Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

1. Overview

Scientific rationale and importance:
Vascular mimicry (VM) enables aggressive tumors to form vessel-like structures independent of endothelial cells, contributing to therapy resistance and poor prognosis. My preliminary data indicate that Midkine (MDK), a secreted growth factor stabilized by USP12, promotes VM in triple-negative breast cancer (TNBC) cells. Proteomic analysis identified Vitronectin (VTN) as a potential downstream target of MDK, and high MDK expression correlates with increased VE-cadherin in patient tumors. This project aims to validate VTN as a bona fide mediator of MDK-induced VM, thereby providing a novel therapeutic target.

Funding/facilities/ethics approvals:
The proposed work will be conducted at Northern Jiangsu People's Hospital, with full access to molecular biology, histology, and imaging facilities. Institutional ethics approval for human tissue use and animal experiments will be secured before study initiation.

I obtained my PhD in Cell Biology from Peking Union Medical College in 2021 and subsequently began working as a physician at Northern Jiangsu People's Hospital, one of the largest and most historic hospitals in Jiangsu Province. Our hospital has well-established research facilities and adequate clinical samples to support both basic and translational research. My research background in cancer biology, supported by a strong publication track record, demonstrates my ability to serve as the PI of this proposal.

This mini grant will enable me to generate preliminary data to secure a full-scale research grant, establish my own research laboratory, and continue investigating the molecular mechanisms and novel therapeutic targets in breast cancer. To facilitate fund transfer, I have obtained approval from our collaborating organization, One Patient One Cure, a US-based nonprofit, to receive the funding.

Estimated timeline:
12 months (September 1, 2025 – August 31, 2026).

Data sharing commitment:
All raw and processed data, including imaging and proteomics datasets, will be deposited in public repositories (e.g., GEO, PRIDE) within six months of publication.

2. Introduction

Background:
VM is a key alternative angiogenesis mechanism in breast cancer, particularly under hypoxic stress, driven by EMT and signaling pathways such as Wnt/β-catenin and TGF-β. Current anti-VEGF therapies fail to suppress VM, underscoring the need for alternative targets.

MDK is overexpressed in various malignancies and promotes proliferation, EMT, angiogenesis, and therapy resistance. In TNBC, MDK overexpression enhances tube formation and VM marker expression. Our proteomics and immunoprecipitation data suggest that MDK may act through VTN, an extracellular matrix glycoprotein implicated in cell adhesion, migration, and angiogenesis.

Research question:
Does VTN mediate MDK-driven VM in breast cancer?

Hypotheses:

H1: MDK promotes VM in breast cancer through upregulation of VTN.
H2: Disruption of VTN abrogates MDK-induced VM in vitro and in vivo.

Impact statement:
Confirmation of the MDK–VTN axis as a driver of VM will open new therapeutic avenues, particularly for TNBC, by targeting VM to improve anti-angiogenic therapy outcomes.

3. Methods

Participants/Sample

Cell lines: TNBC cell lines such as MDA-MB-231 and BT549 with MDK overexpression or knockdown.
Animal models: Orthotopic TNBC xenografts in immunodeficient mice.
Clinical samples: Preserved breast cancer tissues.

Materials and Procedures

Aim 1 – Verify VTN regulation by MDK

Western blot and qRT-PCR to measure VTN levels in MDK-overexpressing and knockdown cells compared to controls.
Immunoprecipitation to assess MDK–VTN interaction.

Aim 2 – Assess functional role of VTN in MDK-induced VM

In vitro tube formation assays: VTN overexpression in MDK-knockdown cells to assess restoration of the VM phenotype (rescue experiment).
In vivo assays: Orthotopic xenografts with manipulated MDK/VTN expression; assess VM structures via PAS staining and VM marker IHC.

Aim 3 – Clinical correlation

Immunohistochemistry for MDK, VTN, and VE-cadherin in TNBC patient tissues.

Statistical correlation of MDK and VTN expression with VM prevalence and patient outcomes.

Planned Analyses

Statistical tests: Two-tailed t-tests or ANOVA for group comparisons; Pearson/Spearman for correlation; Kaplan–Meier survival curves for prognostic value.
Alternative strategy: If VTN overexpression only partially affects MDK-induced VM in vitro, we will screen for cofactors via proteomics.

Ethics and Data Management

4. Pilot Data (Optional)

MDK overexpression in MDA-MB-231 cells increases tubular structure formation.
Proteomics identified VTN as significantly upregulated in MDK-overexpressing cells.

High-MDK tumors in clinical samples show elevated VE-cadherin

5. Budget

Consumable reagents and assay kits: $2,000
Animal studies: $2,300
Imaging and histology: $500
Data analysis software: $200
Total: $5,000

6. References

1 Cerezo-Wallis, D. et al. Midkine rewires the melanoma microenvironment toward a tolerogenic and immune-resistant state. Nat Med 26, 1865-1877 (2020). https://doi.org/10.1038/s41591-020-1073-3

2 Christou, C., Stylianou, A. & Gkretsi, V. Midkine (MDK) in Hepatocellular Carcinoma: More than a Biomarker. Cells 13 (2024). https://doi.org/10.3390/cells13020136

3 Han, B. et al. Cancer incidence and mortality in China, 2022. J Natl Cancer Cent 4, 47-53 (2024). https://doi.org/10.1016/j.jncc.2024.01.006

4 Kadomatsu, K., Tomomura, M. & Muramatsu, T. cDNA cloning and sequencing of a new gene intensely expressed in early differentiation stages of embryonal carcinoma cells and in mid-gestation period of mouse embryogenesis. Biochem Biophys Res Commun 151, 1312-1318 (1988). https://doi.org/10.1016/s0006-291x(88)80505-9

5 Lapkina, E. Z., Esimbekova, A. R. & Ruksha, T. G. [Vasculogenic mimicry]. Arkh Patol 85, 62-69 (2023). https://doi.org/10.17116/patol20238506162

6 Maeda, N. et al. A receptor-like protein-tyrosine phosphatase PTPzeta/RPTPbeta binds a heparin-binding growth factor midkine. Involvement of arginine 78 of midkine in the high affinity binding to PTPzeta. J Biol Chem 274, 12474-12479 (1999). https://doi.org/10.1074/jbc.274.18.12474

7 Muramatsu, T. Midkine, a heparin-binding cytokine with multiple roles in development, repair and diseases. Proc Jpn Acad Ser B Phys Biol Sci 86, 410-425 (2010). https://doi.org/10.2183/pjab.86.410

8 Sheng, B., Wei, Z., Wu, X., Li, Y. & Liu, Z. USP12 promotes breast cancer angiogenesis by maintaining midkine stability. Cell Death Dis 12, 1074 (2021). https://doi.org/10.1038/s41419-021-04102-y

9 Shin, D. H. et al. Midkine Is a Potential Therapeutic Target of Tumorigenesis, Angiogenesis, and Metastasis in Non-Small Cell Lung Cancer. Cancers (Basel) 12 (2020). https://doi.org/10.3390/cancers12092402

10 Wei, X. et al. Mechanisms of vasculogenic mimicry in hypoxic tumor microenvironments. Mol Cancer20, 7 (2021). https://doi.org/10.1186/s12943-020-01288-1

Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

Role and Mechanism of MDK in Promoting Vascular Mimicry in Breast Cancer

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