroxy vitamin D was not successfully measured until after the first dose of prednisone and was found at the upper limit of our reference range 62.0 pg/mL (19.9-79.3).

Conclusion:

Immune checkpoint inhibitors are effective agents in treating various cancers. Adverse effects due to autoimmunity are common and early recognition of life-threatening complications is critical. Although cutaneous and pulmonary sarcoidosis have been described with ICI, to our knowledge, this is the first case report of ICI-related cardiac sarcoidosis presenting with PTH-independent hypercalcemia.

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<![CDATA[SAT-142 Periodic Cushing Syndrome in a Patient with an Intestinal Neuroendocrine Tumor (NET). A Novel Case Report]]> https://www.researchpad.co/article/elastic_article_5945 Introduction A major diagnostic enigma regarding ACTH-dependent Cushing syndrome is the distinction between the source of ACTH, which may have either a pituitary or ectopic origin. We present the first described patient with cyclic Cushing syndrome caused by an intestinal neuroendocrine tumour (NET) detected by 68GA-DOTATATE PET-CT, despite functional tests that were indicative of pituitary Cushing disease. Clinical Case A 53-year old man was admitted to outpatient clinic because of muscle weakness. His phenotype and clinical findings (progressively worsening upper and lower limb weakness, emotional disturbances, easy bruising, a Buffalo hump, prediabetes and leucocytosis) led to the diagnosis of Cushing syndrome. Initial laboratory tests established the diagnosis with an abnormal diurnal cortisol and ACTH secretion (night cortisol F: 22.1μg / dl), absence of suppression with dexamethasone 1mg and increased free urinary free cortisol 24h (243.5μg /24h). Abdominal CT scanning revealed a left-sided adrenal adrenocortical adenoma 1.5 mm in max diameter. Pituitary MRI and somatostatin scintigraphy were normal. Low dexamethasone suppression test was indicative of Cushing (F: 14μg / dl) followed by a combined CRH stimulation test during bilateral inferior petrosal sinus sampling. Pituitary / peripheral ACTH ratio pre-infusion of CRH and 3 min after CRH infusion was compatible with right-sided pituitary origin of ACTH hypersecretion. Pending the results of the laboratory, the patient showed a remission of his symptoms along with a laboratory-confirmed recession of active hypercortisolaemia (LDDST test), and this led to the suspicion of periodic Cushing syndrome. The patient was followed with clinical and laboratory examinations weekly, with recurrence of symptoms 2 months later followed by a new remission 3 months later. A PETGA CT SCAN with 68GA-HA-DOTATATE was performed, which showed an increased uptake of the radioisotope in the small intestine. A surgical excision of the affected small bowel region was performed according to the guidelines for intestinal NETs. Histology confirmed the existence of a well-differentiated neuroendocrine neoplasm of the small intestine of 1.1 cm diameter, grade 1 (WHO 2010). Immunophenotype was positive for serotonin and ACTH. Postoperatively, the patient showed a complete remission of symptomatology and regression of hypercortisolaemia over a 18-month period. Follow-up abdominal MRI and 68GA-HA-DOTATATE revealed no pathological findings. Conclusion: Our patient is the first case of ectopic Cushing disease caused by intestinal NET. The differential diagnosis between pituitary and ectopic Cushing syndrome due to ACTH or CRH hypersecretion is not easy and frequently complicated by the periodicity of the disease. In patients with no visible pituitary lesions on MRI we suggest further investigation for ectopic ACTH- driven Cushing syndrome.

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