ORCID
https://orcid.org/0000-0002-2251-0453
Date of Award
12-9-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Immune checkpoint blockade (ICB) has revolutionized cancer treatment, receiving FDA approval for various previously incurable cancers. In breast cancer, the combination of pembrolizumab (an anti-PD-1 antibody) with chemotherapy has become the standard-of-care for early-stage (Keynote-522) and advance-stage (Keynote-355) triple-negative breast cancer (TNBC). Unfortunately, over 30% of TNBC patients fail to respond to the combined chemo-immune therapy treatment, and the causes are unknown. Furthermore, ICB has demonstrated poor results in hormone-receptor positive breast cancer. While it is well-established that breast cancer cells can evade immune responses, the specific mechanisms driving immune evasion remain unclear. Thus, deciphering the immune system's role in primary and metastatic breast cancer and identifying factors or cells contributing to immune suppression could significantly improve patient outcomes. Dickkopf-1 (DKK1), an inhibitor of the Wnt/β-catenin pathway, is associated with reduced overall survival in breast cancer patients and correlates with the accumulation of suppressive myeloid populations in various cancers. To explore DKK1’s role in primary breast cancer progression, we used orthotopic mouse models consisting of injection PyMT, EO771, or 4T1 breast cancer lines into the mammary fat pad of WT female mice, followed by treatment with a DKK1-neutralizing antibody (mDKN-01). mDKN-01 significantly reduced primary tumor growth in all models tested but was ineffective when the tumor cells were injected into NSG immune-compromised mice, indicating an immune-mediated mechanism. To identify the immune populations affected by DKK1, we conducted experiments combining mDKN-01 with the depletion of various immune cell types, specifically T cells, NK cells, and macrophages. The results showed that only NK cell depletion abrogated the anti-tumor effects of DKK1 neutralization. Further in vitro and ex vivo assays demonstrated that DKK1 impairs NK cell cytotoxicity against tumor cells by downregulating the PI3K/AKT/mTOR signaling pathway. To explore the clinical relevance of these findings, we analyzed DKK1 levels and the percentage of cytotoxic NK cells in circulation in breast cancer patients with progressive or stable metastatic bone disease. We observed that DKK1 levels were increased while cytotoxic NK cells were reduced in patients who progressed. Finally, we found that DKK1 neutralization significantly enhanced the anti-tumor efficacy of chemotherapy + anti-PD-1 in the orthotopic 4T1 tumor model, demonstrating DKK1 targeting can ameliorate the therapeutic response to ICB. DKK1 is also a known regulator of bone loss and increased bone remodeling has been shown to recruit disseminated tumor cells to the bone microenvironment and support their growth into overt metastases. Thus, we tested the efficacy of DKK1 neutralization in mice intratibially injected with breast cancer cells and found significant inhibition of tumor growth in bone. Interestingly, in the metastatic setting analysis of circulating immune populations revealed that mDKN-01 reduced the number of immature immune-suppressive CD11b+Gr1+ myeloid populations, namely myeloid-derived suppressor cells (MDSCs), while increasing anti-tumor CD4 and CD8 T cells. Given that myeloid populations are predominant in the bone marrow, we hypothesized that the accumulation of MDSCs could promote bone metastasis by creating an immune suppressive environment. To test this hypothesis, we depleted MDSC by using anti-Gr1 antibody in mice with 4T1 primary tumors or established metastases to bone. Surprisingly, while anti-Gr1 significantly reduced primary tumor growth and depleted tumor-infiltrating CD11b+Gr1+ cells, it failed to diminish bone metastases, paradoxically leading to the accumulation of CD11b+Gr1+ cells in the bone marrow. Further subset analysis revealed that anti-Gr1 specifically expanded a Ly6CintLy6Gint subset while depleting Ly6Ghigh and Ly6Chigh populations, which possess immune-suppressive capabilities. Notably, Ly6CintLy6Gint cells although lacking immune-suppressive features, had the potential to differentiate into osteoclasts, the bone-resorbing cells that can promote bone metastases by releasing growth factors from the bone matrix. Subsequently, we treated established bone metastases with a combination of anti-Gr1 and Zoledronic Acid, an anti-resorptive drug, achieving significantly reduced tumor growth compared to each treatment alone. These findings suggest the importance of targeting bone immune suppressive populations and osteoclast activity to efficiently reduce bone metastatic tumor growth. In conclusion, our research highlights the key role of DKK1 neutralization in unleashing the killing ability of NK cells and improving the therapeutic response to ICB in primary breast cancer models. We also find that DKK1 neutralization efficiently reduces tumor growth in bone. As DKK1 can induce immune suppression and osteoclast activation, the use of DKK1 neutralizing antibody should be considered for the treatment of breast cancer patients with bone metastases and/or unresponsive to ICB.
Language
English (en)
Chair and Committee
Roberta Faccio
Committee Members
David DeNardo; Deborah Veis; Katherine Weilbaecher; Maxim Artyomov; Sheila Stewart
Recommended Citation
Lee, Seunghyun, "Deciphering Immune Suppression Mechanisms of Primary and Metastatic Breast Cancer" (2024). Arts & Sciences Electronic Theses and Dissertations. 3348.
https://openscholarship.wustl.edu/art_sci_etds/3348