Copper coordination compounds target copper in cancer by diverse mechanisms.

Extracellular adenosine is a potent immunosuppressor that accumulates during tumor growth. We performed proof-of-concept studies investigating the therapeutic potential and mechanism of action of monoclonal antibody (mAb)-based therapy against CD73, an ecto-enzyme overexpressed on breast-cancer cells that catalyzes the dephosphorylation of adenosine monophosphates into adenosine. We showed that anti-CD73 mAb therapy significantly delayed primary 4T1.2 and E0771 tumor growth in immune-competent mice and significantly inhibited the development of spontaneous 4T1.2 lung metastases. Notably, anti-CD73 mAb therapy was essentially dependent on the induction of adaptive anti-tumor immune responses. Knockdown of CD73 in 4T1.2 tumor cells confirmed the tumor-promoting effects of CD73. In addition to its immunosuppressive effect, CD73 enhanced tumor-cell chemotaxis, suggesting a role for CD73-derived adenosine in tumor metastasis. Accordingly, administration of adenosine-5′-N-ethylcarboxamide to tumor-bearing mice significantly enhanced spontaneous 4T1.2 lung metastasis. Using selective adenosine-receptor antagonists, we showed that activation of A2B adenosine receptors promoted 4T1.2 tumor-cell chemotaxis in vitro and metastasis in vivo. In conclusion, our study identified tumor-derived CD73 as a mechanism of tumor immune escape and tumor metastasis, and it also established the proof of concept that targeted therapy against CD73 can trigger adaptive anti-tumor immunity and inhibit metastasis of breast cancer.

Human epidermal growth factor receptor-2 (HER2)-targeted therapies prolong survival in HER2-positive breast cancer patients. Benefit stems primarily from improved control of systemic disease, but up to 50% of patients progress to incurable brain metastases due to acquired resistance and/or limited permeability of inhibitors across the blood-brain barrier. Neratinib, a potent irreversible pan-tyrosine kinase inhibitor, prolongs disease-free survival in the extended adjuvant setting, and several trials evaluating its efficacy alone or combination with other inhibitors in early and advanced HER2-positive breast cancer patients are ongoing. However, its efficacy as a first-line therapy against HER2-positive breast cancer brain metastasis has not been fully explored, in part due to the lack of relevant pre-clinical models that faithfully recapitulate this disease. Here, we describe the development and characterisation of a novel syngeneic model of spontaneous HER2-positive breast cancer brain metastasis (TBCP-1) and its use to evaluate the efficacy and mechanism of action of neratinib. TBCP-1 cells were derived from a spontaneous BALB/C mouse mammary tumour and characterised for hormone receptors and HER2 expression by flow cytometry, immunoblotting and immunohistochemistry. Neratinib was evaluated in vitro and in vivo in the metastatic and neoadjuvant setting. Its mechanism of action was examined by transcriptomic profiling, function inhibition assays and immunoblotting. TBCP-1 cells naturally express high levels of HER2 but lack expression of hormone receptors. TBCP-1 tumours maintain a HER2-positive phenotype in vivo and give rise to a high incidence of spontaneous and experimental metastases in the brain and other organs. Cell proliferation/viability in vitro is inhibited by neratinib and by other HER2 inhibitors, but not by anti-oestrogens, indicating phenotypic and functional similarities to human HER2-positive breast cancer. Mechanistically, neratinib promotes a non-apoptotic form of cell death termed ferroptosis. Importantly, metastasis assays demonstrate that neratinib potently inhibits tumour growth and metastasis, including to the brain, and prolongs survival, particularly when used as a neoadjuvant therapy. The TBCP-1 model recapitulates the spontaneous spread of HER2-positive breast cancer to the brain seen in patients and provides a unique tool to identify novel therapeutics and biomarkers. Neratinib-induced ferroptosis provides new opportunities for therapeutic intervention. Further evaluation of neratinib neoadjuvant therapy is warranted.

The use of copper radioisotopes in cancer diagnosis and radionuclide therapy is possible using chelators that are capable of binding Cu^II with sufficient stability in vivo to provide high tumour-to-background contrast. Here we report the design and synthesis of a new bifunctional chelator, 5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid (MeCOSar), that forms copper complexes of exceptional stability by virtue of a cage amine (sarcophagine) ligand and a new conjugate referred to as SarTATE, obtained by the conjugation of MeCOSar to the tumour-targeting peptide Tyr³-octreotate. Radiolabeling of SarTATE with ⁶⁴Cu^II, a radioisotope suitable for positron emission tomography (PET), was fast (∼20 min), easily performed at room temperature and consistently resulted in high radiochemical purity (>99%). In vitro and in vivo evaluation of ⁶⁴CuSarTATE demonstrated its high selectivity for tumour cells expressing somatostatin receptor 2 (sstr2). Biodistribution and PET imaging comparisons were made between ⁶⁴CuSarTATE and ⁶⁴Cu-labeled DOTA-Tyr³-octreotate (⁶⁴CuDOTATATE). Both radiopharmaceuticals showed excellent uptake in sstr2-positive tumours at 2 h post-injection. While tumour uptake of ⁶⁴CuDOTATATE decreased significantly at 24 h, ⁶⁴CuSarTATE activity was retained, improving contrast at later time points. ⁶⁴CuSarTATE accumulated less than ⁶⁴CuDOTATATE in the non-target organs, liver and lungs. The uptake of ⁶⁴CuSarTATE in the kidneys was high at 2 h but showed significant clearance by 24 h. The new chemistry and pre-clinical evaluation presented here demonstrates that MeCOSar is a promising bifunctional chelator for Tyr³-octreotate that could be applied to a combined imaging and therapeutic regimen using a combination of ⁶⁴Cu- and ⁶⁷CuSarTATE complexes, owing to improved tumour-to-non-target organ ratios compared to ⁶⁴CuDOTATATE at longer time points.

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution

The basement membrane protein, laminin (LM)‐511, is a potent adhesive and migratory substrate for metastatic breast tumor cells in vitro. Its expression correlates with tumor grade and metastatic potential in vivo. These observations suggest that responsiveness to autocrine or paracrine‐derived LM‐511 may be an important property regulating breast cancer metastasis in vivo. To address this, we compared the metastatic potential of 4T1 mammary carcinoma cells to that of 4T1 variants isolated by repeated chemotactic migration toward LM‐511 in vitro (4T1LMF4) followed by serial injection into the mammary gland and recovery of spontaneous metastases from bone (4T1BM2). Variant subpopulations exhibited a distinct morphology on LM‐511 and increased expression of β1 and β4 integrins compared to parental 4T1 cells. Importantly, mice inoculated with 4T1LMF4 and 4T1BM2 variants showed a 2.5‐ to 4‐fold increase in the incidence of spontaneous metastasis to bone compared to 4T1 tumor‐bearing mice. Functionally, 4T1BM2 variants were more adherent and more invasive toward LM‐511 than parental 4T1 cells. Treatment of 4T1BM2 cells with lebein‐1, a disintegrin with selectivity toward LM‐type integrin receptors, potently inhibited their migration and invasion toward LM‐511. Similarly, α3β1 integrin‐dependent migration and invasion of human MDA‐MB‐231 breast carcinoma cells toward LM‐511 were significantly inhibited by lebein‐1. Taken together, these results provide strong evidence that LM‐511 contributes to the metastasis of breast tumors and suggest that targeting integrin‐LM‐511 interactions with lebein‐1 or other inhibitors of LM‐511 receptors may have therapeutic potential for patients with advanced breast cancer.