Mutant p53 gain-of-function and Li-Fraumeni syndrome
Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder characterized by the early onset of various tumors, including soft tissue sarcomas, osteosarcomas, breast cancer, brain tumors, leukemia, and adrenocortical carcinomas. The germline mutation of the TP53 tumor suppressor gene is responsible for LFS. TP53 (p53) encodes a 53 kDa transcription factor that binds DNA as a tetramer. It is well-known for its ability to repress tumor development by regulating genes involved in cell cycle arrest, DNA damage response, apoptosis, senescence, differentiation, and metabolism. However, mutations in p53 not only impair its tumor-suppressor function but also transform it into an oncoprotein, resulting in a gain-of-function phenotype (Trends Pharmacol Sci. 2017 Oct;38(10):908-927).
Missense mutations, particularly at specific hotspots, are prevalent in mutant p53 and provide a selective advantage during cancer progression. Mutant p53s dysregulate metabolic pathways, impair imprinted gene network (Cell. 2015 Apr 9;161(2):240-54; Front Genet. 2021 Jan 15;11:611823), upregulate epigenome and epitranscriptome (Nat Commun. 2023 (accepted)), enhance metastasis, increase angiogenic gene expression (Proc Natl Acad Sci U S A. 2018 Nov 20;115(47):E11128-E11137), and alter mitochondria function (Cancer Discov. 2023 (accepted)). Although several mechanisms have been proposed to explain mutant p53’s gain-of-function, the comprehensive picture of mutant p53-mediated malignant transformation remains unclear. Our laboratory is focused on investigating the gain-of-function effects of mutant p53 and aims to develop therapeutic strategies for treating patients with LFS.
Figure. Mutational landscape of TP53 germline and somatic mutations in human cancer. TP53 missense mutation data is obtained from the International Agency for Research on Cancer (IARC) TP53 database (http://p53.iarc.fr/). Distributions of p53 mutations are plotted over the function of amino acid position, where left side indicating germline mutations and right side indicating somatic mutations. Horizontal axis shows the frequency of any mutation at indicated residues. Vertical axis is the schematic of the p53 protein starting with N-terminal at the top. p53 protein domain contains transcriptional activation domain I and II (TAD 1, 20–40; TAD II, 40–60), the proline domain (PP, 60–90), the sequence-specific core DNA-binding domain (DNA-binding core, residues 100–300), the linker region (L, 301–324), the tetramerization domain (Tet, 325–356), and the lysine-rich basic C-terminal domain (++, 363–393). Most common mutations on “hot spot” are indicated as bold line; R175, G245, R248, R273, R282 residue are the five common hot spots in both germline and somatic mutations (indicated as lollipop). Pie charts illustrate the tumor site distribution of five hotspot TP53 mutations (left, germline; right, somatic). Malignancies located at breast, brain, soft tissues and bone are most commonly seeing over the five hotspot germline mutations; malignancies from these tumor sites are also distributed in the same five hotspot of TP53 somatic mutations (indicated in bold).
Figure is adapted from Trends Pharmacol Sci. 2017 Oct;38(10):908-927.
RB1 tumor suppressor and hereditary retinoblastoma (HRB)
The RB1 tumor suppressor has been widely recognized for its role in inhibiting tumor initiation, development, and progression. A genome-wide association study of 33 cancer types revealed that RB1 is one of the most frequently mutated tumor suppressor genes and is inactivated by various mechanisms across cancers. We explore the epidemiological link between gene mutation and osteosarcomagenesis and study another osteosarcoma-prone genetic disease, hereditary retinoblastoma (HRB). We investigate how HRB patients, caused by autosomal dominant mutations in the RB1 tumor suppressor gene, have a >400-fold increased incidence of osteosarcoma. Since HRB is the first genetic disease to prove the two-hit hypothesis and RB1 is the first tumor suppressor gene to be molecularly defined, the establishment of the HRB iPSC disease model to investigate the RB1 tumor suppressor gene is critical meaningful for the cancer society. More pressingly, no currently available models of RB1 mutation recapitulate the bone malignancy phenotype, severely limiting the ability of researchers to explore treatments for this disease. Our experience in iPSC technology enables us to perfect a model system to study this disease and explore therapeutic interventions.
Osteosarcoma-prone genetic diseases
Hereditary genetic disorders associated with a predisposition to osteosarcoma are relatively rare. However, studies of these diseases have led to important insights that generalize to the broader osteosarcoma population. Eight genetic diseases that predispose to the development of osteosarcoma, collectively informing our understanding of the underlying molecular determinants: Li-Fraumeni syndrome (LFS), Hereditary retinoblastoma (RB), Rothmund-Thomson syndrome (RTS), RAPADILINO syndrome (RAPA), Werner syndrome (WS), Bloom syndrome (BS), Diamond-Blackfan anemia (DBA), and Paget’s disease of bone (PDB).
Figure. Patient-derived iPSC disease models for osteosarcoma. (A) Patient-derived iPSCs are used to model human familial cancer syndromes and reveal a role of the mutant gene in disease development. To apply iPSC methodology to study genetic disease-associated bone malignancy, patient fibroblasts are biopsied from skin and then reprogrammed to iPSCs by the four "Yamanaka factors" (OCT4, SOX2, KLF4 and c-MYC). The iPSCs are then differentiated to MSCs and further to osteoblasts. These iPSC-derived osteoblasts can be examined for osteoblast differentiation defects and tumorigenic ability. Systematic comparison of the genome/transcriptome/interactome between mutant and wild-type osteoblasts can further elucidate pathological mechanisms. (B) Current progress of applying LFS, RB, RTS, RAPA, WS, BS, DBA and PDB patient-derived iPSCs to model disease etiology and dissect disease-associated osteosarcoma.
Figure is modified from Trends Mol Med. 2017 Aug;23(8):737-755.
Elucidate Bone Formation and Regeneration by PSC Platform and Lineage Differentiation
Congenital and acquired bone diseases caused by dysregulation of bone homeostasis constitute a critical public health concern. Bone diseases associated with bone fracture and large bone defects are common causes of disability, and recovery depends on effective bone regeneration. The last decades of applied research in bone biology have led to improved preventive and therapeutic interventions, including multiple classes of drugs, but illness, disability, and mortality caused by bone diseases have remained persistently high.
The human skeleton is a dynamic tissue dedicated to supporting the human frame and permitting the focused application of force that is required for locomotion and spontaneous movement. It also controls various biological processes including (i) osteoblastic lineage commitment of bone marrow multipotent mesenchymal stem cells (MSCs) into bone (osteoblasts (OBs) /osteocytes (OCs)), fat (adipocytes), and cartilage (chondrocytes); and (ii) bone remodeling, a process by which OBs (bone-forming cells) and OCs (bone-resorbing cells) work sequentially to balance bone mass OBs are specialized cells responsible for the formation and maintenance of skeletal architecture. They play a critical role in regulating the production of extracellular matrix proteins (collagen, osteocalcin, and osteopontin) and matrix mineralization, and OC differentiation. Fine-tuning of osteogenesis is necessary for bone homeostasis. Dysregulation of OB-modulating bone homeostasis results in the development of skeletal bone diseases, particularly osteoporosis, osteoarthritis, and osteosarcoma. Therefore, defining OB characteristics and understanding the molecular mechanisms regulating OB differentiation and maturation are extremely important at both individual and population levels. Currently, we integrate hESC/iPSC-derived bone lineage differentiation and systems analysis to elucidate the essential transcription factors in regulating human bone development.