Reducing the Invasiveness of Low- and High-Grade Endometrial Cancers in Both Primary Human Cancer Biopsies and Cell Lines by the Inhibition of Aquaporin-1 Channels

SIMPLE SUMMARY: A major clinical concern for patients with endometrial cancer is the aggressive spread of cancer cells into other tissue regions, worsening symptoms, eluding medical therapy, disrupting organ functions, and increasing risk of relapse. As reported here, drug-like agents that we have found block the ion channel function of a specific membrane protein known as Aquaporin-1 (AQP1) can be used to inhibit invasiveness of endometrial cancer cells. In vitro invasion assays were done with cell lines originally established from endometrial cancers, in parallel with assays on primary endometrial cancer cells isolated from endometrial cancer tissue at the time of surgery. The exciting discovery was that the AQP1 ion channel blockers inhibited invasiveness of both low- and high-grade endometrial cancer types in established cell lines and primary cancer cells at doses that did not cause toxic side effects. Low toxicity is encouraging for potential co-administration with other first-line treatments. Pharmacological agents optimized from lead compounds could be invaluable for slowing the progression of both low- and high grade cancers, including a challenging subset which are unresponsive to current hormone-based therapies. Further work on refining structures of the drugs, their distribution and clearance will be needed for progress towards bench-to-bedside clinical translation. ABSTRACT: Aquaporin (AQP) channels in endometrial cancer (EC) cells are of interest as pharmacological targets to reduce tumor progression. A panel of compounds, including AQP1 ion channel inhibitors (AqB011 and 5-(phenoxymethyl) furan-2-carbaldehyde, PMFC), were used to test the hypothesis that inhibition of key AQPs can limit the invasiveness of low- and high-grade EC cells. We evaluated the effects on transwell migration in EC cell lines (Ishikawa, MFE-280) and primary EC cells established from surgical tissues (n = 8). Quantitative PCR uncovered classes of AQPs not previously reported in EC that are differentially regulated by hormonal signaling. With estradiol, Ishikawa showed increased AQPs 5, 11, 12, and decreased AQPs 0 and 4; MFE-280 showed increased AQPs 0, 1, 3, 4, 8, and decreased AQP11. Protein expression was confirmed by Western blot and immunocytochemistry. AQPs 1, 4, and 11 were colocalized with plasma membrane marker; AQP8 was intracellular in Ishikawa and not detectable in MFE-280. AQP1 ion channel inhibitors (AqB011; PMFC) reduced invasiveness of EC cell lines in transwell chamber and spheroid dispersal assays. In Ishikawa cells, transwell invasiveness was reduced ~41% by 80 µM AqB011 and ~55% by 0.5 mM 5-PMFC. In MFE-280, 5-PMFC inhibited invasion by ~77%. In contrast, proposed inhibitors of AQP water pores (acetazolamide, ginsenoside, KeenMind, TGN-020, IMD-0354) were not effective. Treatments of cultured primary EC cells with AqB011 or PMFC significantly reduced the invasiveness of both low- and high-grade primary EC cells in transwell chambers. We confirmed the tumors expressed moderate to high levels of AQP1 detected by immunohistochemistry, whereas expression levels of AQP4, AQP8, and AQP11 were substantially lower. The anti-invasive potency of AqB011 treatment for EC tumor tissues showed a positive linear correlation with AQP1 expression levels. In summary, AQP1 ion channels are important for motility in both low- and high-grade EC subtypes. Inhibition of AQP1 is a promising strategy to inhibit EC invasiveness and improve patient outcomes.