Dose-dependent toxicity and acquired resistance are two major challenges limiting the efficacious treatment of mammary carcinoma (MC) with doxorubicin. Herein, we investigated the function of Trefoil Factor 3 (TFF3) in the sensitivity and acquired resistance of estrogen receptor positive (ER+) MC cells to doxorubicin. Doxorubicin treatment of ER+MC cells increased TFF3 expression. The depletion of TFF3 by siRNA or inhibition with a small molecule TFF3 inhibitor (AMPC) synergistically enhanced the efficacy of doxorubicin in ER+MC through the suppression of doxorubicin-induced AKT activation and enhancement of doxorubicin-induced apoptosis. Elevated expression of TFF3 and increased activation of AKT were also observed using a model of acquired doxorubicin resistance in ER+MC cells. AMPC partially re-sensitized the doxorubicin resistant cells to doxorubicin-induced apoptosis. Indeed, doxorubicin resistant ER + MC cells exhibited increased sensitivity to AMPC as a single agent compared to doxorubicin sensitive cells. In vivo, AMPC attenuated growth of doxorubicin sensitive ER+MC xenografts whereas it produced regression of xenografts generated by doxorubicin resistant ER+MC cells. Hence, TFF3 inhibition may improve the efficacy and reduce required doses of doxorubicin in ER+MC. Moreover, inhibition of TFF3 may also be an effective therapeutic strategy to eradicate doxorubicin resistant ER+MC.

Increased expression of trefoil factor 3 (TFF3) has been reported in colorectal carcinoma (CRC), being correlated with distant metastasis and poor clinical outcomes. Amongst the CRC subtypes, mesenchymal (CMS4) CRC is associated with the worst survival outcome. Herein, the functional roles of TFF3 and the pharmacological inhibition of TFF3 by a novel specific small molecule TFF3 inhibitor-2-amino-4-(4-(6-fluoro-5-methylpyridin-3-yl)phenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (AMPC) in CMS4 CRC was explored. Forced expression of TFF3 in CMS4 CRC cells promoted cell proliferation, cell survival, foci formation, invasion, migration, cancer stem cell like behaviour and growth in 3D Matrigel. In contrast, siRNA-mediated depletion of TFF3 or AMPC inhibition of TFF3 in CMS4 CRC cells decreased oncogenic behaviour as indicated by the above cell function assays. AMPC also inhibited tumour growth in vivo. The TFF3-stimulated oncogenic behaviour of CMS4 CRC cells was dependent on TFF3 activation of the p44/42 MAPK (ERK1/2) pathway. Furthermore, the forced expression of TFF3 decreased the sensitivity of CMS4 CRC cells to 5-fluorouracil (5-FU); while depleted TFF3 expression enhanced 5-FU sensitivity in CMS4 CRC cells. 5-FU treatment induced TFF3 expression in CMS4 CRC cells. AMPC, when used in combination with 5-FU in CMS4 CRC cells exhibited a synergistic inhibitory effect. In summary, this study provides functional evidence for TFF3 as a therapeutic target in CMS4 CRC.