Combined mTORC1/mTORC2 inhibition blocks growth and induces catastrophic macropinocytosis in cancer cells

Abstract

The mammalian target of rapamycin (mTOR) pathway, which plays a critical role in regulating cellular growth and metabolism, is aberrantly regulated in the pathogenesis of a variety of neoplasms. Here we demonstrate that dual mTORC1/mTORC2 inhibitors OSI-027 and PP242 cause catastrophic macropinocytosis in rhabdomyosarcoma (RMS) cells and cancers of the skin, breast, lung, and cervix, whereas the effects are much less pronounced in immortalized human keratinocytes. Using RMS as a model, we characterize in detail the mechanism of macropinocytosis induction. Macropinosomes are distinct from endocytic vesicles and autophagosomes in that they are single-membrane bound vacuoles formed by projection, ruffling, and contraction of plasma membranes. They are positive for EEA-1 and LAMP-1 and contain watery fluid but not organelles. The vacuoles then merge and rupture, killing the cells. We confirmed the inhibition of mTORC1/mTORC2 as the underpinning mechanism for macropinocytosis. Exposure to rapamycin, an mTORC1 inhibitor, or mTORC2 knockdown alone had little or reduced effect relative to the combination. We further demonstrate that macropinocytosis depends on MKK4 activated by elevated reactive oxygen species. In a murine xenograft model, OSI-027 reduced RMS tumor growth. Molecular characterization of the residual tumors was consistent with the induction of macropinocytosis. Furthermore, relative to the control xenograft tumors, the residual tumors manifested reduced expression of cell proliferation markers and proteins that drive the epithelial mesenchymal transition. These data indicate a role of mTORC2 in regulating tumor growth by macropinocytosis and suggest that dual inhibitors could help block refractory or recurrent RMS and perhaps other neoplasms and other cancer as well.

Characterization of mTOR Activity and Metabolic Profile in Pediatric Rhabdomyosarcoma

“…Meanwhile, the results of the latest clinical trial suggest the benefit of additional temsirolimus therapy in comparison with bevacizumab [ 18 ]. Further improvement is expected via using dual inhibitors, since these can overcome rapalog resistance by targeting both the mTORC1 and the C2 complexes or the PI3K-mTOR pathway simultaneously [ 5 , 35 , 36 , 37 ]. Some investigations have reported the strong inhibitory effect of mTORC1/2 dual inhibitors in vitro as well.…”

Rapamycin and mTORC2 inhibition synergistically reduce contraction‐stimulated muscle protein synthesis

“…Moreover, a previous study in rhabdomyosarcoma cells indicated that while mTORC1 inhibition (rapamycin treatment) or mTORC2 inhibition (Rictor knockdown) alone had little effect, combined inhibition of mTORC1 and mTORC2 effectively inhibited tumour growth (Srivastava et al . 2019). Interestingly, muscle contraction increased SGK1 expression, and this effect was attenuated by combined Rictor mKO and rapamycin treatment.…”

The Dual Role of Macropinocytosis in Cancers: Promoting Growth and Inducing Methuosis to Participate in Anticancer Therapies as Targets

“…The mTOR pathway is often disrupted in cancers, and the intersection between mTOR and TPCs suggests intriguing therapeutic possibilities (197). In cancers associated with skin, breast, lung and cervix, and rhabdomyosarcoma (RMS), Srivastava et al found that the use of dual mTORC1/mTORC2 inhibitors, OSI-027 and PP242, resulted in over-activation of macropinocytosis, inhibiting tumor survival (24). In further experiments, xenograft RMS in mice treated with OSI-027 showed reduced tumor growth, reduced proliferation, and excessive macropinocytosis in cancer cells.…”

“…In particular, Kim et al discovered that macropinocytosis can promote the survival of malnourished PTEN-deficient prostate cancer cells (21). In recent years, studies have reported that plasma membrane vacuolar ATPase (V-ATPase) (22), Syndecan 1 (SDC1) (23), mTORC1/mTORC2 (24), and epidermal growth factor receptor (EGFR) pathways (25) are closely associated with the molecular mechanism of macropinocytosis in tumor cells. In addition, the C-Jun N-terminal kinase (JNK) signaling pathway played a key role in methuosis (26).…”