Sunitinib treatment had clinical benefits in both carriers of *SDHB* mutations and patients with apparently sporadic tumors (62). The progression-free survival (PFS) (the length of response

Figure 1 | The 10 hallmarks of cancer and metastatic pheochromocytomas and paragangliomas (MPPGs). This figure describes the 10 hallmarks of cancer and several mechanisms identified to date in patients with MPPG that contribute to their tumor development.

duration) was, however, not very impressive (4.1 months). The study was in fact an intention-to-treat analysis that included the three nonevaluable patients in the final evaluation of PFS. Some patients, nonetheless, exhibited a durable response to sunitinib; the longest response lasted 3 years. The study concluded that some patients with MPPG may benefit from antiangiogenic therapies such as sunitinib (62). However, the dose of sunitinib must be carefully chosen, and adverse events should be prevented or treated aggressively with dose adjustment and/or interventions to prevent exacerbation of hypertension or symptoms such as pain. The FIRSTMAPPP trial (NCT01371201) is a multinational phase 2 study evaluating sunitinib in patients with MPPG. The intervention group receives 37.5 mg sunitinib daily. This dose of sunitinib is lower than the dose currently approved for the treatment of patients with kidney cancer (50 mg daily, 4 weeks on, 2 weeks off). The lower dose may be associated with a better safety profile.

Pazopanib was tested in a phase 2 clinical trial involving patients with MPPG (63). The intervention group received 400 mg pazopanib daily for 2 weeks of the first cycle, then 800 mg for 2 weeks of the second cycle, followed by 800 mg daily for the whole of the subsequent cycles. Patients needed to have measurable disease, as the primary endpoint was ORR. Of the seven recruited patients, only one patient exhibited a confirmed partial response of −57%. This patient's response to pazopanib lasted for approximately 2 years. Four patients had disease progression. Almost every patient exhibited hypertension; severe hypertension was noted in 50% of patients, including one patient who developed Takotsubo cardiomyopathy. The serious cardiovascular adverse events happened once the dose of pazopanib was titrated to 800 mg daily. The trial was terminated because of poor accrual (63).

This trial aimed to evaluate pazopanib as a potential therapeutic option for MPPG patients because previous comparative studies in patients with kidney cancer suggested that pazopanib was better tolerated than sunitinib (64, 65). Later, however, it was recognized that these studies had several pitfalls related to patient selection and quality-of-life assessment (66); more importantly, clinical practice indicated that patients treated with sunitinib and pazopanib had, in reality, similar compliance patterns. Thus, clinical considerations, including the physician's experience, should determine treatment choices. Pazopanib could prove to be an effective medication to treat MPPG. Nevertheless, it is important to remember that MPPGs are very challenging tumors; in addition to a large tumor burden, MPPGs frequently secrete excessive amounts of catecholamines that predispose patients to cardiovascular disease. A pazopanib dose of 800 mg daily was likely too high to tolerate.

## Inhibition of Angiogenesis: Axitinib

Pseudohypoxia activates angiogenesis through stimulating the synthesis and secretion of VEGFs by MPPG cells. Axitinib is a potent antiangiogenic medication that has been approved by regulatory agencies for the treatment of patients with kidney cancer. Axitinib inhibits VEGFR-2 but does not inhibit other receptors involved in angiogenesis such as PDGFR-β. For this reason, axitinib is expected to cause fewer adverse events than multi-tyrosine kinase inhibitors that also target angiogenesis (67).

A phase 2 clinical trial of axitinib (NCT01967576) enrolled 11 patients (68). The primary endpoint was PFS, and secondary endpoints included ORR and safety. The intervention was 5 mg axitinib given twice daily. The dose of axitinib was increased to 7–10 mg twice daily in patients who did not experience side effects more severe than grade 2 hypertension. Approximately 36% of patients achieved a partial response, and 54% had stable disease. Of those with stable disease, half had some degree of regression. Only one patient exhibited disease progression. The ORR was 36% (68). No patients tolerated the starting dose of 5 mg twice daily for a long period of time because they developed hypertension. Hypertension was common and frequently serious. About 82% of patients had grade 3–4 hypertension and required dose reduction or discontinuation of therapy (68). The trial is currently closed for recruitment.

## Inhibition of Angiogenesis, Proliferative Signaling, and Invasion and Metastasis: Cabozantinib

Cabozantinib is a multi-tyrosine kinase inhibitor approved for the treatment of patients with medullary thyroid and clear cell renal cell carcinomas. Cabozantinib is perhaps, the most potent antiangiogenic medication available in clinical practice. Cabozantinib inhibits VEGFR-2 as well as the RET and c-Met receptor pathways (69, 70). Although the inhibition of the RET receptor pathway may not be of interest for the treatment of the great majority of patients with MPPG (malignant pheochromocytomas are an exceptional phenotype of patients with multiple endocrine neoplasia type 2, which is associated with RET mutations) (71), the inhibition of the c-Met pathway may indeed be of interest. MET activation is a universal mechanism that drives cell survival, invasion, and metastasis in many cancers and cooperates with the VEGFR pathways to promote tumor angiogenesis (72). Upregulation of the c-MET pathway occurs as a consequence of the VEGFR inhibition, leading to tumor resistance to antiangiogenic medications and escape from VEGFR inhibition (72). Inhibition of the c-Met pathway may delay the development of tumor resistance and improve clinical outcomes (73). Patients with kidney cancer treated with cabozantinib exhibit significantly longer PFS than do patients treated with sunitinib (74). Cabozantinib is also an interesting medication to study in MPPG patients because of its potential impact on the bone microenvironment. Cabozantinib has been associated with palliation of bone pain, improvement of anemia, modulation of bone turnover, and bone scan resolution in patients with malignancies frequently associated with bone metastases (75). MPPG frequently spreads to the bones, predisposing patients to overwhelming skeletal-related events (58, 76).

A phase 2 clinical trial of cabozantinib is currently ongoing (NCT02302833). The primary endpoint of this study is ORR. The trial includes an exploratory branch of patients with MPPG with predominant bone metastases. The intervention is 60 mg cabozantinib daily with dose titration to 40 or 20 mg depending on patients' toleration of adverse events. Patients require objective evidence of disease progression to be included in the trial. Preliminary results in 10 patients with measurable disease showed an ORR of 40% (77). Half of patients had stable disease, and only one patient did not have a response to therapy. All patients with stable disease had tumor regression; the clinical benefit rate was 90% (77). In addition, all patients with bone metastases exhibited a reduction of glucose uptake as demonstrated by FDG-PET. Patients with hormonally active tumors associated with partial responses or stable disease exhibited improvement of symptoms of catecholamine excess, including diabetes mellitus and hypertension (77). No patients experienced severe hypertension. However, most patients required dose reduction because of grade 2 fatigue or hand-foot syndrome. Two patients required dose reduction because of asymptomatic grade 3 elevation of pancreatic enzymes and formation of a rectal fistula, respectively (77). This clinical trial is actively recruiting participants.

## Induction of Cell Death and Prevention of Replicative Immortality: Iobenguane 131I and 177Lu-DOTATATE

The radiopharmaceutical metaiodobenzylguanidine (MIBG) was created in 1979 (78). MIBG is labeled with 131I at the metaposition and is taken up by the noradrenaline transporter. Once inside the tumor cell, MIBG releases lethal radiation that causes severe DNA damage, inhibiting cell proliferation and causing cell death. Up to 80% of MPPG patients have tumors that express the noradrenaline transporter in the cell membrane (79). Responses to MIBG are, however, limited, with only 30% of MPPG patients seeing a clinical benefit (80). The limited benefits associated with MIBG are likely attributable in part to its manufacturing process (81). MIBG is produced by a simple isotope exchange that leaves a large amount of unlabeled MIBG, called cold MIBG, in each dose. Cold MIBG may compete with labeled MIBG for the noradrenaline transporter, preventing the uptake of labeled MIBG and decreasing the tumor's exposure to radiation. MIBG delivers low levels of radioactivity per dose (~1.59 MBq/μg) (82). In addition, cold MIBG may compete with noradrenaline for the noradrenaline transporter, increasing the concentrations of circulating noradrenaline and predisposing to cardiovascular events during or shortly after the drug's administration (83). Iobenguane 131I is also MIBG labeled with 131I at the meta-position. Unlike conventional MIBG, iobenguane 131I is produced from a solidphase ultratrace precursor that eliminates the presence of cold MIBG. Iobenguane 131I is, then, a highly specific radiopharmaceutical agent that delivers very high levels of radioactivity per dose (~92.5 MBq/μg). Furthermore, iobenguane 131I may be associated with a lower rate of cardiovascular events than conventional MIBG (84).

A phase 1 dose-escalation study of iobenguane 131I in patients with MPPG determined the maximum tolerated dose to be 296 MBq/kg (8 mCi/kg) (85). A pivotal phase 2b clinical trial of iobenguane 131I was then developed, with an intervention of 2–500 mCi doses of iobenguane 131I separated by a period of at least 3 months depending on bone marrow toxicity. This trial recruited 81 patients, 68 of whom had MIBG-avid tumors and received at least one therapeutic dose of iobenguane 131I. Fifty patients received two doses of iobenguane 131I (86). The trial's primary endpoint was clinical: the number of patients who had at least a 50% reduction in the dose and number of antihypertensives for at least 6 months. Secondary endpoints included ORR, OS, and safety (86). One-quarter of patients achieved the primary endpoint, and many of the patients who did not achieve the primary endpoint nonetheless had improvement of hypertension with a reduction of less than 50% in the dose and number of antihypertensives (86). Almost all patients had a tumor response. Partial responses and stable disease were noted in 30 and 68%, respectively, of patients treated with two doses (86). The proportion of patients who experienced a partial response increased over time, suggesting that iobenguane 131I has persistent antitumor effects (86). Overall, 90% of patients treated with two doses continued to have a partial response or stable disease 12 months after receiving the initial dose (86). The most common treatment-emergent adverse events were consistent with expected radiation-related risks: bone marrow suppression, nausea and vomiting, fatigue, and dizziness. Hematological toxicities resolved within 4–8 weeks and without the need for stem cell transplantation (86). On the basis of these findings, the United States Food and Drug Administration granted breakthrough therapy and fast-track designation to iobenguane 131I for the treatment of MPPG.

Peptide receptor radionuclide therapy (PRRT) is a molecular therapy used to treat neuroendocrine tumors. Examples of PRRT agents include 177Lu-DOTATATE and 90Y-DOTATE. 177Lu-DOTATATE is approved for the treatment of patients with somatostatin receptor-positive gastroenteropancreatic neuroendocrine tumors. This radiopharmaceutical binds to the somatostatin receptors present at the tumor cell membrane, delivering lethal radiation. MPPGs usually express somatostatin receptors. In fact, the sensitivity of 68Ga-DOTATATE positron emission tomography/computed tomography imaging in patients with MPPG seems to be higher than that of MIBG scans (87, 88). This observation makes 177Lu-DOTATATE an interesting medication to evaluate in clinical trials. Initial prospective studies of 177Lu-DOTATATE and 90Y-DOTATE included occasional patients with MPPG. Response rates were disappointing, with less than 10% of these patients exhibiting a clinical benefit (89, 90). This observation contrasted with the higher sensitivity of octreotide scintigraphy compared with MIBG scans. Investigators hypothesized that MPPGs may have inappropriate expression of somatostatin receptor subtypes and/or processing errors that caused the lack of response to octreotide and its analogs (91). In fact, molecular studies in a few MPPG specimens have found minimal or no expression of the somatostatin receptor 2 (92). DOTATATE mainly targets this receptor (93). Recently, however, the interest in 177Lu-DOTATATE for the treatment of patients with MPPG has been reactivated. A retrospective study of patients with MPPG treated with MIBG (*n* = 16), 90Y-DOTATE (*n* = 12), or 177Lu-DOTATATE (*n* = 2) suggested that PRRT offered better OS and PFS than did conventional MIBG (94). Nevertheless, this study had several limitations. The sample size was very small, the treatment groups for comparison were quite heterogeneous, and the authors did not conduct a multivariate or propensity score analysis to reduce bias. Nevertheless, individual clinical observations suggested that some patients benefited from PRRT (94). In a more recent retrospective study of 20 patients with MPPG treated with 177Lu-DOTATATE, 29% had partial responses and 62% had stable disease 3 months after therapy (95). Fourteen patients had hypertension, and only six patients had disease progression before treatment was provided. Nine patients received radiosensitizing chemotherapy. Some reduction in the dosage of antihypertensive medications was observed in 62% of the patients with hypertension (95). However, the small and heterogeneous sample of this study, the fact that most patients had stable disease before treatment, and the simultaneous use of chemotherapy hinder the interpretation of this study's results. Therefore, a prospective clinical trial is required to prove the efficacy of PRRT in patients with MPPG.

## Regulation of Cellular Energetics: HIF Inhibitors

Several medications that inhibit the HIF-2α pathway have been tested in patients with cancer. Most of these medications have been shown not to be very effective and have not moved from phase 1 to phase 2 clinical trials (96). Crystallography identified a large protein cavity within the HIF-2α PAS-B domain. This cavity is the target of potent 130 HIF-2α inhibitors (PT2385 and PT2977) (97). Recently, PT2385 was evaluated in a phase 2 clinical trial for heavily pretreated patients with progressive renal cancer (98). Several patients had partial responses, and one patient had a complete response. Interestingly, side effects were minimal (98). These drugs have not been tested in patients with MPPG. An investigator-initiated proposal for a phase 2 clinical trial of PT2977 for MPPGs is under evaluation by regulatory agencies.

## Enhancement of Immune System Tumor Recognition: Interferon and Pembrolizumab

Pseudohypoxia causes inactivation of cytotoxic T-cell lymphocytes, activation of immune-suppressive monocytes, increased adenosine production, and increased expression of the immune checkpoint protein programmed death-ligand 1 (PD-L1) and its receptor, among many other immune system disarrangements (55, 99, 100).

One of the first immunotherapies introduced to clinical practice was interferon alpha-2b. Interferon alpha-2b activates natural killer cells that can recognize and destroy cancer cells and has been used for the treatment of patients with gastroenteropancreatic neuroendocrine tumors, melanoma, and kidney cancer. Some of these patients had clinical benefits, with disease stabilization and occasional partial responses (101). These findings correlated with histological evidence of tumor necrosis. For many years, interferon alpha-2b and octreotide analogs were considered the pillars of treatment for patients with gastroenteropancreatic neuroendocrine tumors (102). As MPPGs are also neuroendocrine tumors, occasionally patients were also treated with interferon alpha-2b. A recent retrospective study of 14 patients with progressive MPPG who were treated with interferon alpha-2b showed that 12 patients had disease stabilization and 2 had partial responses (103). Baseline PFS was 9.4 months, while the PFS of patients treated with interferon alpha-2b was 17.2 months. This study suggested that immunotherapy could have a positive impact on patients with MPPG (103). Prospective studies of interferon alpha-2b in other malignancies had several methodological problems (101); furthermore, the side effects of this drug, including fatigue, depression, flu-like syndrome, renal failure, and liver toxicity, substantially alter patients' quality of life and frequently lead to therapy discontinuation (104). Consequently, interest in interferon alpha-2b has declined.

Over the last decade, several novel immune therapies have been developed. These medications target immune-related molecular pathways such as the cytotoxic T-lymphocyte-associated protein 4 and PD-L1/PD-1 pathways. These pathways, among others, play an important role in the recognition of the cancer cell by the immune system. These immune checkpoint inhibitors are approved for the treatment of several malignancies, including melanoma, kidney cancer, and non-small cell lung cancer. Prospective studies have demonstrated disease stabilization, partial responses, and sometimes disease resolution (105–107). Although serious adverse events—mainly autoimmune events have been described, for the most part patients tolerate these medications well (105–107). A phase 2 clinical trial of the PD-1 inhibitor pembrolizumab for patients with MPPG is currently underway. This study hypothesizes that the administration of single-agent pembrolizumab to patients with PD-L1-positive MPPG will result in a nonprogression rate of greater than 20% (based on RECIST 1.1 criteria) at 27 weeks (nine cycles). The study is actively recruiting patients (NCT02721732).

## WHERE ARE WE GOING?

Clinical and basic research studies have revealed that replicative immortality, upregulated and sustained proliferative signaling, genome instability (single mutations), inflammation, deregulation of cellular energetics, angiogenesis, and activation of mechanisms responsible for invasion and metastasis are hallmarks of the development of MPPG that could be effectively targeted with available treatments. MPPG treatments, then, can be categorized on the basis of their actions on one or more of these hallmark capabilities. As detailed above, each potential therapy for patients with MPPG typically targets only one or two hallmark capabilities. Preliminary results from prospective clinical trials and observations derived from retrospective studies strongly suggest that angiogenesis is a predominant druggable hallmark of MPPG. Patients treated with angiogenesis inhibitors have indeed exhibited clinical benefits such as tumor size reduction and improvement of hormonal symptoms. However, although individual clinical responses may sometimes last for several years, these responses are, in general, transitory (108). The development of resistance is not exclusive to therapies that target angiogenesis; it is also expected with other therapies because they target a limited number of MPPG hallmark capabilities (109). In fact, cancer cells may, over time, acquire other capabilities that lead to treatment resistance, tumor recurrence, and disease progression.

In addition, each of the hallmark capabilities is regulated by partially redundant molecular pathways, and individual targeted therapies may not fully downregulate all of the molecular pathways responsible for a specific hallmark capability. Therefore, some MPPG cells may survive and adapt over time to the biological impositions of targeted therapies. These adaptive mechanisms may include the development of new mutations, the

remodeling of cells' epigenetic characteristics, and modifications of their microenvironment. Single medications or combined therapies that target several hallmark capabilities at the same time may produce a higher rate of response and more durable benefits. However, patients receiving such treatments might be more prone to develop severe adverse events than are patients treated with medications that target only one or two capabilities. Therefore, the dose and administration of these drugs must be carefully calibrated in order to achieve the best possible therapeutic response while minimizing the severity of side effects. The phase 2 clinical trial of cabozantinib discussed above, for instance, is exploring this concept. It is also important to develop clinical trials that combine therapies with different and complementary mechanisms of action. There are currently no such trials for patients with MPPG, and any such trial will likely need to be preceded by a phase 1 trial to evaluate dosage and safety.

Our current knowledge about the hallmarks of cancer suggests that exploring other therapeutic options for patients with MPPG may be helpful. Telomerase inhibitors that can modulate replicative immortality, poly (ADP-ribose) inhibitors that can stabilize the genome, selective anti-inflammatory medications, inhibitors of the hepatocyte growth factor and c-Met pathways that can stop invasion and metastasis, proapoptotic Bcl-2 homology domain 3 inhibitors that can prevent resistance to cell death, cyclin-dependent kinase inhibitors that can enhance the activity of growth suppressors, aerobic glycolysis inhibitors, and epidermal growth factor receptor inhibitors that can prevent sustained proliferative signaling all are potential medications to evaluate in clinical trials (**Figure 2**).

Metastatic pheochromocytomas and paraganglioma is an orphan disease with no Food and Drug Administration-approved therapies. The final results of the phase 2 pivotal clinical trial with iobenguane 131I are sound and impressive (86). Given the rarity of MPPG, it would be very difficult to develop a phase 3 clinical trial, so regulatory agencies should consider approving iobenguane 131I for the treatment of patients with MIBG-avid MPPG. If such approval is granted, iobenguane 131I may become the first-line treatment for many patients with MPPG. Clinical trials with multityrosine kinase inhibitors, HIF inhibitors, 177Lu-DOTATATE, and pembrolizumab would become therapeutic options to explore in patients with non-MIBG-avid MPPG, patients with MIBG-avid tumors that do not respond to iobenguane 131I, and patients who have contraindications for iobenguane 131I therapy.

## CONCLUSION

The available treatments for MPPG are, so far, not curative; research is needed to evaluate therapies with novel mechanisms of action. The use of tyrosine kinase inhibitors, radionuclide agents, and immune therapy may improve the outcomes of patients with MPPG and should be studied in clinical trials. It is always important to treat and prevent hormonal complications and symptoms that derive from direct drug toxicity, so drug doses must be carefully selected. Clinical trials combining therapies that target several hallmarks of MPPG in a simultaneous or sequential manner are an essential goal of MPPG research.

## AUTHOR CONTRIBUTIONS

CJ has written this invited review manuscript. CJ created the manuscript structure, abstract, sections, figures, and chose the references. CJ is the only author of this manuscript.

## ACKNOWLEDGMENTS

The author acknowledges Dr. Amy Ninetto from the Department of Scientific Publications at The University of Texas MD

## REFERENCES

Anderson Cancer Center for the thorough editing provided to this manuscript. The author would like to thank very much Ms. Natalie Papadam, Mr. Peter Liu, the Team Nat Foundation, Ms. Margaret Cazalot, Ms. Marle Granek, and Dr. Catherine Cotton for their immense support to develop clinical trials for patients with malignant pheochromocytomas and paragangliomas. Thanks to their inspiration and support, patients have new hope.

## FUNDING

This publication was supported by the Team Nat Foundation and the Cazalot Fund for research on pheochromocytoma and paraganglioma.

literature review. *J Clin Endocrinol Metab* (2017) 102:4013–22. doi:10.1210/ jc.2017-00562

and paragangliomas. *Clin Cancer Res* (2016) 22:2301–10. doi:10.1158/1078- 0432.CCR-15-1841

**Conflict of Interest Statement:** The author declares that the submitted work was not carried out in the presence of any personal, professional, or financial relationships that could potentially be construed as a conflict of interest.

*Copyright © 2018 Jimenez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Jing Ma1,2 and Adrian Vella2 \**

*1Division of Endocrinology and Metabolism, Shanghai Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China, 2Division of Endocrinology, Diabetes and Metabolism, Mayo Clinic College of Medicine, Rochester, NY, United States*

#### *Edited by:*

*Derek LeRoith, Icahn School of Medicine at Mount Sinai, United States*

#### *Reviewed by:*

*Yukihiro Fujita, Asahikawa Medical University, Japan Burkhard Göke, Medical School Hamburg, Germany*

*\*Correspondence:*

*Adrian Vella<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Diabetes, a section of the journal Frontiers in Endocrinology*

*Received: 19 April 2018 Accepted: 31 May 2018 Published: 26 June 2018*

#### *Citation:*

*Ma J and Vella A (2018) What Has Bariatric Surgery Taught Us About the Role of the Upper Gastrointestinal Tract in the Regulation of Postprandial Glucose Metabolism? Front. Endocrinol. 9:324. doi: 10.3389/fendo.2018.00324*

The interaction between the upper gastrointestinal tract and the endocrine system is important in the regulation of metabolism and of weight. The gastrointestinal tract has a heterogeneous cellular content and comprises a variety of cells that elaborate paracrine and endocrine mediators that collectively form the entero-endocrine system. The advent of therapy that utilizes these pathways as well as the association of bariatric surgery with diabetes remission has (re-)kindled interest in the role of the gastrointestinal tract in glucose homeostasis. In this review, we will use the changes wrought by bariatric surgery to provide insights into the various gut–pancreas interactions that maintain weight, regulate satiety, and limit glucose excursions after meal ingestion.

Keywords: incretin hormones, bariatric surgery, gastric emptying, gastric accommodation, insulin secretion, insulin action, vagus nerve

## BACKGROUND

In the United States, the prevalence of obesity is rapidly increasing with 65% of adults and 17% of adolescents and children classified as being overweight or obese (1). Obesity is associated with multiple diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and osteoarthritis, as well as being associated with an increased frequency of the risk factors for cardiovascular disease (2). Approximately 9% of national health-care costs have been attributed to excess weight (3). Because of the evidence that weight reduction ameliorates or corrects the comorbidities of obesity, the US Preventive Services Task Force has recommended that body mass index (BMI) is routinely assessed and weight management recommended for obese patients (4).

Behavioral intervention with lifestyle and dietary modification usually achieves modest weight loss (4). While generally safe, most regain the weight lost within 5 years. Pharmacotherapy for obesity is considered for patients who have failed efforts at lifestyle modification and who have a BMI ≥ 30 kg/m2 or a BMI ≥ 27kg/m2 in the presence of comorbidities such as diabetes (5). However, there have been significant concerns about the long-term safety of such medications and many of the currently available medications have limited efficacy (6).

Bariatric surgery, sometimes referred to as metabolic surgery, is usually considered for patients who have a BMI ≥ 40 kg/m2 or a BMI ≥ 35 kg/m2 associated with comorbidities such as type 2 diabetes (5). Restrictive surgeries such as adjustable gastric banding (AGB) and sleeve gastrectomy (SG) limit the capacitance of the stomach. Rouxen-Y gastric bypass (RYGB) is the most commonly performed bypass procedure and produces gastric restriction together with selective malabsorption. RYGB involves creation of a gastric pouch by separating the stomach across the fundus. Drainage of this 10–30 ml pouch is achieved by a gastrojejunostomy. The distal end of the jejunum is anastomosed ~150 cm below the gastrojejunostomy effectively bypassing the distal stomach, duodenum, and proximal jejunum. Duodenal switch (DS) is a variation of biliopancreatic diversion and involves a SG with division of the duodenum below the pylorus. The distal ileum is anastomosed to the short stump of the duodenum producing a ~100 cm channel for nutrient absorption. The other end of the duodenum is closed and the remaining small bowel connected onto the enteral limb 75–100 cm from the ileocecal valve (2).

Observational and prospective studies have suggested that bariatric surgery is the most effective intervention for weight loss producing an average weight loss of 30–35% that is maintained in ~60% of patients at 5 years (7). This has led to a dramatic increase in the number of procedures performed annually from 13,365 in 1998 (8) to 216,000 in 2016 according to the data released by American Society for Metabolic and Bariatric Surgery (9). In a meta-analysis of 136 studies of bariatric surgery, which included a total of 22,094 patients, Buchwald et al. reported that within studies examining type 2 diabetes after bariatric surgery, 1,417 of 1,846 (76%) patients experienced complete resolution. When categorized by operative procedure, there were clear differences in efficacy. Diabetes resolved in 98.9% of patients undergoing biliopancreatic diversion or DS. In contrast, the rate was 83.7% for RYGB and 47.9% for AGB (10). A retrospective review of 257 patients who underwent the long-limb modification of RYGB (400–500 cm Roux limb length) at our institution reported resolution of type 2 diabetes in 94% of patients (11). Recent prospective, randomized controlled trials have, however, reported lower remission rates for diabetes with RYGB, although it remains superior to medical therapy (12–14). Setting aside the superiority of one procedure over the other in terms of inducing diabetes remission [which is likely related to residual β-cell function at the time of the procedure (15, 16) as well as the magnitude of weight loss (17)], obvious differences between procedures can be used to explore the role of the gastrointestinal tract in metabolism. RYGB is sometimes complicated by the occurrence of hyperinsulinemic hypoglycemia (18). Its incidence is uncertain although it has been suggested that excessive glucagon-like peptide-1 (GLP-1) secretion after RYGB (19) may be the cause of this phenomenon, but this is unlikely (20). The condition has been the subject of an extensive review recently (21) (**Figure 1**).

The anatomic differences among bariatric procedures result in differences in enteroendocrine secretion (**Table 1**): postprandial GLP-1 concentrations are lower after SG compared to RYGB in the comparative studies undertaken in humans (22–26). On the other hand, a liquid meal, especially after gastric restriction, may

#### Table 1 | Efficacy of different bariatric surgeries.

*Increase,* ↑*; decrease,* ↓*; no change,* ↔*; NA, no available evidence; SG, sleeve gastrectomy; RYGB, Roux-en-Y gastric bypass; AGB, adjustable gastric banding; DS, duodenal switch; GLP-1, glucagon-like peptide-1; GIP, gastric inhibitory polypeptide; CCK, cholecystokinin.*

not recreate conditions present after a solid meal (27). Indeed, liquid emptying especially after restrictive gastric surgery is dependent on fasting gastric volume (28, 29). SG removes the capacitance function of the stomach and decreases ghrelin concentrations to a greater extent than does RYGB (24, 25, 30). This is more apparent when acyl-ghrelin is measured (22, 31, 32). Whether these differences can explain metabolic outcomes will be explored in detail below (**Figure 2**).

## CALORIC RESTRICTION—CHANGES IN GASTRIC VOLUME, COMPLIANCE, AND ACCOMMODATION

Restrictive procedures reduce gastric volume—indeed, the postoperative period of any form of bariatric surgery is characterized by a significant degree of caloric restriction (33). Fasting blood glucose and insulin resistance improves within 6 days of gastric bypass and occurs before any weight loss (34). This had been observed previously with very low-calorie diets outside of bariatric surgery (35), suggesting that caloric restriction at least partially, explains the acute improvement in glucose control after bariatric surgery (14, 36, 37). Six weeks of caloric restriction (700–900 kcal/day) decreases fasting and postprandial glycemia by lowering fasting endogenous glucose production and improving β-cell function (38). Jackness et al. showed that very low caloric restriction (500 kcal/day) produced a similar improvement in β-cell function as those who underwent post-RYGB (39). Indeed, caloric restriction after RYGB outweighs the effect of GLP-1 on glucose metabolism [as studied by the use of a competitive antagonist of GLP-1 at its receptor (40)]. Of course outside of the mechanical restrictions induced by bariatric surgery, there are difficulties with long-term compliance with regimens of caloric restriction. However, in an open-label, cluster-randomized trial 24% patients achieved weight losses of 15 kg or more in 12 months, and 86% experienced diabetes remission (41).

Multiple factors influence satiation including stomach capacitance and emptying. The stomach increases in volume in anticipation of food ingestion (42). In the normal stomach, this volume expansion is not associated with an increase in gastric wall stiffness (or increased intraluminal pressure) suggesting a change in the mechanical properties of the stomach wall (increased compliance) to accommodate the capacitive function of the stomach (43). This function is primarily fulfilled by the proximal stomach, which serves as a food reservoir, while the distal stomach triturates food to a size that can pass the pylorus. The physical nature, particle size, fat, and caloric content of food alter emptying rate (27). Although nutrient and non-caloric liquids empty rapidly, solids are initially retained in the stomach while antral contractions propel particles toward the closed pylorus. Food particles are emptied once they are ~2 mm in diameter (27). Restrictive procedures eliminate the function of the proximal stomach displacing food to the distal stomach and accelerating emptying. It is uncertain if restriction of accommodation alone alters gastric emptying (44). In SG, a functioning pylorus is retained and gastric volume is usually larger than the pouch created after RYGB. Post-RYGB it has been assumed that there is little neuromuscular control on gastric emptying since the pylorus is bypassed [although this may not be correct (45)]. Surgical vagotomy (which occurs in RYGB when the gastric pouch is created) alters gastric accommodation but may not change emptying (46) and typically does not have durable effects on weight (47).

Gastric emptying plays an important role in determining the magnitude of change in glucose concentrations after nutrient ingestion (48). Indeed, variation in the rate of gastric emptying alters peak insulin response after 75 g oral glucose, in both healthy subjects and patients with type 2 diabetes (49, 50). Fasting gastric volume affects the rate of emptying of a liquid challenge (51). Interventions that delay gastric emptying have the potential to regulate glycemia in patients with diabetes. Accelerating nutrient flow to the small intestine with erythromycin increases the postprandial glycemic response (52), whereas slowing gastric emptying with Xenin-25, a 25-amino acid neurotensin-related peptide, reduces postprandial blood glucose (53). Although it is important to match the rate of gastric emptying and the onset and offset of insulin action, significantly lower insulin requirements are observed in patients with type 1 diabetes with gastroparesis than those without, during the first hour of the postprandial period (54). Delaying gastric emptying is also a mechanism of action of some antidiabetic medications, such as GLP-1 analogs and pramlintide (55, 56).

Gut hormones can modulate food intake over and above that caused by mechanical restriction after bariatric surgery (22). Ghrelin increases food intake after esophagectomy or gastrectomy (57, 58). Neuronal GLP-1R mediates the anorectic effects of GLP-1 (59). Inhibition of GLP-1 action with Exendin-9,39 after RYGB accelerates gastric emptying (45). Taken together, these observations suggest that factors other than anatomy contribute to the upper gastrointestinal response to food ingestion. The attraction of certain foods decreases after RYGB (60) and appetite may be altered by enteroendocrine secretion (61, 62). A potential mechanism is *via* GLP-1, which alters gastrointestinal transit, gastric accommodation (45, 46, 63), and has direct effects on hypothalamic nuclei outside of the blood–brain barrier (64). GLP-1 and GLP-1 receptor agonists decrease food intake and cause weight loss (65, 66). GLP-1 also modulates taste sensitivity in rodents (67–70). The peripheral concentrations of GLP-1 observed in the early postprandial period in subjects post-RYGB, exceed concentrations observed after infusion at 0.75 pmol/kg/ min, and are similar to those observed after infusion at 1.5 pmol/ kg/min—both infusion rates that alter gastrointestinal function (71). It is, therefore, reasonable to consider that the postprandial rise in GLP-1 might affect feeding behavior after RYGB, and to a lesser extent SG, where the increase in GLP-1 is less marked (22–26). The elevated postprandial concentrations of GLP-1 observed after RYGB are unlikely to be the cause of diabetes remission after bariatric surgery. We (45) and others (72) have shown that inhibition of GLP-1 actions in the postprandial period has limited effects on glucose concentrations in people after RYGB. This is in agreement with data from mice deficient in the GLP-1 receptor that lost the same amount of weight as wild-type mice (73). This is also the case after SG in humans (74) and in mice deficient in the GLP-1 receptor (75). On the other hand, SG decreases acyl-ghrelin concentrations, presumably due to excision of a large part of the ghrelin-secreting stomach, which should decrease appetite (22, 31, 32). Fasting after SG is not associated with a rise in (low) ghrelin concentrations, in contrast to RYGB (22, 24, 25, 30–32).

In an effort to circumvent the costs and complications associated with bariatric surgery, various attempts have been made to develop endoscopically placed devices that might cause weight loss. One such device is a synthetic sleeve placed post-pylorus under endoscopic control. The rationale underlying such a device is to ensure that nutrients are prevented from coming in contact with the absorptive surfaces of the proximal small bowel (76). Unfortunately, such a device is prone to migration, bleeding, and bolus obstruction. A placebo-controlled study utilizing the device as treatment for type 2 diabetes was terminated prematurely because of a ~3% incidence of hepatic abscess in subjects using the device (77). Other devices such as intra-gastric balloons to induce early satiety are under study.

## GHRELIN

Ghrelin is a 28-amino acid peptide and is the only orexigenic hormone recognized in humans. It is secreted from the gastric mucosa and hypothalamus in both rodents and humans. There are two forms of circulating ghrelin, unacylated and acylated ghrelin (AG) (78). In the fasting state, AG is elevated (~110 pM) and decreases (~70 pM) in response to food ingestion. Patients with Prader–Willi syndrome—a syndrome characterized by excessive feeding behavior—have high concentrations of circulating ghrelin (79). Fasting and postprandial acyl-ghrelin levels are decreased following SG, compared to Roux-en-Y gastric bypass (RYGB), which may play a role in weight loss (26). SG involves removal of the gastric fundus—the primary source of ghrelin synthesis and secretion. Exogenous ghrelin administration increases energy intake in both rodents (80) and humans (81). Infusion of ghrelin in patients after esophagectomy (58) or gastrectomy (57) increases caloric intake and appetite. Although the contribution of ghrelin to normal physiology is unclear, it has been demonstrated that ghrelin can directly inhibit insulin secretion (82). Pharmacologic concentrations of ghrelin or ghrelin receptor agonists accelerate gastric emptying, suppress insulin secretion, and increase glucagon secretion (83). In a randomized controlled phase Ib clinical trial, ghrelin accelerated gastric emptying and improved gastrointestinal symptoms in patients with type 2 diabetes (84). Ghrelin receptor agonists are being developed as potential therapies for gastroparesis (85).

## INCRETIN HORMONES

The incretin effect is a phenomenon first observed several decades ago when intravenous glucose produced lower insulin concentrations, despite higher glucose concentrations than observed after ingestion of an equivalent amount of glucose (86). This observation has subsequently been confirmed with isoglycemic infusion studies (87). The subsequent discovery of glucagon-like immunoreactivity in the gut led to the realization that proglucagon is synthesized in enteroendocrine cells intercalated between enterocytes and distributed throughout the intestine. GLP-1 and gastric inhibitory polypeptide (GIP) are two incretin hormones, which stimulate postprandial insulin secretion (88). GLP-1 is released from L-cells, most densely located in the distal small intestine and colon, although they are also located more proximally in the duodenum and jejunum (89). There is some evidence in rodent models of paracrine GLP-1 secretion within pancreatic islets (90). GIP is secreted from K cells (which reside mainly in the duodenum and upper jejunum) in response to nutrient ingestion. The early secretion of GLP-1 might involve an indirect neural or hormonal mechanism (91). The later secretion of GLP-1 is dependent on direct contact of nutrients in the small intestine with L-cells (92). Targeted delivery of lauric acid in enteric-coated pellets to the ileum and colon can stimulate substantial endogenous GLP-1 release and attenuate postprandial glycemia (93). To stimulate its receptor, GLP-1 requires the presence of 2 N-terminal amino acids, which are cleaved by the enzyme, dipeptidyl peptidase-4 (DPP-4), rendering the truncated form (GLP-1-9,36) inactive. Because of the widespread distribution of DPP-4, the active form of GLP-1 has a short half-life in the circulation (94). GLP-1 receptor agonists that are not substrates of DPP-4 and DPP-4 inhibitors are approved for the treatment of type 2 diabetes. They lower fasting and postprandial glucose concentrations (66). In addition to stimulating insulin secretion, pharmacologic concentrations of GLP-1 (and GLP-1 receptor agonists) inhibit gastric emptying, and suppress glucagon secretion. Moreover, GLP-1 and GLP-1 receptor agonists increase satiety, leading to a reduction in weight (95).

Although GIP secretion is preserved, the insulinotropic effect of GIP is diminished in type 2 diabetes. Unlike GLP-1, GIP stimulates glucagon secretion during hypoglycemia (96, 97) and has no effect on gastric emptying. Circulating concentrations of GIP are related to BMI (98), which suggests a role of GIP in energy metabolism. In mice, high GIP concentrations promote obesity and insulin resistance (99). However, recent study shows that there is a synergistic effect of GIP and GLP-1 co-agonists in weight lowering (100) and glycemic improvement in patients with type 2 diabetes than monoagonist (101). Addition of a dual GIP/GLP-1 receptor agonist (NNC0090-2746) to metformin improved glycemic control with accompanying reductions in body weight and circulating cholesterol (102). The molecular mechanism underneath the metabolic improvements is not known. The effects of GIP on glucose metabolism are an area of ongoing investigation, which will hopefully be accelerated by the development of a specific GIP receptor antagonist (103).

## CHOLECYSTOKININ (CCK)

Cholecystokinin is secreted from the I-cells by exposure to nutrients in the duodenum and upper jejunum. Fat is a strong stimulus for CCK secretion, followed by protein, whereas carbohydrate is a weaker stimulus of CCK secretion. CCK concentrations increase from fivefold to tenfold after ingestion of a mixed meal and inhibit gastric emptying through activation of CCK-1 receptors (104). Physiological concentrations of CCK delay entry of glucose into the duodenum, reducing postprandial glucose excursions (105). In rats, CCK decreased hepatic glucose production to maintain glucose homeostasis by inhibiting CCK-A receptors and triggering a gut–brain–liver neuronal axis (106). In humans, CCK dose-dependently presents early satiety and reduces the energy intake at a buffet style meal, which was attenuated by the CCK-1 antagonist, loxiglumide (107). However, the long-term effects of CCK administration in humans and its role in obesity therapy are not clear.

## ROLE OF THE VAGUS—VAGAL BLOCKADE/VAGOTOMY

The gastrointestinal tract is innervated by the parasympathetic and sympathetic divisions of the autonomic nervous system. The parasympathetic innervation originates from the dorsal motor nucleus of the vagus (DMV) in the medulla (108), while the sympathetic supply derives from the prevertebral ganglia (109). Gastric motility is partially controlled by the vagus nerve, a mixed motor, and sensory nerve. The sensory axons of the vagus receive afferent inputs from gastrointestinal receptors and then project to the nucleus of the solitary tract (110). Nucleus of the solitary tract (NTS) neurons activate vagal motor neurons in the nucleus ambiguus and the dorsomedial nucleus to regulate the smooth muscle contractions in the stomach and duodenum, with these neural loops being known as vagovagal reflexes (111). Bilateral truncal vagotomy (112), aiming for treating of peptic ulcer surgery, and electrical vagal blockade (113) results in delayed gastric emptying, and weight loss—at least in the short term. The gastric vagal branches are often damaged during bariatric surgery (114). It remains controversial whether vagal innervation of the portal hepatis contributes to the beneficial effects of RYGB on food intake, energy expenditure, and body weight (115). Electrical vagal blockade does not seem to have significant effects on glucose metabolism (116).

Obese subjects exhibit decreased heart rate variability likely due to an imbalance of sympathetic and parasympathetic activity (117). Overactivity of the sympathetic nervous system is more significant in obese subjects with type 2 diabetes than in those subjects without diabetes (118). Weight reduction following RYGB and AGB in severely obese patients is associated with an increase in heart rate variability (119). The underlying mechanism(s) remain unknown but the improvement in autonomic function does not appear to be related to improved insulin action (120). It has been posited that these changes in autonomic function could arise from crosstalk between the gastrointestinal tract and the central nervous system (121) generated by a neuro-inflammatory reflex (122) arising from the gut microflora.

## THE TASTE SIGNALING SYSTEM

It is increasingly recognized that bariatric surgery may alter food preference and taste, in particular, the perception of sweet taste. This likely contributes to the reduction in energy intake after surgery (123). Both SG and RYGB result in a reduction of the frequency of food craving and the hedonic component of taste perception (124). Subjects experience a decreased desire to consume sweet and fatty flavors after RYGB (60) and SG (125).

The sweet taste signaling system includes heterodimeric G protein-coupled receptors, composed of the taste receptors (TRs), T1R2 + T1R3 heterodimers, which are activated by the binding of sweet compounds such as monosaccharides and disaccharides (126). These receptors are G-protein coupled (gustducin), and activation increases phospholipase C-β2 activity, which ultimately results in the release of Ca2+ from intracellular stores and the opening of a transient receptor potential ion channel TRPM5. The resulting membrane depolarization activates gustatory afferents (127). Sweet TRs are found in the tongue, gastrointestinal tract, pancreas, adipose tissue, brain, and bone (128). Expression of T1R2 + T1R3 also occurs in the entero-endocrine L cells (129), suggesting that the sweet sensing system in the gut is involved in incretin secretion. T1R3 knockout mice exhibit impaired GLP-1 secretion and glucose intolerance (130). Intragastric infusion of nutrients with lactisole, a T1R2/T1R3 blocker, attenuates GLP-1 and peptide YY secretion in humans (131, 132). The expression of sweet taste receptors and downstream molecule transcripts are disordered in models of type 2 diabetes (133). T1R2 expression is reciprocally regulated by luminal glucose in health, but not in patients with type 2 diabetes; during acute hyperglycemia, T1R2 transcript levels decrease in response to duodenal glucose infusion in healthy subjects, but increase in subjects with type 2 diabetes (134).

In addition to changes in oral taste sensitivity, the expression of T1R2 and T1R3 is decreased in the small intestine of rats after bariatric surgery; this occurs in parallel with elevation of GLP-1 (135). Functional magnetic resonance imaging or positron emission tomography demonstrates a decrease in neural activity in the brain reward areas in response to high-calorie foods (136).

## PERMEABILITY AND GLUCOSE TRANSPORT

The proximal small intestine initiates carbohydrate absorption after digestion. Glucose absorption is mediated by the sodium glucose co-transporter-1 (SGLT1) across the apical cell membrane and partially by the glucose transporter 2 (GLUT-2) at high glucose concentrations (137). The small intestine has a maximal capacity of glucose absorption of about 0.5 g/min (or 2 kcal/min) per 30 cm (138). The absorptive rate depends on the exposure rate of glucose, region, and length of the small intestine, and the expression of glucose transporters (139). The inhibition of motility and blood flow in the small intestine also attenuates glucose absorption (140). Plasma concentration of 3-O-methylglucose, a glucose analog that is not metabolized, is normally used to measure the absorption rate of glucose. Physiologically, enhanced glucose absorption in the proximal gut would increase blood glucose concentrations; acute hyperglycemia itself appears to enhance glucose absorption (141). Rodent models of diabetes exhibit small intestinal hyperplasia and increased absorption of glucose from intestinal mucosa (142). It is unclear to what extent inhibition of SGLT-1 can alter glucose absorption in a way that is relevant to postprandial glycemic control in diabetes.

Active glucose transport and intestinal permeability are increased in obesity and diabetes. For a given caloric intake, this could alter the nutrient load entering the portal circulation (143–146). Changes in intestinal thickness and transcription of SGLT-1 and GLUT-2 occur after RYGB (147, 148). Foregut exclusion decreases glucose absorption in rodents (149). However, it is currently not known, and if so, the extent to which RYGB and SG alter the rate of active intestinal glucose absorption or the rate of passive intestinal permeability.

Intestinal integrity provides a physical barrier to luminal bacteria, toxins, and antigens from the external environment. In health, it allows the passage of water and nutrients. Increased paracellular permeability, following disruption of the intestinal tight junctions enables bacteria to leak out of the intestinal lumen into the blood stream (150). Factors that influence permeability include the gut microbiome and fatty acids (whether ingested directly or as products of bacterial fermentation) (151). Bile acids could alter gut permeability through the G-proteincoupled bile acid receptor (TGR5), a cell surface receptor, which occurs at a high level expression in the human placenta and spleen and is also found in multiple tissues such as the lung, liver, adipocytes, and the gastrointestinal tract (152). A systematic review of 14 studies suggests that that fasting and postprandial lipopolysaccharide (LPS) are increased in patients with diabetes (153). LPS is the core component of the outer membrane of Gram-negative bacteria. Metabolic endotoxemia is defined by a twofold to threefold increase in plasma LPS concentration (154). Rosiglitazone is the most effective in the lowering the LPS in patients with type 2 diabetes, but the extent to which this contributes to the glucose-lowering effects of this compound are unknown (155).

## BILE ACID METABOLISM

Bile acids are synthesized in hepatocytes *via* cytochrome P450 mediated oxidation of cholesterol and then secreted into the intestinal lumen through the biliary system. 95% of intestinal bile acids are reabsorbed in the distal gut and transported back to the liver by the enterohepatic circulation (156, 157). CCK induces production of bile, contraction of the gall bladder, and relaxation of the sphincter of Oddi, to deliver bile into the duodenum (158). Bile acids promote digestion and absorption of lipids in the gastrointestinal tract as well as participate in the regulation of glucose and energy homeostasis (159), acting through two specific receptors, the farnesoid X receptor (FXR) and TGR5.

FXR is expressed in the liver and the intestine in humans and is a member of the nuclear receptor super-family. It can be activated by both primary and secondary conjugated bile acids (160, 161). Similar to other nuclear receptors, FXR translocates to the cell nucleus and subsequently induces expression of the small heterodimer partner (SHP). SHP is involved in bile acids synthesis by downregulating the gene transcription of cholesterol 7 alpha-hydroxylase (CYP7A1), a rate-limiting enzyme in bile acid synthesis. The activation of TGR5 triggers the production of intracellular cAMP and secondary active the mitogen activated protein kinase signaling pathway to perform different functions in various organs. For instance, TGR5 is expressed in rodent and human pancreatic islets and regulates insulin secretion (162). TGR5 in enteroendocrine L cells stimulates secretion of GLP-1 (163). In addition, TGR5 may regulate energy homeostasis through activating deiodinases to convert the prohormone thyroxine (T4) into the active hormone triiodothyronine (T3) (164, 165).

Circulating bile acids' concentrations after meal ingestion are decreased in obese subjects compared to lean controls (166). This difference is no longer significant after bariatric surgery (167, 168). The effects of SG on body weight and glucose tolerance are attenuated in the absence of FXR (169) and in TGR5 knock-out mice (170). In a diet-induced obesity mouse model, diversion of bile flow to the ileum produces similar metabolic benefits to RYGB (171), while the ability of RYGB to decrease body weight and improve glucose tolerance is substantially reduced in the absence of FXR. Bile acids may stimulate insulin secretion *via* activation of FXR and inhibition of ATP-dependent K + channels (172). It has been suggested that the changes in bile acid composition and concentrations induced by bariatric surgery can contribute to metabolic changes *via* FXR and TGR5-signaling pathways.

However, in humans, the contribution of bile acid changes to metabolic improvements after bariatric procedures is less clear. One study reported that total plasma bile acid concentrations increased twofold after RYGB but decreased after AGB, despite similar weight loss (173). Longitudinal study suggests that there are two phasic increases in plasma bile acid concentrations in a cohort of RYGB patients at 1 month and up to 24 months after surgery (168). This time course differs from the time course of metabolic resolution suggesting that they are unrelated phenomena.

## THE GUT MICROBIOME

The human gut microbiome consists of 10–100 trillion of microorganisms, primarily bacteria, in the digestive tract (174). The composition of the gut microbiome influences digestion, absorption, inflammation, and intestinal motility. Over the past decade, several studies have demonstrated that gut microbial populations are closely associated with metabolic disorders such as dyslipidemia, obesity, and diabetes (175). The gut microbiome is established early in life (176). Exposure to antibiotics alters the normal distribution of intestinal flora and is associated with changes in metabolism in some (177) but not all studies (178). Diet and lifestyle and geography are the primary influencers of the distribution of intestinal flora (179).

In humans, gut microbiota produce glycoside hydrolases and polysaccharide lyases, which facilitate digestion of sucrose, lactose, and starch (180). Undigested polysaccharides are subject to fermentation by intestinal bacterial leading to the production of short-chain fatty acids, which can provide 5–10% energy consumption (181). Gut microbiota is also involved in signaling of FXR and TGR5 by modifying the bile acid pool (182). In fact, bile acids interact with gut microbiota by direct effects on the mucosal defense, membrane integrity, oxidative and pH stress to increase the growth of bile acid-metabolizing bacteria (183).

d-lactate acidosis is a rare complication of jejuno-ileal bypass surgery or patients with short bowel syndrome (SBS) (184). d-lactate production is mainly dependent on the colonic microbiome (184). Notably, in patients with SBS or after jejuno-ileal bypass surgery, delivery of an increased amount of undigested carbohydrates to the colon can result in excess d-lactate accumulation (185, 186). *Bacteroides thetaiotaomicron* abundance is decreased in obese subjects compared to lean individuals (187). Patients with type 2 diabetes may exhibit decreased abundance of butyrate-producing bacteria and an increase in various opportunistic pathogens (188). Dietary fiber intake in patients with type 2 diabetes increases acetate and butyrate-producing bacteria improves glycemic control (189). Use of metformin is accompanied by increased abundance of *Escherichia* and a decrease of *Intestinibacter* (190). Impaired glucose tolerance is reversed after the transfer of metformin-altered microbiota to germ-free mice (191). Acarbose alters bile acid metabolism through changes in gut microbial populations (192). This results in great interest in microbiota alteration on improvement of metabolic parameters. Recently, transplantation of fecal microbiota or "bacteriotherapy" seems a promising therapeutic method for metabolic syndrome (193).

Individuals with obesity exhibit markedly decreased abundance of *B. thetaiotaomicron*. However, the abundance of this microbe increased after SG despite similar metabolic outcomes suggesting that this is incidental to the improvements in glucose metabolism after bariatric surgery (187). Randomized trials are warranted in the future to further assess the gut mechanism after bariatric surgeries in humans.

## CONCLUSION

The upper gastrointestinal tract plays a primary role in the regulation of glucose excursions in response to meal ingestion by determining the rate of gastric emptying and indirectly by regulating appetite and satiation, barrier integrity, and nutrient absorption. Bariatric surgery has helped improve our knowledge of the mechanisms underlying gut–pancreas interactions and may enable development of effective dietary or pharmacological strategies in the management of diabetes.

## AUTHOR CONTRIBUTIONS

JM wrote the paper with input from AV regarding content and layout. AV edited the draft for clarity and content.

## ACKNOWLEDGMENTS

AV's research is supported by the Mayo Clinic General Clinical Research Center (UL1 TR000135) and by the National Institutes of Health (DK78646, DK116231).

## REFERENCES

antidiabetic tool. *Surg Obes Relat Dis* (2016) 12(3):702–4. doi:10.1016/j. soard.2015.08.503

glucagon responses to hypoglycemia in type 1 diabetes. *Diabetes* (2015) 64(1):72–8. doi:10.2337/db14-0440

metabolism in nondiabetic subjects. *Diabetes Metab Syndr Obes* (2014) 7:305–12. doi:10.2147/DMSO.S65733

**Conflict of Interest Statement:** AV is an investigator in an investigator-initiated study sponsored by Novo Nordisk. He has consulted for vTv Therapeutics, XOMA, Sanofi-Aventis, Novartis, and Bayer in the past 5 years. JM has no relevant disclosures.

*Copyright © 2018 Ma and Vella. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Cryopreservation of Ovarian Tissue: Opportunities Beyond Fertility Preservation and a Positive View Into the Future

### Stine G. Kristensen\* and Claus Y. Andersen

Laboratory of Reproductive Biology, Faculty of Health Science, The Juliane Marie Centre for Women, Children and Reproduction, University Hospital of Copenhagen, University of Copenhagen, Copenhagen, Denmark

In current years, ovarian tissue cryopreservation (OTC) and transplantation is gaining ground as a successful method of preserving fertility in young women with primarily cancer diseases, hereby giving them a chance of becoming biological mothers later on. However, OTC preserves more than just the reproductive potential; it restores the ovarian endocrine function and thus the entire female reproductive cycle with natural levels of essential hormones. In a female population with an increased prevalence in the loss of ovarian function due to induced primary ovarian insufficiency (POI) and aging, there is now, a need to develop new treatments and provide new opportunities to utilize the enormous surplus of follicles that most females are born with and overcome major health issues associated with the lack of ovarian hormones. Cell/tissue-based hormone replacement therapy (cHRT) by the use of stored ovarian tissue could be one such option comprising both induction of puberty in prepubertal POI girls, treatment of POI and premature menopause, and as primary prevention at the onset of menopause. In the current review, we explore known and entirely new applications for the potential utilization of OTC including cHRT, social freezing, culture of immature oocytes, and a modern ovarian resection for women with polycystic ovaries, and discuss the indications hereof.

Keywords: ovarian tissue cryopreservation, fertility preservation, cell/tissue-based hormone replacement therapy, induction of puberty, ovarian resection, social freezing, in vitro maturation

## INTRODUCTION

The follicle constitutes the functional unit of the ovary and produces steroids and peptide hormones to regulate the female reproductive cycle. During the follicular phase more than 90% of the available oestradiol is produced by a single selected preovulatory follicle, which in the luteal phase is transformed to the corpus luteum that secretes progesterone in high concentrations. The unique physical distribution of the follicular reserve within the ovary, with the vast majority of small resting follicles located in the outer cortical region and the growing stages of follicles located in the inner medullary region, represents a perfect opportunity to preserve an organ function without freezing the entire organ. By isolating the cortical region, containing 90% of the follicular reserve, human ovarian tissue has been successfully cryopreserved for fertility preservation in young women with cancer diseases for over two

### Edited by:

Jeff M. P. Holly, University of Bristol, United Kingdom

#### Reviewed by:

Jung Ryeol Lee, Seoul National University College of Medicine, South Korea Shahar Kol, IVF Unit at Elisha Hospital, Haifa, Israel Andreas Norbert Schüring, Universitätsklinikum Münster, Germany

#### \*Correspondence:

Stine G. Kristensen<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Reproduction, a section of the journal Frontiers in Endocrinology

Received: 13 April 2018 Accepted: 11 June 2018 Published: 28 June 2018

#### Citation:

Kristensen SG and Andersen CY (2018) Cryopreservation of Ovarian Tissue: Opportunities Beyond Fertility Preservation and a Positive View Into the Future. Front. Endocrinol. 9:347. doi: 10.3389/fendo.2018.00347 decades. Subsequent transplantation of thawed ovarian tissue has restored ovarian endocrine function in 95% of the patients and resulted in the birth of over 130 children worldwide (1, 2). Half of all the children born have been conceived by natural conception, which highlights the exceptional ability of this procedure to actually restore an entire organ function in contrast to conventional fertility preserving strategies like oocyte and embryo freezing, in which the fertility potential is fixed by the number of oocytes retrieved and IVF is the only option to conceive. Moreover, ovarian tissue cryopreservation (OTC) can be performed from day to day and is therefore the only fertility preserving strategy for girls and young women who needs to undertake acute gonadotoxic treatment, and for prepubertal girls who do not yet produce mature oocytes for freezing.

Primary ovarian insufficiency (POI) is defined as the cessation of the ovarian function before the age of 40 years, and is a common cause of infertility in women. Its incidence is estimated to be as high as 1 in 100 by the age of 40, and 1 in 1,000 by the age of 30 years (3, 4). Spontaneous POI include genetic, autoimmunological, inflammatory, enzyme deficiency, metabolic, or very often idiopathic causes, whereas induced POI occurs mainly due to oncological treatment such as surgery (bilateral oophorectomy), chemotherapy and radiotherapy (5).

Up until now fertility preservation has been the primary goal of OTC in young girls and women diagnosed with a malignancy or genetic disease threatening to destroy their ovarian reserve. However, the restoration of an organ function, the steroidogenic capacity of the transplanted ovarian tissue and the unique access to immature oocytes from small antral follicles have now evoked new perspectives and ideas to expand the utilization of OTC beyond its traditional purpose of fertility preservation for medical reasons. These novel ideas approach a broader population of women and include utilization of OTC for cell/tissue-based hormone replacement therapy (cHRT), non-medical reasons, optimizing culture systems for immature oocytes, and performing a modern ovarian resection for women with polycystic ovaries (**Figure 1**). This kind of ideas which are technical possible, but not yet proven, will undoubtedly raise controversy in the field and a plethora of questions concerning the ethics, safety, cost-effectiveness, superiority, and implications of the proposed procedures. In this paper, we aim to explore known and entirely new applications for the utilization of OTC and discuss the indications for such procedures.

## CELL/TISSUE-BASED HORMONE REPLACEMENT THERAPY (cHRT)

More efficient cancer treatments combined with a general increase in life expectancy have led to a higher frequency of POI in a growing population of cancer survivors and aging women. It is widely accepted that the pillar of treatment of POI is pharmacologic hormone replacement therapy (pHRT), at least until the average age of natural menopause. Pharmacologic HRT with estrogen alone or estrogen and progestogens in combination is known to effectively compensate for the loss of ovarian hormone production, but only when it is delivered at an optimal dosage, frequency, and at an appropriate time (8, 9). Unfortunately, pharmacological delivery methods of HRT are not integrated into the hypothalamic-pituitary (HP) axis which would facilitate feedback and regulation of dosage and timing of circulating hormone levels. As such, pharmacological hormone delivery results in consistently higher serum concentrations as compared to those associated with normal functional ovaries.

New approaches using cHRT offer a potential physiological solution to timely control of hormone delivery and the ability to restore the functionality of the HPO axis. OTC can be used as cHRT as thawed ovarian tissue grafted into the pelvis cavity restores the natural hormone milieu and endocrine function of the ovary which have been consistently documented by a rise in estradiol and a decrease in FSH and LH levels returning to premenopausal levels 4–5 months after transplantation leading to cessation of menopausal symptoms and renewed menstrual cycles in the vast majority of transplanted patients (1, 10, 11). Restored hormone production may even be the desired effect rather than fertility restoration in some patients (10), and a more appealing alternative to pHRT for some women. In Denmark around a dozen young women who entered menopause due to cancer treatment have had frozen/thawed tissue transplanted only to become a "normal woman" again and avoid menopause (10), The average duration of graft function is approximately 5 years, but the function can persist for over 9 years (10), depending on the follicular density at the time of OTC (2). The transplantation procedure may be repeated multiple times in order to stretch the longevity of the tissue, if sufficient amounts of ovarian tissue has been stored, hereby extending the lifespan of ovarian endocrine activity up to 11 years or more (12, 13). If patients are merely in the need of sex steroid production, a heterotopic graft site like the abdominal wall or a subcutaneous site may be preferred over an orthotopic site as it requires less invasive surgery and would obviate the need for general anesthesia and a laparoscopic procedure. A cHRT approach combining a heterotopic graft site with multiple transplantations extending the longevity of banked ovarian tissue can therefore facilitate prolonged ovarian endocrine function and have implications way beyond fertility preservation.

## Inducing Puberty in Pre-pubertal POI Girls

Survivors of childhood cancer represent a rapidly growing population of patients in which temporary or permanent POI is a common side effect of the cytotoxic treatments. In addition, POI often has a genetic background with more than 50 genes in which mutations can be causative and many other genes that may be implicated (14). These genes can affect various processes such as gonadal development, DNA replication/ meiosis and DNA repair, hormonal signaling, immune function, and metabolism, and include conditions like Turner syndrome, sickle cell anemia, thalassemia, and

galactosemia. These young girls often experience delayed puberty or need pHRT to induce puberty to enable the pubertal growth spurt as well as development of secondary sexual characteristics. However, pharmacological delivery of increasing doses of estrogen followed with progesterone only address some aspects of puberty and does not completely match the physiological complexity of the hormonal milieu during puberty. Moreover, there is only limited data on the long-term safety of exogenous hormones in childhood cancer survivors and pHRT can cause potentially significant side effects (15, 16).

Cell/tissue-based HRT can therefore be an attractive approach to provide a more physiological hormonal milieu for induction of puberty in pre-pubertal POI girls. In 2012, Poirot and coworkers published the first case report showing that stored ovarian tissue from pre-pubertal girls can provide adequate endogenous sex steroid hormone levels to induce puberty (17). In this case, a 13-year old girl developed POI after being treated for sickle-cell disease at the age of 10, and after having 3 out of 23 stored ovarian pieces transplanted, she entered puberty. A second case report was published shortly after with another 13-year old girl who had developed POI following treatment for Ewing sarcoma at the age of 9 (18). In both cases, a unilateral oophorectomy was performed before treatment and a small proportion of the frozen ovarian cortical fragments (i.e., 10–20% of the stored tissue) were transplanted to a heterotopic location in the former case and to the remaining ovary in the latter. Puberty was successfully induced in both girls although the graft function failed after nearly 2 years.

It can be discussed whether or not young girls should choose to save their ovarian tissue for later use to achieve fertility instead (19), but using only a small percentage (i.e., 10–20%) of the stored tissue might be worth it for some young girls to feel "normal" during a critical period of their emotional and social life as the physical maturation process directly affects body and brain to alter children's needs, interests, and moods. Several studies have suggested that young childhood cancer survivors with POI face significant psychosexual dysfunction and that the symptoms of POI could severely stunt social growth and exacerbate anxiety and feelings of isolation (20, 21). Therefore, cHRT could potentially be beneficial in the long run at both the physiological and psychological level, and the preferred choice for some pre-pubertal POI girls in order for them to experience the life of every other teenager and becoming a woman with physiological levels of sex hormones. However, the preferred treatment for young girls with POI is still pHRT, as it is cheap, simple, and ready available and does not include any surgical intervention. Moreover, the theoretical superiority of physiological hormone levels over the pharmacological levels for induction of puberty has not been proven in any human clinical trials and awaits further research.

## Achieving or Postponing Natural Menopause

One century ago the average life expectancy corresponded to the natural age of menopause around the age of 50 years, however, nowadays the majority of women live beyond 80 years in many developed countries, and the demographic structure is changing toward an increasingly aging population in which women will spend 30–40% of their lives being postmenopausal. In combination with the increasing population of cancer survivors experiencing POI these developments in society calls for preventive strategies to alleviate and decrease short- and longterm consequences and health risks associated with the lack of ovarian endocrine function.

The use of pHRT has been vigorously debated for several decades. In the 1980's and 90's pHRT was administered to millions of women to relieve menopausal symptoms and by the mid-1990s estrogen became the biggest-selling prescription drug in the US. However, it all changed with the publication of the results from the Women's Health Initiative (WHI) randomized clinical trials in 2002 (22), suggesting pHRT to cause an increased risk of breast cancer, after which many women discontinued pHRT or avoided starting pHRT at all ages, including before age 50 years (23, 24). One of the big misunderstandings was that the results from the WHI studies were inappropriately extrapolated from women in the late postmenopausal stage (aged >60 years) to younger women in the early stage of postmenopause (50– 59 years), and even further to women experiencing premature or early natural menopause. The WHI study was built upon numerous observational studies and clinical trials consistently demonstrating benefits in the prevention of chronic diseases, which include reduced coronary heart disease (CHD) and mortality, when pHRT was initiated near the onset of menopause (25–30). The WHI study was therefore designed to test the effects of pHRT in women initiating treatment a decade or more after menopause. This is where the "timing hypothesis" comes into play and suggest that different clinical effects occurs if hormones are initiated close to the onset of menopause compared with several years later (31). Basic and animal studies together with clinical studies have now shown that the timing of pHRT can be crucial with respect to especially CHD, cognitive decline, and dementia (8, 31–34). In 2005, the Multi-Institutional Research on Alzheimer Genetic Epidemiology study showed that the risk of dementia was reduced in women who initiated hormonal therapy at age 50–63 years, but was not reduced in women who started hormonal treatment at ages 64–71 or 72–99 years (35).

Reassessment of clinical trials in women initiating pHRT close to the onset of menopause together with more recent studies and meta-analyses now show a long list of benefits with estrogen alone therapy, and risks are considered rare. Beyond symptomatic relief, improvements in quality of life and a reduction in osteoporosis, estrogen-based therapy has now been shown to be cost-effective and have a very favorable risk– benefit profile in healthy women under the age of 60. Specifically, estrogen alone therapy have been shown to consistently decrease CHD in women under 60 years of age by up to 40% and to decrease all-cause mortality by 20–40% (36–38). Moreover, a long-term follow up on the WHI study showed that estrogen alone therapy resulted in a significant decrease in the total risk of cancer by 20% in women aged 50–59 years (39).

The majority of studies show benefits with estrogen alone rather than with estrogen plus progestogen, however, no particular HRT regimen can currently be advocated (31). Moreover, the use of age-appropriate estrogen doses has been reported to be crucial to maximize cardiovascular benefits while minimizing risk of adverse effects such as venous thromboembolism and stroke (32). This is where cHRT comes into the picture and we hypothesize that transplantation of stored ovarian tissue could be used to restore endocrine ovarian function at the onset of menopause or for women entering menopause prematurely. In 2015, we proposed that ovarian tissue frozen in the young years could be transplanted back at the time of menopause to provide continued endogenous, physiological levels of steroid production to postpone menopause (40). A small portion of stored ovarian tissue may be transplanted subcutaneously during local anesthesia multiple times providing menstrual cycles for a prolonged period, using the woman's own tissue and follicles to sustain menstrual cycles with natural variations in the whole armamentarium of hormones. In this way, natural fertility will also be avoided and prevent senior women to conceive spontaneously. Recent animal studies have demonstrated that supraphysiological plasma levels of estrogen were required for pHRT to achieve benefits in bone health that were comparable to those achieved by cHRT (using ovarian cell constructs) at much lower plasma hormone concentrations (41). Such studies suggest a potential benefit of physiological secreted sex steroid hormones under the control of the HPO axis at which effects can be obtained at lower and safer plasma levels.

In theory, cHRT using OTC at a young age could be used as a physiological solution to prevent the massive medical legacy of osteoporosis and menopause-related conditions in the large population of aging women, however, currently the majority of women would probably not subject themselves to an elective surgery purely for delaying menopause and it can be argued whether or not any women would prefer to have regular menstrual cycles up to 60 years of age, when readily available pHRT compounds can deliver the hormones without the bleeding. Nevertheless, OTC could potentially be justified in some women already undergoing pelvic surgery for other reasons for example, a Caesarean section or an appendectomy where ovarian tissue could be collected and frozen as an adjunct. This is, however, also an approach which is highly debatable and may not be ethically and medically appropriate as no studies have been conducted that directly or indirectly compare the benefit and disadvantage of ovarian tissue removal for future cHRT.

The group of women for whom the use of cHRT are most likely to become a clinical option initially is the thousands of women worldwide who have already had ovarian tissue frozen for fertility preservation due to different malignancies and genetic conditions. For various reasons, a large number of these women will not have used their stored tissue by the time they reach menopause, either prematurely or at the natural age of menopause. Some of these women might wish to use their store tissue for postponing menopause or to achieve natural menopause which is nowadays recommended by many scientific societies (8, 31). Studies have shown that women who become postmenopausal before the age of 50 years and do not receive any treatment have an increased risk for cardiovascular diseaserelated mortality compared to women receiving pHRT (42–46). Other studies have shown an almost doubled long-term risk of cognitive impairment or dementia in women who underwent oophorectomy before menopause (47). A risk that was eliminated if estrogen therapy was initiated after the surgery and continued up to age 50 years or longer (47), which is advertising a need for sustained ovarian function up to at least the age of natural menopause.

However, many concerns and unanswered questions exist; Is it safe? To whom is the risk-benefit profile actually favorable? Are physiological hormones better than pharmaceutical? Careful consideration of risks and benefits, individually structured counseling and close monitoring are needed for each woman who may want to use their ovarian tissue for primary prevention or postponing menopause. To summarize, cHRT in the form of stored ovarian tissue or ovarian cell constructs (41, 48, 49) could potentially be used at the onset of natural or induced menopause as an ideal time to institute preventative strategies that could potentially increase the quality and length of women's lives. However, this is again technical possible but not proven in a clinical setting with women in this age group, and it can be argued that pHRT should be preferred in all cases as it is the cheapest and simplest way to provide hormones, and no real evidence for the superiority of physiological hormone levels exists. Therefore, more research in the area of physiological and pharmacological hormones is needed in order for this potential application to be adopted clinically in the future.

## OTC FOR NON-MEDICAL REASONS

In many countries women also have the opportunity to preserve their fertility due to personal reasons and hereby postpone childbearing. The increasing age of childbearing observed in most high-income countries is often depicted as a result of women selectively choosing to pursue a career and other life goals before having children, however, evidence now suggests that the primary reason is the lack of a partner who is willing to commit to parenthood (50, 51). The traditional method of fertility preservation for these social indications is vitrification of mature oocytes. In recent studies, it has become evident that most of the women who cryopreserve oocytes for non-medical reasons are in their mid to late 30 s, well educated, socio-economically advantaged and single (52–54). Data concerning the reproductive experiences and outcomes of this growing group of women is still very limited, but two severe issues with today's policy for nonmedical oocyte freezing have emerged. First of all, women are too old when they decide to store oocytes as the mean age at the time of freezing is around 37 years (53, 55), which may be deemed suboptimal (56). Additionally, one in five patients in this group is a low responder and combined with the advanced maternal age this results in one quarter of the women having fewer than 8–10 mature oocytes frozen which is considered necessary for a reasonable chance of success (57). The second issue is that the majority of the women with frozen oocytes do not come back to collect them and the utilization rate of the stored oocytes is currently below 10% (53, 55). Collectively, it appears that especially reproductive aged women may only to a limited extend benefit from this approach.

A proposition would be to use OTC as an alternative to oocyte vitrification in connection with fertility preservation for non-medical reasons. In contrast to mature oocytes, transplanted ovarian tissue will give the woman the opportunity to conceive spontaneously without IVF, which many women and their potential future partners would probably appreciate. If the woman ends up not needing the stored ovarian tissue for fertility, then the tissue is not wasted, but could instead be used as cHRT to alleviate postmenopausal symptoms later on or avoiding POI. Thus, OTC may serve multiple purposes depending on the need for either fertility or endocrine function and provide a better justification of such intervention. In addition, by storing ovarian tissue, and not just a fixed number of mature oocytes, a range of potential new treatments, which are currently being developed, might become available for these patients in the future. These treatments include in vitro follicle activation (58), culture of human preantral follicles (48, 59, 60), autologous transfer of mitochondria to oocytes (61), and in vitro maturation of immature oocytes (62).

OTC requires at least two surgical procedures to collect and graft ovarian tissue and it might be overwhelming and excessive for some patients. Therefore, OTC may only be a preferable option over oocyte vitrification for some groups of patients in particular women of advanced maternal age with a low ovarian reserve or poor responders which require multiple cycles of controlled ovarian stimulation. For these groups of patients, their future use of stored ovarian tissue could potentially be enhanced by in vitro follicle activation (IVA) in which the resting pool of follicles is activated mechanically and/or biochemically prior to transplantation. In 2013, Kawamura et al. were the first to test IVA in a clinical setting and enabled POI patients without menstrual cycles for several years to conceive with their own oocytes by activating a residual pool of resting follicles (58). This technique has so far resulted in three live births in Japan and two pregnancies in other clinics (58, 63–65).

Taken together, oocyte cryopreservation is the clinical available option for non-medical freezing, but it can be argued that women seeking fertility preservation for non-medical reasons should be presented with both fertility preserving options and the potential beneficial applications for cHRT and future use, and then the woman can help decide which method is most suitable for her. It should be highlighted that age is the most limiting and critical factor for both methods, and the success rate always depends hereof, which means that the issue with low ovarian reserve will apply for both methods, and low return rates are probably an inevitable factor in this group of women.

## A MODERN OVARIAN RESECTION FOR ANOVULATORY PCOS WOMEN

A completely new way of utilizing OTC is in the context of surgical treatment for anovulatory women with polycystic ovaries. Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders among women in their reproductive age and is a predominant cause of anovulatory infertility. In 1930s, Stein and Leventhal developed the ovarian wedge resection (OWR) in which the ovarian volume was surgically reduced during laparotomy (66). For decades OWR was the only treatment for PCOS as it effectively restored regular menstrual cyclicity for a period of time in 80–90% of women with PCOS and allowed conception in 45–65% of those who underwent surgery (66). However, the procedure later became less popular due to significant side effects; primarily a significant incidence of postoperative pelvic adhesion formation adding to the infertility-issue (67–69). Furthermore, follow-up studies revealed that OWR was not always curative and a significant number of women, 30–35%, reverted into a state of anovulation or oligo-ovulation (69, 70). With the introduction of clomiphene citrate (CC) and human menopausal gonadotropins in the 1960s the surgical treatment of PCOS was less used, and today CC is used as the first-line therapy for ovulation induction in women with anovulatory PCOS (71, 72). However, 10–15% of women are CC-resistant and will not ovulate in response to CC, and in those who do respond, not all will conceive. Laparoscopic ovarian drilling (LOD) using a unipolar electrode is currently recommended as a successful second-line treatment for ovulation induction in women with CC-resistant PCOS, as it has been shown to be just as effective as gonadotropin treatment, but less expensive and not associated with an increased risk of multiple pregnancy, ovarian hyperstimulation syndrome (OHSS) or pregnancy loss (71, 73–75). In the 1980's the first studies on LOD showed that ovulation was restored in 92% of patients, with a pregnancy rate of up to 80% (76). Furthermore LOD allows multiple attempts of conception, but without intensive monitoring, and is preferred by the majority of patients (75). The main shortcomings of LOD are postoperative adhesion formations and the potential risk of affecting the ovarian reserve in case of excessive damage (75).

We therefore hypothesize that the development of a modern version of an ovarian resection in which ovarian biopsies are excised surgically during laparoscopy and subsequently frozen could potentially provide an alternative to traditional LOD and OWR in CC-resistant women with PCOS. In this case, the surgical intervention should be performed in a way in which whole pieces of cortical and medullary tissue is removed in amounts similar to what is destroyed during LOD, and the ovarian cortex could then be isolated and frozen to preserve the follicles for potential later use instead of wasting them when performing LOD or OWR. Thus, in cases were too much tissue is removed during the surgical procedure the stored ovarian tissue can be grafted as back-up, or potentially be used for cHRT if the patient later on enters menopause prematurely or for fertility at an advanced reproductive age. In 1970's it was incidentally noted that ovarian biopsy alone (taken for diagnostic purposes) could induce regular menstrual cyclicity and conception in some women with PCOS (77), and in 1980's OWR was also performed through multiple ovarian biopsies and reported successful using a laparoscopic approach with less adhesion formations (78). Like LOD the proposed modern version of an ovarian resection through excision of ovarian biopsies would restore regular ovulation and allow spontaneous pregnancy, and in patients who remain anovulatory following the procedure most of them would have an increased responsiveness of the ovary to CCtreatment or respond less aggressively to exogenous hormone stimulation. Taken together, the proposed procedure using OTC to advance a surgical treatment for PCOS patients is completely theoretical and has not been tested in a clinical setting, however, this approach could potentially utilize the excessive—and in this case harmful—follicle pool in a beneficial way by relieving PCOS symptoms immediately and at the same time securing ovarian endocrine function later on in life. The efficacy and advantages of the proposed procedure now needs to be proven and compared to LOD in a clinical setting to justify any potential use in the future.

## OPTIMIZING CULTURE SYSTEMS FOR IMMATURE OOCYTES

By freezing ovarian cortical tissue for fertility preservation, it is possible to gain access to a wide range of immature oocytes, including early preantral and small antral stage follicles, which can be isolated and collected from surplus or donated ovarian tissues (62, 79). This provides a rare opportunity to characterize the basic molecular mechanisms controlling and regulating human follicular growth and maturation in unstimulated ovaries of fertile women (80, 81), and to develop and optimize culture conditions for immature oocytes (59, 62), which could provide additional fertility for young women with a wide range of indications. Thus, utilization of immature oocytes could in this way augment the overall fertility from the OTC procedure. Moreover, young cancer survivors with a risk of ovarian involvement from the underlying cancer cannot have their stored ovarian tissue transplanted safely, and current advances in culture systems for the earliest stages of follicles could potentially provide fertility for these women in the future. In a recent paper, Telfer et al. succeeded with in vitro growth of unilaminar follicles (IVG) in a multi-step culture model which supported the development and maturation to the Metaphase II stage (60).

Several studies have now shown that a considerable number of immature oocytes can be collected from small antral follicles visible on the surface of the ovary or released to the dissection medium during the preparation for OTC (62, 82–84). These oocytes can be matured, vitrified and used to augment fertility to the patient (62, 84–86). The maturation rate has been shown to vary between ∼30 and 60% (87), and the first three live births resulting from a cryopreserved embryo obtained from in vitro matured (IVM) oocytes has been reported (82, 88, 89). However, there is plenty of room for improvement and currently IVM is mainly used as an additional fertility preserving option in combination with OTC or to avoid OHSS in PCOS patient. A more widely use of IVM is currently not accepted due to the fact that implantation and developmental potential of embryos derived from IVM oocytes has consistently been reported to be significantly lower in comparison to in vivo matured oocytes (90, 91).

One explanation for the inefficiency of IVM is that IVM protocols have changed little, if any, since the first reported birth obtained by IVM oocytes in 1994 using a non-specific medium (MEM) with addition of FSH, LH and estradiol as hormones and maternal serum as supplements (92). In recent reports, commercial maturation media have been used for IVM in both patients with PCOS and women undergoing fertility preservation, but there is no real evidence that the formula is appropriate for oocyte maturation in vitro (91). Thus, IVM in connection with OTC provides a unique platform to compare multiple culture media compositions and improve basal maturation conditions for IVM. Another explanation for the decreased implantation and pregnancy rate with the use of IVM derived embryos is insufficient endometrial receptivity. The limited time between oocyte recovery and embryo transfer is potentially insufficient to allow full completion of the endometrial proliferative phase, which could compromise the formation of a secretory endometrium and the chances of embryonic implantation (91). A study by De Vos et al. has shown that implantation and pregnancy rates were much higher in replacement cycles using frozen embryos obtained after IVM compared to cycles in which embryos were transferred fresh after IVM (93). Thus, the relatively lower clinical efficiency that currently characterizes IVM might derive not exclusively from reduced oocyte quality caused by inadequate IVM conditions, but also from inadequate endometrial receptivity. Therefore, the notion that IVM oocytes are intrinsically less developmentally competent will need to be reconsidered.

Despite the current limitations, IVM continue to attract growing interest in consideration of potential novel applications in human ART. However, to advance more sophisticated IVM systems able to reproduce more physiologically and efficiently the process of oocyte maturation, we need more research material and settings such as provided by OTC, in which we can gain a better understanding of the biological mechanisms transforming a fully-grown oocyte into a mature fertilizable gamete.

## CONCLUSION

OTC in prepubertal girls and young women is becoming an increasingly well-established method of fertility preservation in

## REFERENCES

many clinics worldwide. However, by storing ovarian tissue more than just the reproductive potential is preserved. The steroidogenic capacity and endocrine function of the tissue could potentially expand the utilization of OTC and the ovarian reserve to other beneficial applications throughout the female lifespan and several indications beyond cancer. With an increasing prevalence in loss of ovarian function due to POI and aging we now need to explore these opportunities and outweigh the cost-benefit and risk-benefit ratios associated with potential new treatments. Nonetheless, it is important to recognize that fertility and endocrine function restored with ovarian tissue go hand in hand and that both are the result of follicular activity.

In conclusion, the described and suggested applications for OTC in this review are technically possible, however they have not been validated clinically (with the exception of puberty induction), and therefore the suggested applications should be regarded as an optimistic view on the potential future use of OTC. Moreover, the suggested applications for OTC are thought provoking to many people and the controversial opinions hereof should of course be recognized. Finally, the burden of repeated surgical interventions in connection with OTC must in all cases be weighed against the simple, cheap, and readily available pHRT medications which already exist and are administered to millions of women worldwide.

## AUTHOR CONTRIBUTIONS

SK and CA both contributed to the conception and writing of this paper.

## ACKNOWLEDGMENTS

The Research Pools of Rigshospitalet, The ReproUnion network, and the Novo Nordisk foundation is thanked for financial support.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kristensen and Andersen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neuropeptide G Protein-Coupled Receptors as Oncotargets

Terry W. Moody 1 \*, Irene Ramos-Alvarez 2 and Robert T. Jensen2

1 Department of Health and Human Services, National Cancer Institute, Center for Cancer Research, National Institute of Diabetes, Digestive, and Kidney Disease (NIDDK), Bethesda, MD, United States, 2 Digestive Diseases Branch, National Institute of Diabetes, Digestive, and Kidney Disease (NIDDK), Bethesda, MD, United States

Neuropeptide G protein-coupled receptors (GPCRs) are overexpressed on numerous cancer cells. In a number of tumors, such as small cell lung cancer (SCLC), bombesin (BB) like peptides and neurotensin (NTS) function as autocrine growth factors whereby they are secreted from tumor cells, bind to cell surface receptors and stimulate growth. BB-drug conjugates and BB receptor antagonists inhibit the growth of a number of cancers. Vasoactive intestinal peptide (VIP) increases the secretion rate of BB-like peptide and NTS from SCLC leading to increased proliferation. In contrast, somatostatin (SST) inhibits the secretion of autocrine growth factors from neuroendocrine tumors (NETs) and decreases proliferation. SST analogs such as radiolabeled octreotide can be used to localize tumors, is therapeutic for certain cancer patients and has been approved for four different indications in the diagnosis/treatment of NETs. The review will focus on how BB, NTS, VIP, and SST receptors can facilitate the early detection and treatment of cancer.

\*Correspondence: Terry W. Moody<EMAIL_ADDRESS>
Carsten Grötzinger,

Edited by: Derek LeRoith,

Reviewed by: Erika Peverelli,

Germany

Icahn School of Medicine at Mount Sinai, United States

Università degli Studi di Milano, Italy

Charité Universitätsmedizin Berlin,

#### Specialty section:

This article was submitted to Systems and Translational Endocrinology, a section of the journal Frontiers in Endocrinology

Received: 15 May 2018 Accepted: 11 June 2018 Published: 29 June 2018

#### Citation:

Moody TW, Ramos-Alvarez I and Jensen RT (2018) Neuropeptide G Protein-Coupled Receptors as Oncotargets. Front.  The activated GPCRs undergoes a conformation change dissociating the G-protein into a GTP-bound α subunit and β, γ dimer. GPCRs for bombesin (BB) and neurotensin (NTS) interact with Gq/11, whereas receptors for vasoactive intestinal peptide (VIP) interact with Gs and somatostatin (SST) receptors interact with Gi/0 (2). BB and NT receptors cause phosphatidylinositol (PI) turnover resulting in the elevation of cytosolic Ca2+ and activation of protein kinase (PK) C. VIP receptors activate adenylyl cyclase resulting in elevated cAMP whereas SST receptors reduce the elevation of cAMP stimulation caused by VIP.

Neuropeptides modulate neural activity in the brain in a paracrine manner, however, they function as autocrine growth factors in cancer (3). BB, NTS, and VIP stimulate the growth of small cell lung cancer (SCLC) cells whereas SST inhibits growth (3). BB and the structurally related gastrin-releasing peptide (GRP) bind with high affinity to the GRP receptor or BB2R; NTS binds high affinity to NTSR1; VIP binds with high affinity to VPAC1/VPAC2 and SST as well as octreotide/lanreotide bind with high affinity to SSTR2/SSTR5 but reduced affinity to SSTR1, SSTR3, and SSTR4. The agonist-GPCR complex is internalized and the GPCR recycle to the membrane but the peptide is metabolized in lysosomes. Because cancers frequently over-express GPCRs, the cancer GPCRs can be used to deliver neuropeptide-drug conjugates into the cancer cell (4). In contrast, GPCR antagonists bind to the GPCR at the cell surface but are not internalized. PD176252 (GRPR antagonist) and SR48692 (NTSR1 antagonist) inhibit cancer growth (5–7). This review will focus on how neuropeptide GPCRs are oncotargets for the early detection and treatment of cancer.

## GRPR, NEUROMEDIN B RECEPTOR AND BBR SUBTYPE 3

SCLC has high levels of the BB-like peptide GRP (8, 9). GRP is derived from a 148 amino acid prepropeptide (10). After removal of the N-terminal 23 amino acid signal sequence, pro-GRP1−125 is metabolized by a prohormone convertase to GRP, which contains 27 amino acids and has an amidated C-terminal. GRP (proGRP1−27) is readily metabolized in the blood but SCLC patients have elevated pro-GRP (11). Antibodies to proGRP31−98 have been used to detect high concentrations of proGRP (>100 pg/ml) in the serum of patients with SCLC. Because proGRP is elevated in the serum of 71% of the SCLC patients it may be a biomarker for SCLC (12). BB or GRP, but not proGRP bind with high affinity to the GRPR. The C-terminal octapeptide of BB or GRP can be neutralized by mAb 2A11. mAb2A11 inhibits the growth of SCLC in vitro and in mouse models in vivo (13). In a clinical trial, 2A11 was well tolerated and one patient had SCLC remission whereas four patients had stable disease out of 13 patients treated (14). The results indicate that the GRP precursor may be a biomarker for SCLC.

**Table 1** shows that the GRPR, which is localized to chromosome xp22, contains 384 amino acids and is a member of the class A/Rhodopsin-like GPCR (15, 16). The neuromedin B (NMB) R or BB1R, which is localized to chromosome 6q24, contains 390 amino acids whereas BB receptor subtype-3 (BRS-3), which is localized to chromosome xq26, contains 399 amino acids. The NMBR and BRS-3 have about 50% sequence homology with the GRPR (17, 18). The GRPR binds GRP and NMB with high and low affinity, respectively. The NMBR binds GRP and NMB with low and high affinity, respectively. The orphan receptor BRS-3 binds both GRP and NMB with low affinity but MK5046 binds with high affinity (7). The universal agonist BA1, (D-Tyr6 , β-Ala11, Phe 13, Nle14) BB6−14, binds with high affinity to the GRPR, NMBR, and BRS-3. Numerous amino acids in TM domains 6 and 7 as well in extracellular loops (EL) 1, 2, and 3 of the GRPR are essential for high affinity binding of GRP (4). While the GPCRs of each family have a similar sequence, the pharmacological profile is different.

BB-drug conjugates were synthesized which are cytotoxic for lung cancer cells. The topoisomerase-1 inhibitor camptothecin (CPT) was coupled with a linker to the N-terminal of BA1. Surprisingly, the resulting CPT-L2-BA1 bound with higher affinity to the GRPR, NMBR, and BRS-3 than did BA1 (19). CPT-L2-BA1 was an agonist which increased PI turnover and was internalized. The CPT-L2-BA1 was metabolized in the lysosome leading to the release of CPT (20). Also, BB agonists have been coupled to paclitaxel (21), doxorubicin (22), marine toxins (23), magainin II (24), and siRNA to the GRPR (25) resulting in decreased cancer cellular proliferation. Doxorubicin was coupled to a GRPR antagonist and the resulting AN-215 was cytotoxic for gastric, colon, lung, ovarian, endometrial, breast, and pancreatic cancer (26). RC-3095, a GRPR antagonist, was tested in 25 patients with solid tumors. RC-3095 had minimal toxicity but a partial response was only seen in 1 patient (27).

BBR antagonists were developed which inhibit the growth of cancer cells. Peptide antagonists such as RC-3095 or (Psi13,14 , Leu14)BB blocked the GRPR, and they inhibited the growth of cancer cells (27, 28). Small molecule antagonists such as PD168368 were synthesized which inhibit the growth of cancer cells which have NMBR (6). Also, bantag-1 is a peptide antagonist for BRS-3 (29). The BB receptor antagonists inhibited the growth of lung cancer cells in vitro and in vivo using nude mice bearing lung cancer xenografts. GRPR, NMBR, and BRS-3 mRNA was detected in 11/13 lung cancer cell lines (7). All lung cancer cell lines tested had at least 1 type of BBR and many cell lines had all 3 receptors.

In contrast, a high density of GRPR but not NMBR or BRS-3 were detected in most prostate and breast cancer cells (30). GRPR agonists were labeled with 111In, 64Cu, 99mTc, 68Ga, 18F for imaging studies. Using a 99mTc-BB2−14 analog 14 prostatic lesions were visualized in patients (31). Using a 99mTc-RGD-BB analog, tumors were visualized in 6/6 breast cancer patients (32). Using a 64Cu-BB6−14 analog, tumors were visualized in 3 of 4 prostate cancer patients (33). It remains to be determined if the imaging of GRPR will be useful in the early detection of breast and/or prostate cancer.

Many of the growth effects of BB-like peptides on non-SCLC (NSCLC) cells may result from transactivation of receptor tyrosine kinases (RTK) such as the epidermal growth factor receptor (EGFR). Activation of the NMBR in NSCLC cells causes PI turnover leading to increased phosphorylation of the EGFR (**Figure 1**). Addition of NMB to NSCLC cells increases the tyrosine phosphorylation of the EGFR after 1 min leading to the tyrosine phosphorylation of ERK after 2 min (34). The transactivation of the EGFR that is regulated by the NMBR is inhibited by the tyrosine kinase inhibitor (TKI) gefitinib or the NMBR antagonist PD168368. The transactivation process in NSCLC cells is mediated by the EGFR ligand transforming growth factor (TGF)α (**Figure 1**). The inactive precursor pro-TGFα is metabolized by matrix metalloprotease (MMP) enzymes in the membrane to biologically active TGFα which is secreted and binds to the EGFR. The transactivation of the EGFR caused by addition of NMB to NSCLC cells is inhibited by GM6001 (MMP inhibitor) or anti-TGFα Ab. The transactivation process requires reactive oxygen species (ROS). Addition of N-acetyl cysteine (antioxidant) or tiron (superoxide scavenger) impaired the ability of NMB to increase EGFR tyrosine phosphorylation. The ROS may oxidize Cys773 of the EGFR increasing its tyrosine kinase activity and/ or oxidize protein tyrosine phosphatases (PTP) impairing their ability to metabolize phosphotyrosine (35, 36). The results indicate that GPCRs

#### TABLE 1 | Peptide GPCRs (human).

PACAP interact with both Gq and Gs. GPCR activate Src and MMP resulting in the production of EGFR ligands such as TGFα. When TGFα binds to the EGFR, tyrosine kinase activity is increased leading to phosphorylated EGFR homodimers or EGFR-HER2 heterodimers. The RTK activates the Ras-Raf-MEK-ERK pathway leading to increased cellular proliferation. The RTK activates the PI3K-PKD-AKT-mTOR pathway leading to increased cellular survival. The phosphorylated EGFR/HER2 is dephosphorylated by protein tyrosine phosphatase (PTP).

regulate the transactivation of receptor tyrosin kinases (RTKs) in NSCLC cells.

The EGFR contains 1,186 amino acids with a 621 and extracellular domains I and III bind EGF or TGFα with high affinity (37). The EGFR has a 23 amino acid TM domain and a 542 intracellular domain with tyrosine kinase activity. Lys721 is essential for binding ATP and the phosphorylation of protein substrates. Upon binding of ligand, the EGFR can undergo homodimerization resulting in the phosphorylation of Tyr1068 , Tyr1086, Tyr1148, and Tyr1174. Alternatively, the EGFR can form heterodimers with HER2. The MAPK and PI3K/Akt pathways are downstream of the EGFR and are important for EGFR mediated proliferation and cancer cellular survival, respectively. Currently, we are investigating if GPCRs transactivate additional RTK such as HER2, HER3 or HER4 in cancer cells.

NMB increases the proliferation of NSCLC cells. In contrast, PD168368 and gefitinb inhibit the growth of NSCLC cells (34). Surprisingly, combinations of the NMBR antagonist with the EGFR TKI reduced the proliferation of NSCLC cells in a synergistic manner. The results indicate that GPCR antagonists potentiate the action of TKI in NSCLC. Traditionally NSCLC which kills 130,000 U.S. citizens annually is treated with combination chemotherapy, however, the 5 year survival rate is only 15%. The EGFR is mutated is approximately 13% of the NSCLC patients and those with the L858R mutation have increased tyrosine kinase activity and sensitivity to TKI such as gefitinib or erlotinib (38, 39). Traditionally NSCLC patients are treated with combination chemotherapy, however, the 5 year survival rate is only 15% (40).

## NEUROTENSIN RECEPTORS

NTS is present in numerous SCLC cell lines (9, 41). NTS is derived from a 170 amino acid precursor and metabolized to a biologically active peptide which contains 13 amino acids (42). NTS and its C-terminal fragment NTS8−13 bind with high affinity to the NTSR1, which is localized to chromosome 20q13, contains 418 amino acids, and is a class A/Rhodopsin-like GPCR. **Table 1** shows that the NTSR2 which is on chromosome 2p25 contains 410 amino acids and binds NTS and levocabastine with high affinity. The NTS−-NTSR1 complex has been crystallized and NTS8−13 sits on top of the NTS binding pocket and interacts with TM domains 6 as well as 7 and EL 2 as well as 3 (43). Both NT and BB receptors have short N-terminals which have little effect on ligand binding (44). The nonpeptide NTSR1 antagonist SR48692 binds deep into the NTSR1 binding pocket and blocks the effects of NTS agonists. Also, SR142948A blocks both the NTSR1 and NTSR2. The NTSR3 is not a GPCR but is sortilin.

NTS binds with high affinity to SCLC cells (45). Addition of NTS to cancer cells causes PI turnover leading to increases PKC activity and elevation of cytosolic Ca2+ (46–48). In contrast, the NTSR2 agonist levocabastin has little effect on lung cancer cells. The effects of NTS on second messenger production and proliferation was antagonized by SR48692 (5). NTS addition to cancer cells causes phosphorylation of various proteins such as focal adhesion kinase (FAK) or ERK (49, 50). The phosphorylated ERK increasesthe expression of c-fos and c-jun leading to cellular proliferation (51). NTS stimulates proliferation whereas SR48692 inhibits the proliferation of lung cancer cells (5). NT addition to NSCLC cells increased EGFR tyrosine phosphorylation 5-fold (52). NT (5 nM) half-maximally increased EGFR transactivation after 2 min. NTS or NTS8−13 but not NT1−8 or levocabastine increase EGFR tyrosine phosphorylation. The NTSR1 regulation of EGFR transactivation is inhibited by SR48692, gefitinib, PP1, GM6001, TGFα antibodies and antioxidants. SR48692 and gefitinib inhibit the proliferation of NSCLC cells in a synergistic manner. Previously, JMV449, a NT8−13 analog, was found to increase expression of the EGFR, HER2, and HER3 after 24 h (53). JMV449 addition to cells increase MMP activity resulting in HB-EGF and neuregulin 1 release, which activates the EGFR and HER3, respectively.

NTSR1 regulates the EGFR transactivation in numerous cancers including colon, foregut neuroendocrine, lung, and prostate cancer (47, 52, 54, 55). Lung cancer and gastric cancer patients whose tumors had high densities of NTSR1 had decreased survival (53, 56). Addition of NTS to NSCLC cells caused tyrosine phosphorylation of the EGFR in a PLCdependent manner (52). Phosphorylated β-catenin dissociates from E-cadherin and increases the expression of NTSR1. Wnt/βcatenin signaling increases the expression of E-cadherin leading to epithelial to mesenchymal transitions and cancer metastasis (57). Recently, 3BP-227, a SR142948A analog, was radiolabeled and used to image tumors containing NTSR1. In nude mice containing HT29 colon cancer tumors 177Lu-3BP-227 localized to the tumors with high tumor-to-kidney or tumor-to-liver ratios using whole-body SPECT/CT techniques (58). In 5 out of 6 patients with ductal pancreatic adenocarcinoma tumor uptake of 177Lu-3BP-227 was observed (59). It remains to be determined if 177Lu-3BP-227 will improve survival of patients whose tumors are enriched in NTSR1.

## VIPRs AND PITUITARY ADENYLATE CYCLASE ACTIVATING POLYPEPTIDE RECEPTOR

The biological activities of the VIP and PACAP family of peptides are mediated by 3 GPCR (VPAC1, VPAC2, and PAC1), which are members of the classB/secretin-like receptors (60). **Table 1** shows that VPAC1, which is localized to chromosome 3p22, contains 457 amino acids with a 112 amino acid N-terminal. VPAC2, which is localized to chromosome 7q36, contains 438 amino acids with a 103 amino acid N-terminal. PAC1, which is localized to chromosome 7p14, contains 468 amino acids with a 125 amino acid N-terminal. PAC1 has about 50% sequence homology with VPAC1 or VPAC2 (60). The large N-terminal extracellular domain of PAC1 has antiparallel β-sheets and binds to the C-terminal of PACAP (61, 62). The PAC1 receptor has 3 closed transitional states (G1-G3) and one open state named G4 (63). The N-terminal of PACAP, which activates PAC1 binds to EL and TM domains (64). VPAC1, VPAC2, and PAC1 interact with Gs resulting in elevated cAMP, however, PAC1 interacts with Gq as well resulting in PI turnover (65). VIP, which contains 28 amino acids, is derived from a 170 amino acid precursor protein. PACAP-27 as well as PACAP-38 is derived from a 176 amino acid precursor protein and 67% of the amino acids in PACAP-27 and VIP are identical (60). VPAC1 and VPAC2 binds VIP and PACAP-27 or PACAP-38 with high affinity, whereas PAC1 binds PACAP-27 or PACAP-38 with high affinity but VIP with low affinity. Maxidillin, a 61 amino acid peptide isolated from sand fly, binds with high affinity to PAC1 but not VPAC1 or VPAC2 (66). Recently, a number of PACAP-38 analogs were synthesized which prefer PAC1 relative to VPAC1 or VPAC2 by over an order of magnitude (67). VIPhybrid is a peptide antagonist which binds with moderate affinity to VPAC1 or VPAC2, whereas, PACAP(6– 38) is a peptide antagonist for PAC1 (68). Selective non-peptide antagonists for VPAC1, VPAC2 or PAC1 remain unknown.

VPAC1 is present in numerous cancers including breast, colon, liver, lung, neuroblastoma, pancreatic, and prostate cancers in high densities (69). VPAC2 is present in moderate densities in gastric pancreatic adenocarcinomas, gastric leiomyomas, thyroid cancer, and sarcomas (70). PAC1 is present in brain, breast, colon lung, neuroendocrine, pancreatic, pituitary, and prostate cancer as well as neuroblastoma/pheochromocytoma (71). In SH1SY5Y neuroblastoma cells, numerous PAC1 splice variants (SV) were detected in the N-terminal and intracellular loop (IL) 3 (72). PAC1 has 18 exons and deletion of exons 5,6 or 4-6 reduce the N-terminal by 7, 21 (short) or 57 amino acids (very short) (73). The short PAC1 but not the very short PAC1 bind PACAP-38 with high affinity and elevate cAMP (74). Alternative splice variants (SV) of IL3 result in the addition of an additional 28 amino acid segment (hip) to PAC1null (75). Addition of a different set of 28 amino acids to IL3 of the PAC1 results in the hop SV. Finally, both SVs can be added resulting in PAC1hiphop. The order of potency to increase PI turnover was PAC1hop > PAC1null = PAC1hiphop > PAC1hip (76). Thus binding of PACAP and second messenger production can be altered by PAC1 deletions and SVs.

VPAC1 can be utilized to image cancer tumors. 18F(Arg15,21)VIP localized to T47D breast cancer cells in nude mice (77) and 64Cu-TP3982 localized to mammary tumors in MMTVneu transgenic mice (78). 99mTc-TP3982 was used to image breast tumors in 5 patients (79). The VPAC1-agonist complex internalizes in cancer cells and the ligand is metabolized in lysosomes. VPAC1 has been used to deliver VIP analogs containing cytotoxic CPT, paclitaxel, ellipticin or geldanomycin to cancer cells (80–83). The actions of VIP are antagonized by peptides such as VIP10−28 or VIPhybrid (84). Addition of VIP to cancer cells results in elevated cAMP which activates PKA. Activation of PKA results in CREB phosphorylation which increases nuclear oncogene expression of c-Myc leading to increased proliferation (**Figure 1**). VIP increases the proliferation of lung cancer cells whereas VIPhybrid inhibits proliferation (84). Addition of PACAP-27 or PACAP-38 to lung cancer cells containing VPAC1, VPAC2 or PAC1 increases cAMP, however, it causes PI turnover in cells containing PAC1. When PI is metabolized, ERK becomes phosphorylated. Phosphorylated ERK increased the expression of the nuclear oncogenes c-fos and c-jun leading to increased cancer cellular proliferation. PACAP(6–38) inhibits the proliferation of lung cancer cells in vitro and in vivo (85).

VIP may be a promoter of carcinogenesis. VPAC1 density is higher in mammary cancer than adjacent normal tissue using rat and mouse models (86). Specific binding of 125I-VIP to mouse mammary tumors was inhibited with high affinity by (Lys15, Arg16, Leu17) VIP1−7GRF8−27 (VPAC1 peptide agonist) but not Ro25-1553 (VPAC2 peptide agonist).  Finally VIPhybrid inhibits mammary carcinogenesis in C3(1)SV40T antigen mice (89).

Addition of PACAP-27 or PACAP-38 but not VIP causes transactivation of the EGFR in NSCLC cells (90). The PAC1 regulation of EGFR tyrosine phosphorylation is inhibited by PACAP(6–38), gefitinib, PP2, GM6001, and ROS inhibitors. Diphenyleneiodonium (DPI), a NADPH oxidase (NOX) inhibitor impaired the ability of PACAP to increase EGFR tyrosine phosphorylation. NOX-4, which produces ROS, is present in NSCLC cells (91). PACAP-27 addition to NSCLC cells increased ROS which was inhibited by DPI. VIP addition to breast cancer cells increased EGFR and HER2 phosphorylation (92). The EGFR which dimerizes, may form homodimers with itself or heterodimers with HER2. VPAC1 regulation of EGFR transactivation was blocked by JV-1-53 (VPAC1 antagonist), PP2 or H89 (PKA inhibitor). In contrast, the PAC1 regulation of EGFR transactivation in NSCLC cells was inhibited by U73122 (phospholipase C inhibitor) but not H89. The results indicate that the EGFR can be transactivated by GPCR which interact with Gq or Gs.

## SST RECEPTORS

SST occurs endogenously in two principal forms (SST-14, SST-28) and their action is mediated by 5 related subtypes of GPCRs (SSTR1-5) (93, 94). SST receptors are not only widely expressed on normal tissues, but also are frequently overexpressed by many neoplasms, particularly NETs [i.e., carcinoids/pancreatic neuroendocrine tumors (panNETs)] (93–95). SST has a wide range of physiological actions and they are primarily inhibitory (93, 94).

SST and its receptors represent the prototype for a clinically successful peptide/peptide receptor oncotarget. It is the only peptide-GPCR system which has multiple approved indications (four different indications) for the treatment of a class of human neoplasms, NETs. Furthermore, its results in NETs have potential applicability for its clinical utility in a number of other neoplasms.

The initial approved indication for SST analogs was its use in hormone-excess states. Depot long-acting formulations of synthetic SST analogs (octreotide-LAR, lanreotide autogel) (**Figure 2**) are the drugs of choice to control various functional NET syndromes due to the ectopic release of a biologically active peptides by the NETs (93, 96–99). This includes the control of such widely different functional NET syndromes as the carcinoid syndrome (diarrhea, flushing) due to metastatic carcinoid tumor; severe diarrhea due to VIPomas; rash due to glucagonomas; acromegaly due to excessive growth hormone release primarily by pituitary adenomas, and a number of others (93, 96–100). Almost all (>90%) of the well-differentiated forms of these NETs (>95%) overexpress somatostatin receptor subtype 2 which has high affinity for octreotide and therefore it is generally effective in controlling the hormone-excess state and in many, their growth. Some NETs such as pituitary adenomas do not overexpress SSTR2 with the result that octreotide/lanreotide are only effective in 20–70% of patients (101). One solution to this has been the development of next generation somatostatin analogs such as pasireotide, which has high affinity for multiple subtypes (SSTR5 > SSTR2 > SSTR3 > SSTR1), which has been shown to be effective in these patients and is approved for use in the treatment of a acromegaly.

The second approved indication for SST analogs is for its antiproliferative activity on NETs. In numerous pre-clinical studies and animal studies, it was shown that SST analogs have antiproliferative effects on NETs, as well as number of other human tumors (102–107) Two double-blind Phase 3 studies (108, 109) in patients with advanced NETs treated with lanreotide/octreotide increased the patient's progressive free survival (PFS), which lead to FDA approval. Recent meta-analyses (106, 110) of SST analogs anti-proliferative effects in all publications (106) or the above two studies (110), in patients with advanced NETs, demonstrate

good anti-proliferative activity, significant benefit from their use resulting in disease control (HR 0.51, p < 0.01), with the response rates vs. placebo being 58 vs. 32% and a good safety profile. In general, these studies demonstrate that SST analog treatment in patients with advanced NETs result in a tumoristatic effect primarily, rather than a decrease in the tumor size. SST analogs are now recommended in recent guidelines as well as expert reviews as one of the initial treatments for controlling tumor growth in patients with advanced NETs, especially those with well-differentiated NETs, and slower growth rates (99, 111–115). Numerous in vitro and animal studies report that SST receptors are expressed on a number of other non-endocrine tumors, and that SST analogs have anti-proliferative activity in these tumors (93, 94, 97, 103, 104, 107, 116, 117). No controlled trials have established the use of SST analogs for anti-proliferative effects in patients with non-endocrine tumors.

The third approved indication for SST analogs in patients with NETs is for imaging of the tumor. SST receptor imaging (SRI) was originally approved for the use of 111In-labeled pentetreotide with SPECT/CT scanning (Octreoscan), which is now replaced by the use of 68Ga-DOTATATE PET/CT scanning, which has greater resolution, sensitivity and high specificity (118–122). Almost all well differentiated NETs overexpress the somatostatin receptor subtypes (SSTR2 > SSTR5 > SSTR3) that bind this radiolabeled agonist with high affinity (118, 119, 123). A systematic review (122) demonstrated that the use of 68Ga-DOTATATE PET/CT scanning changed the management of the patient in a mean of 44% (range, 16–71%). SRI is now essential for the staging and management of NET patients and is the most sensitive method to allow whole body scanning rapidly to present a complete assessment of the extent of the tumor (112, 118, 121).