Primary Hyperparathyroidism Cell Models for Research

Primary hyperparathyroidism (PHPT) is the most common cause of hypercalcemia in the outpatient setting, with a prevalence of approximately 1-7 per 1000 adults in the United States (WHO, 2020). It is more common in women, with a female-to-male ratio of 3:1, and incidence increases with age, peaking between 50 and 60 years. Most cases are sporadic, but familial forms occur in about 10% of patients. The disease is characterized by excessive secretion of parathyroid hormone (PTH) from one or more parathyroid glands, leading to hypercalcemia and hypophosphatemia. Clinical manifestations include nephrolithiasis, osteoporosis, and neuropsychiatric symptoms, but many patients are asymptomatic. Surgical removal of the abnormal gland(s) is the only curative treatment, with a success rate of over 95% in experienced hands. However, persistent or recurrent disease occurs in 5-10% of cases, and there is a need for better medical management for patients who are not surgical candidates. The disease serves as an excellent model for studying calcium homeostasis, endocrine tumorigenesis, and the role of specific genes in parathyroid cell proliferation and function.

PHPT is an ideal model for mechanistic studies due to its well-defined genetic basis and the availability of normal and tumor parathyroid tissues. Key research areas include: understanding the molecular pathways that drive parathyroid tumorigenesis, identifying biomarkers for disease progression, developing targeted therapies for inoperable cases, and exploring the role of calcium-sensing receptor (CASR) in regulating PTH secretion. Public datasets such as TCGA and GEO provide transcriptomic and genomic data from parathyroid tumors, enabling integrative analyses. Open questions include the identification of driver mutations in sporadic tumors, the mechanisms of resistance to calcimimetics, and the development of non-invasive diagnostic tools. Gene-edited cell models are crucial for functional validation of candidate genes and for drug screening.

Primary hyperparathyroidism arises from dysregulation of parathyroid cell proliferation and PTH secretion. Key pathways include:

• • Wnt/β-catenin pathway: Aberrant activation promotes cell proliferation. Mutations in CTNNB1 or loss of APC have been reported in some parathyroid adenomas.
• • Cyclin D1/CCND1 pathway: Overexpression of cyclin D1 due to chromosomal rearrangement or amplification drives G1-S transition.
• • MEN1 pathway: The MEN1 gene encodes menin, a tumor suppressor that regulates gene transcription and genome stability. Loss of menin leads to parathyroid tumorigenesis.
• • CASR signaling: The calcium-sensing receptor (CASR) regulates PTH secretion. Inactivating mutations in CASR cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, but somatic mutations are rare in sporadic adenomas.
• • CDC73/HRPT2 pathway: Mutations in CDC73 (parafibromin) are associated with hyperparathyroidism-jaw tumor syndrome and some sporadic adenomas, leading to loss of tumor suppressor function.

Data from TCGA and COSMIC databases.

Multiple signaling networks are deregulated in PHPT:

• • Wnt/β-catenin: Activation leads to transcription of proliferative genes (e.g., MYC, CCND1).
• • MAPK pathway: Mutations in RAS or BRAF are rare but can activate ERK signaling, promoting proliferation.
• • PI3K/AKT/mTOR: Activation enhances cell survival and growth.
• • Calcium signaling: CASR mutations disrupt intracellular calcium homeostasis, affecting PTH secretion.
• • Cell cycle regulators: p53, Rb, and CDK inhibitors are often altered, leading to uncontrolled proliferation.

Organoids derived from parathyroid tissue are emerging as more physiologically relevant models, preserving the 3D architecture and cellular heterogeneity. They can be used for drug testing and studying cell-cell interactions.

• • PDX models: Patient-derived xenografts of parathyroid tumors in immunodeficient mice retain the genetic and phenotypic features of the original tumor.
• • GEMMs: Genetically engineered mouse models with targeted mutations in Men1, Cdc73, or Casr recapitulate aspects of PHPT.
• • Induced models: Use of chemical carcinogens or viral vectors to induce parathyroid tumors.

CRISPR-based gene editing enables the creation of isogenic cell lines with precise genetic modifications, such as knockout of tumor suppressors (e.g., MEN1, CDC73) or knock-in of oncogenic mutations (e.g., CTNNB1). These models are invaluable for studying gene function and drug response. Commercially available, sequence-verified models accelerate research by providing consistent and reproducible tools. For example, a MEN1 knockout cell line can be used to study the effects of menin loss on parathyroid cell proliferation and PTH secretion, while a CASR knock-in cell line can help investigate calcium-sensing mechanisms.

Knockout and knock-in lines are used to validate the role of genes implicated in PHPT. For instance, CRISPR-mediated knockout of MEN1 in parathyroid cells can confirm its tumor suppressor function, while overexpression of CCND1 can demonstrate its oncogenic potential. These models allow for high-throughput screening to identify genetic modifiers and downstream effectors.

Isogenic pairs (e.g., wild-type vs. MEN1 knockout) are used to screen for compounds that selectively inhibit mutant cells. This approach can identify drugs that target specific genetic vulnerabilities. Additionally, gene-edited models can be used to study resistance mechanisms to calcimimetics or other therapies by introducing resistance mutations and monitoring cell growth.

CRISPR synthetic lethality screens can identify genes that are essential only in the context of specific mutations, revealing potential therapeutic targets. For example, in MEN1-deficient cells, synthetic lethal partners could be targeted to selectively kill tumor cells. Gene-edited models also enable the identification of secreted biomarkers by comparing the secretome of mutant and wild-type cells.