PURPOSE: Triple-negative breast cancer (TNBC) is one of the deadliest forms of cancer, tending to strike earlier and metastasize faster than other breast cancers. Unfortunately, due to its lack of progesterone, estrogen, or HER2 receptors, there are no targeted therapies available to patients with TNBC. However, in 2019 Shen et al found that TNBC tumors with higher levels of tubulointerstitial nephritis antigen-like 1 (Tinagl1) had better prognoses, and that treating tumors with free Tinagl1 protein can suppress TBNC tumor growth by interrupting EGFR and integrin signaling.1 We sought to expand upon this work in two ways. First, we sought to improve the delivery of Tinagl1 by transitioning from the recombinant protein used in Shen (2019) to lipid-nanoparticle based gene therapy. Second, we sought to characterize the changes to the tumor microenvironment that Tinagl1 treatment induces.
METHODS: Using our lipid-protamine-DNA nanoparticles (LPDs), we encapsulated Tinagl1-expressing plasmid DNA (Tinagl1 pDNA) and targeted them to tumors through sigma-receptor targeting moiety aminoethyl anisamide (AEAA).2 Briefly, Tinagl1 pDNA is condensed into a solid core with protamine. This protamine-pDNA core is encapsulated into liposomes decorated with DSPE-PEG(2000) and DPSE-PEG-AEAA (10:1). Tinagl1 LPDs can be injected intravenously into Balb/C mice bearing 4T1 tumors in the mammary fat pad. Tumor growth is measured with calipers three times a week, with tumor volume calculated as length x width x width/2. AKT phosphorylation was measured by Western blotting; angiogenesis, measured by CD31 and VEGF, as well as Hif1a expression, were measured using immunofluorescence staining.
RESULTS: Tinagl1 LPDs injected intravenously suppress tumor growth in a transient, controllable fashion. Confirming the results shown in Shen (2019), Tinagl1 reduces the phosphorylation of AKT. Shen (2019) showed that this is a combined result of interfering with integrin binding and EGFR dimerization on the surface of TNBC cells, resulting in poor activation of the MAPK and FAK pathways. However, our second goal for this study was to further analyze the effects of Tinagl1 expression on the tumor microenvironment. In addition to suppressing tumor cell proliferation, Tinagl1 significantly promotes angiogenesis in the tumor. The increase in blood flow appears to reduce the expression of Hif1a in the tumor, indicating less hypoxia and potentially improving the susceptibility of tumors to treatment.
CONCLUSION: In addition to halting the growth of TNBC tumor cells, Tinagl1 is able to remodel the tumor microenvironment by increasing angiogenesis. Because Tinagl1 suppresses tumor proliferation and metastasis simultaneously, angiogenesis results in reduced Hif1a and hypoxia without the typical detrimental effects.
