IRES inhibition induces terminal differentiation and synchronized death in triple-negative breast cancer and glioblastoma cells

Abstract

Internal ribosome entry site (IRES)-mediated translation is a specialized mode of protein synthesis which malignant cells depend on to survive adverse microenvironmental conditions. Our lab recently reported the identification of a group of compounds which selectively interfere with IRES-mediated translation, completely blocking de novo IGF1R synthesis, and differentially modulating synthesis of the two c-Myc isoforms. Here, we examine the phenotypic consequences of sustained IRES inhibition in human triple-negative breast carcinoma and glioblastoma cells. A sudden loss of viability affects the entire tumor cell population after ∼72-h continuous exposure to the lead compound. The extraordinarily steep dose-response relationship (Hill-Slope coefficients −15 to −35) and extensive physical connections established between the cells indicate that the cells respond to IRES inhibition collectively as a population rather than as individual cells. Prior to death, the treated cells exhibit prominent features of terminal differentiation, with marked gains in cytoskeletal organization, planar polarity, and formation of tight junctions or neuronal processes. In addition to IGF1R and Myc, specific changes in connexin 43, BiP, CHOP, p21, and p27 also correlate with phenotypic outcome. This unusual mode of tumor cell death is absolutely dependent on exceeding a critical threshold in cell density, suggesting that a quorum-sensing mechanism may be operative. Death of putative tumor stem cells visualized in situ helps to explain the inability of tumor cells to recover and repopulate once the compound is removed. Together, these findings support the concept that IRES-mediated translation is of fundamental importance to maintenance of the undifferentiated phenotype and survival of undifferentiated malignant cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5161-4) contains supplementary material, which is available to authorized users.

Translational control of the undifferentiated phenotype in ER‑positive breast tumor cells: Cytoplasmic localization of ERα and impact of IRES inhibition

“…7E ), very different from the morphology observed with terminal differentiation at high cell density ( Fig. 3 ) ( 12 ).…”

“…The dominant isoform of Myc (p64) is a major contributor to breast oncogenesis ( 20 ). However, the p67 isoform of Myc, which is known to be generated via use of an alternative upstream initiation codon, has been attributed potent growth inhibitory and pro-death properties ( 21 , 22 ) and was also observed in the triple-negative breast tumor cells subjected to IRES inhibition, where it correlated with terminal differentiation and comprehensive death of those cells ( 11 , 12 ). These decreases in IGF1R, CDK1 and ERα and the modulation of Myc translation (including shift to the p67 isoform) may be key molecular events that contributed to the stochastic death of low-density tumor cells in which IRES-mediated translation has been inhibited.…”

“…We previously reported ( 12 ) that highly undifferentiated triple-negative breast tumor cells (as well as glioblastoma and osteosarcoma cells) were forced into terminal differentiation when continuously exposed to IRES inhibitor cpd_P for >72 h. The triple-negative breast tumor cells did not tolerate this transition to a fully differentiated phenotype and underwent a synchronized population-wide cell death event which was non-apoptotic and shared a number of features with cornification. In the ER-positive breast tumor cells, as little as 15–24 h exposure to cpd_W led to a terminal differentiation outcome which very closely resembled that observed in the triple-negative cells, with similar increases in polarity and structural organization.…”

“…We recently described ( 11 ) the first of three IRES-inhibitor lead compounds (cpd_P), comparing and contrasting the effects on the IGF1R and MYC IRESs. We found that sustained IRES inhibition induced terminal differentiation in triple-negative breast cancer and other highly undifferentiated tumor types ( 12 ).…”

Translation acrobatics: how cancer cells exploit alternate modes of translational initiation

“…By regulating expression of these oncogenes, ITAFs impinge on several hallmarks of cancer . The examples presented above suggest that pharmacological interventions that increase or decrease expression or activity of ITAFs could be a potential targeted therapy in certain types of cancers . However, since most IRESs that aid in translating oncogenic mRNAs appear to be recognized by single ITAFs , one valid avenue of drug therapy could be to disrupt the interaction or recognition of IRESs by their respective ITAF.…”