Identification and understanding the mechanism-of-action of critical genes in bone metastasis colonization and drug resistance
Cancer is a major health threat world-wide. The majority of cancer-related death is not caused by primary cancer, but by metastasis, the spread of tumor cells to critical distal organs. Unfortunately, metastatic tumor cells are also very refractory to traditional therapies like chemotherapy and radiation therapy. Recent researches also start to appreciate the importance of tumor microenvironment, the composition of many stromal cells, immune infiltrated cells, and the extracellular matrix in regulating these processes. The lab is integrating sophisticated mouse models, bioinformatic analysis, and molecular and cellular tools to dissect out the detailed molecular cross talk in cancer metastasis and therapy resistance. The discovered results could potentially provide the foundation for improving the treatment of metastatic cancer and therapy resistance.
Post-translational regulation of transcription factors in breast cancer migration and metastasis.
To gain migratory capacity and as the first step in metastasis, tumor cells need to down-regulate E-cadherin expression to reduce cell-cell adhesion, switch its cell shape from cobble-stone like epithelial cells to fibroblast-like mesenchymal cells to realize its invasive ability. A few transcriptional factors including SNAIL family members are major factors which promote EMT and cancer metastasis. Identifying the regulatory mechanisms for SNAIL family proteins might be able to help identify new therapeutic targets for blocking cancer metastasis. Building on my previous experience in the study of E3 ligase and proteasomal degradation pathway during my earlier studies, the lab developed an innovative project to identify E3 ligases that are crucial for SNAIL protein regulation in breast cancer metastasis.
We established a novel dual-luciferase screening system for identifying ubiquitin ligases that control the degradation of protein-of-interests (SNAIL/SNAI2 proteins as in our study). The coding sequence of human SNAIL gene is fused in-frame with the firefly luciferase gene coding sequence (SNAIL-Luc) to produce the SNAIL-Luciferase fusion reporter protein. A breast cancer cell line was then transduced with a lentiviral vector containing the Renilla luciferase (R-Luc) control (Fig. 1A).
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Figure 1. Luciferase based E3 ligase - protein substrate screening systemA.Illustration of the dual luciferase reporter screening system for SNAIL-targeting E3 ligases. CMV, cytomegalovirus; LTR, long terminal repeat. The coding sequence of the human SNAIL gene is fused in-frame with the firefly luciferase gene coding sequence to produce the SNAIL-Luciferase fusion reporter protein (SNAIL-Luc). Renilla luciferase (R-Luc) serves as an internal control.B.Schematic of PKD1-dependent SNAIL protein ubiquitylation and degradation by FBXO11. Using the system established in A, we screened a siRNA library against all human E3 ligases (555 genes in our |
library) and identified FBXO11 as a bona fide E3 ligase targeting the SNAIL protein. SNAIL protein is first phosphorylated by recognized and ubiquitylated by the SCF-FBXO11 E3 ligase complex. Polyubiquitylated SNAIL protein is then degraded through the 26S proteasomal degradation pathway. |
Therapeutic Antibody Targeting Tumor- and Osteoblastic Niche-Derived Jagged1 Sensitizes Bone Metastasis to Chemotherapy.The system was then used to efficiently screen a siRNA library against all human E3 ligases. We successfully identified SCF-FBXO11 as a novel E3 ligase targeting SNAIL degradation; while ASB13 as a novel E3 ligase against SNAIL2 protein (Fig. 1B and model not shown here). Our study provided an important proof-of-principle to apply a similar screening strategy to identify other E3 ligase-protein substrate pairs (2014 Cancer Cell, PMID: 25203322; 2020 Genes & Development, PMID: 32943576).
Bone is one the most frequent sites for metastasis in many solid cancers. More than 70% suffer from bone metastasis, which is often accompanied by severe bone pain, fracture and potentially lethal complications such as hypercalcemia. Although chemotherapy and anti-osteolytic agents such as bisphosphonate and RANKL antibody denosumab can reduce morbidity associated with bone metastasis, these treatments often do not significantly extend the survival time of the patients or provide a cure, as metastatic cancers often acquire resistance to these treatments.
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Figure 2. Therapeutic Antibody Targeting Tumor- and Osteoblastic Niche-Derived Jagged1 Sensitizes Bone Metastasis to Chemotherapy A.Tumor derivedJagged1interacts with osteoblasts to induce IL-6, which feeds back to tumor cells for tumor cell proliferation.Jagged1 also activates osteoclast differentiation and bone resorption, leading to osteolytic bone metastasis. Jagged1 neutralizing antibody 15D11 is sufficient in blocking both interactions and inhibiting bone metastasis progression.B.Chemotherapy induces Jagged1 expression in osteoblast cells, which feeds back to tumor cells to promote bone metastatic tumor cell survival in chemotherapy. Combinational treatment of both chemo-agents with 15D11 synergistically kills tumor cells in the bone and might provide benefit to breast cancer patients with bone metastatic disease. |
Previous studies identified tumor-derived Jagged1 (one of the major Notch signaling ligands) as a bone metastasis-promoting factor (Fig. 2A).We and our collaborators utilized the XenoMouse technology to generate fully human monoclonal antibodies against Jagged1(named as 15D11 antibody).When administratedinvivo, 15D11 protected the bone structure by preventing excessive bone absorption caused by tumor-derived Jagged1. Interestingly,synergistically treatment of the bone metastasis with chemotherapy agent (i.e., Paclitaxel) and 15D11 neutralizing antibody resulted in up to 100 folds reduction of bone metastasis burden in our preclinical mouse models. This synergistic effect is independent of Jagged1 expression level in tumor cells. Mechanistically, we discover that this effect is caused by another layer of tumor-stroma interaction mediated by osteogenic Jagged1. During chemotherapy against bone metastasis, Jagged1 is significantly induced in osteoblast cells and its precursor cells – mesenchymal stem cells (MSCs), which feeds back to tumor cells to activate Notch signaling and promote tumor cell survival in the bone microenvironment. Blocking osteogenic Jagged1 induced survival signaling with 15D11 administration thus potentiates chemotherapeutics’ cytotoxic effect on tumor cells. Our results thus clearly suggest a new treatment scenario in which breast cancer patients receiving chemotherapy should also opt for JAGGED1 blockade therapy. The paper reporting these discoveries were recently published inCancer Cell(PMID: 29232552) and was highlighted by expert preview in the same issue of cancer Cell and by other top cancer journals, with further news report by several news media platforms
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1.Fan H.; Wang X.; Li W.; Shen M.; Wei Y.;Zheng H.*;Kang Y.*; ASB13 inhibits breast cancer metastasis through promoting SNAI2 degradation and relieving its transcriptional repression of YAP,Genes & Development, 2020, 34(19-20): 1359-1372
2.Li W.; Shen M.; Jiang Y.Z.; Zhang R.;Zheng H.; Wei Y.; Shao Z.M.; Kang Y.*; Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2,Genes & Development, 2020, 34(19-20): 1310-1315
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4.Shen M*, Jiang YZ, Wei Y, Ell B, Sheng X, Esposito M, Kang J, Hang X,Zheng H, Rowicki M, Zhang L, Shih WJ, Celià-Terrassa T, Liu Y, Cristea I, Shao ZM, Yibin Kang#(2019) Tinagl1 suppresses triple-negative breast cancer progression and metastasis by simultaneously inhibiting integrin/FAK and EGFR signalingCancer Cell, 35(1): 64-80.e7 PMID: 30612941
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