Alex C. Kim

Preclinical Targeting of the Type I Insulin-Like Growth Factor Receptor in Adrenocortical Carcinoma

CONTEXT Drug therapy for adrenocortical carcinoma (ACC), a rare and lethal malignancy, is largely empirical and ineffective. New treatments directed at molecular targets critical to the pathophysiology of ACC may prove more efficacious. OBJECTIVE The objective of the study was to profile human adrenal tumors and ACC cell lines to assess activated IGF signaling and determine the efficacy of two IGF receptor (IGF-1R) antagonists alone and in combination with mitotane. EXPERIMENTAL DESIGN ACC cell lines that display or lack activated IGF signaling are used to assess the effects of two IGF-1R antagonists in cultured cells and ACC xenograft tumors. RESULTS Transcriptional profiling data derived from DNA microarray analysis of human adrenal tumors implicate IGF2 as the single highest up-regulated transcript in the vast majority of carcinomas. We show that the majority of ACC cell lines tested display constitutive IGF ligand production and activation of downstream effector pathways. Both IGF-1R antagonists cause significant dose-dependent growth inhibition in ACC cell lines. Furthermore, we observe that mitotane, the first-line adrenolytic drug used in patients with ACC, results in enhanced growth inhibition when used in combination with the IGF-1R antagonists. We next examined the activity of IGF-1R antagonists against ACC xenografts in athymic nude mice. IGF inhibition markedly reduced tumor growth greater than that observed with mitotane treatment, and combination therapy with mitotane significantly enhanced tumor growth suppression. CONCLUSION These findings establish a critical role of IGF signaling in ACC pathophysiology and provide rationale for use of targeted IGF-1R antagonists to treat adrenocortical carcinoma in future clinical trials.

Targeted disruption of β-catenin in Sf1-expressing cells impairs development and maintenance of the adrenal cortex

The nuclear receptor steroidogenic factor 1 (Sf1, Nr5a1) is essential for adrenal development and regulates genes that specify differentiated adrenocortical function. The transcriptional coactivator β-catenin reportedly synergizes with Sf1 to regulate a subset of these target genes; moreover, Wnt family members, signaling via β-catenin, are also implicated in adrenocortical development. To investigate the role ofβ -catenin in the adrenal cortex, we used two Sf1/Cre transgenes to inactivate conditional β-catenin alleles. Inactivation of β-catenin mediated by Sf1/Crehigh, a transgene expressed at high levels, caused adrenal aplasia in newborn mice. Analysis of fetal adrenal development with Sf1/Crehigh-mediated β-catenin inactivation showed decreased proliferation in presumptive adrenocortical precursor cells. By contrast, the Sf1/Crelow transgene effected a lesser degree of β-catenin inactivation that did not affect all adrenocortical cells, permitting adrenal survival to reveal age-dependent degeneration of the cortex. These results define crucial roles for β-catenin - presumably as part of the Wnt canonical signaling pathway - in both embryonic development of the adrenal cortex and in maintenance of the adult organ.

In search of adrenocortical stem and progenitor cells.

Scientists have long hypothesized the existence of tissue-specific (somatic) stem cells and have searched for their location in different organs. The theory that adrenocortical organ homeostasis is maintained by undifferentiated stem or progenitor cells can be traced back nearly a century. Similar to other organ systems, it is widely believed that these rare cells of the adrenal cortex remain relatively undifferentiated and quiescent until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Historical studies examining cell cycle activation by label retention assays and regenerative potential by organ transplantation experiments suggested that the adrenocortical progenitors reside in the outer periphery of the adrenal gland. Over the past decade, the Hammer laboratory, building on this hypothesis and these observations, has endeavored to understand the mechanisms of adrenocortical development and organ maintenance. In this review, we summarize the current knowledge of adrenal organogenesis. We present evidence for the existence and location of adrenocortical stem/progenitor cells and their potential contribution to adrenocortical carcinomas. Data described herein come primarily from studies conducted in the Hammer laboratory with incorporation of important related studies from other investigators. Together, the work provides a framework for the emerging somatic stem cell field as it relates to the adrenal gland.

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and β-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal β-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal β-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized β-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized β-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.

Adrenocortical cells with stem/progenitor cell properties: Recent advances

The existence and location of undifferentiated cells with the capability of maintaining the homeostasis of the adrenal cortex have long been sought. These cells are thought to remain mostly quiescent with a potential to commit to self-renewal processes or terminal differentiation to homeostatically repopulate the organ. In addition, in response to physiologic stress, the undifferentiated cells undergo rapid proliferation to accommodate organismic need. Sufficient adrenocortical proliferative capacity lasting the lifespan of the host has been demonstrated through cell transplantation and enucleation experiments. Labeling experiments with tritium, BrdU, or trypan blue, as well as transgenic assays support the clonogenic identity and location of these undefined cells within the gland periphery. We define undifferentiated adrenocortical cells as cells devoid of steroidogenic gene expression, and differentiated cells as cells with steroidogenic capacity. In this review, we discuss historic developmental studies together with recent molecular examinations that aim to characterize such populations of cells.

Genetic p53 deficiency partially rescues the adrenocortical dysplasia phenotype at the expense of increased tumorigenesis.

Telomere dysfunction and shortening induce chromosomal instability and tumorigenesis. In this study, we analyze the adrenocortical dysplasia (acd) mouse, harboring a mutation in Tpp1/Acd. Additional loss of p53 dramatically rescues the acd phenotype in an organ-specific manner, including skin hyperpigmentation and adrenal morphology, but not germ cell atrophy. Survival to weaning age is significantly increased in Acd(acd/acd) p53(-/-) mice. On the contrary, p53(-/-) and p53(+/-) mice with the Acd(acd/acd) genotype show a decreased tumor-free survival, compared with Acd(+/+) mice. Tumors from Acd(acd/acd) p53(+/-) mice show a striking switch from the classic spectrum of p53(-/-) mice toward carcinomas. The acd mouse model provides further support for an in vivo role of telomere deprotection in tumorigenesis.

Inhibition of Protein Kinase Cβ by Enzastaurin Enhances Radiation Cytotoxicity in Pancreatic Cancer

Purpose: Aberrant activation of protein kinase Cβ (PKCβ) by pancreatic cancer cells facilitates angiogenesis and tumor cell survival. Targeting PKCβ with enzastaurin, a well-tolerated drug in clinical trials, would be expected to radiosensitize pancreatic tumors through direct antitumor and antivascular effects. Experimental Design: We tested the hypothesis that enzastaurin radiosensitizes pancreatic cancer cells in culture and in vivo through inhibition of PKCβ. We analyzed pancreatic cancer xenografts for growth delay and microvessel density after treatment with enzastaurin, radiation, or both. We determined the effect of radiation and enzastaurin on glycogen synthase kinase 3β, a mediator of cell death in culture and in vivo. Results: At concentrations attained in patients, enzastaurin reduced levels of active PKCβ measured by phosphorylation at Thr500 in culture and in xenografts. Enzastaurin alone did not affect pancreatic cancer cell survival, proliferation, or xenograft growth. However, enzastaurin radiosensitized pancreatic cancer cells in culture by colony formation assay. Enzastaurin alone decreased microvessel density of pancreatic cancer xenografts without appreciable effects on tumor size. When combined with radiation, enzastaurin increased radiation-induced tumor growth delay with a corresponding decrease in microvessel density. Enzastaurin inhibited radiation-induced phosphorylation of glycogen synthase kinase 3β at Ser9 in pancreatic cancer cells in culture and in tumor xenografts, suggesting a possible mechanism for the observed radiosensitization. Conclusions: Enzastaurin inhibits PKCβ in pancreatic cancer cells in culture, enhancing radiation cytotoxicity. Additional antivascular effects of enzastaurin were observed in vivo, resulting in greater radiosensitization. These results provide the rationale for a clinical trial in locally advanced pancreatic cancer combining enzastaurin with radiation.

Wnt/βcatenin signaling in adrenocortical stem/progenitor cells: Implications for adrenocortical carcinoma

A. Kim a, T.-J. Giordano b, R. Kuick c, K. Serecky d, G.D. Hammer a,d,e,∗ a Cellular and Molecular Biology Training Program, University of Michigan, 109 Zina Pitcher Place, 1502 BSRB, Ann Arbor MI 48109-2200, United States b Department of Pathology, University of Michigan, 109 Zina Pitcher Place, 1502 BSRB, Ann Arbor MI 48109-2200, United States c Department of Pediatrics, University of Michigan, 109 Zina Pitcher Place, 1502 BSRB, Ann Arbor MI 48109-2200, United States d Department of Molecular and Integrative Physiology, University of Michigan, 109 Zina Pitcher Place, 1502 BSRB, Ann Arbor MI 48109-2200, United States e Department of Internal Medicine, Division of Metabolism, Endocrinology, and Diabetes, University of Michigan, 109 Zina Pitcher Place, 1502 BSRB, Ann Arbor MI 48109-2200, United States

The discovery of vemurafenib for the treatment of BRAF-mutated metastatic melanoma

ABSTRACT Introduction: In the era of precision medicine and sophisticated modern genetics, the discovery of the BRAFV600 inhibitor, vemurafenib, quickly became the model for targeted therapy in melanomas. As early as 2002, the majority of metastatic melanomas were described to harbor the BRAFV600 mutation, setting the stage for an explosion of interest for targeting this protein as a novel therapeutic strategy. The highly selective BRAFV600 inhibitor, vemurafenib, was identified initially through a large-scale drug screen. Areas covered: Here we examine vemurafenib’s journey from discovery to clinical use in metastatic melanoma. Topics covered include preclinical data, single agent Phase 1,2 and 3 clinical trials, resistance issues and mechanisms, adverse effects including the development of squamous cell cancers, and combination trials. Expert opinion: Due to its tolerance, low toxicity profile, rapid tumor response, and improved outcomes in melanoma patients with BRAFV600 mutations, vemurafenib was advanced rapidly through clinical trials to receive FDA approval in 2011. While its efficacy is well documented, durability has become an issue for most patients who experience therapeutic resistance in approximately 6–8 months. In addition, a concerning toxicity observed in patients taking the drug include development of localized cutaneous squamous cell carcinomas (SCCs). It is hypothesized that drug resistance and SCC development result from a similar paradoxical activation of protein signaling pathways, specifically MAPK. Identification of these mechanisms has led to additional treatment strategies involving new combination therapies.