Targeted inhibition of mammalian target of rapamycin signaling inhibits tumorigenesis of colorectal cancer

Abstract

Purpose: The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism, and cytoskeleton. Because approximately 60% of sporadic colorectal cancers (CRC) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs.

Experimental design: HCT116, KM20, Caco-2, and SW480 human CRC cells were used to determine the effects of pharmacologic (using rapamycin) or genetic (using RNAi) blockade of mTOR signaling on cell proliferation, apoptosis, cell cycle progression, and subcutaneous growth in vivo.

Results: We show that the mTOR complex proteins mTOR, Raptor, and Rictor are overexpressed in CRC. Treatment with rapamycin significantly decreased proliferation of certain CRC cell lines (rapamycin sensitive), whereas other cell lines were resistant to its effects (rapamycin resistant). Transient siRNA-mediated knockdown of the mTORC2 protein, Rictor, significantly decreased proliferation of both rapamycin-sensitive and rapamycin-resistant CRC cells. Stable shRNA-mediated knockdown of both mTORC1 and mTORC2 decreased proliferation, increased apoptosis, and attenuated cell cycle progression in rapamycin-sensitive CRCs. Moreover, stable knockdown of both mTORC1 and mTORC2 decreased proliferation and attenuated cell cycle progression, whereas only mTORC2 knockdown increased apoptosis in rapamycin-resistant CRCs. Finally, knockdown of both mTORC1 and mTORC2 inhibited growth of rapamycin-sensitive and rapamycin-resistant CRCs in vivo when implanted as tumor xenografts.

Conclusions: Targeted inhibition of the mTORC2 protein, Rictor, leads to growth inhibition and induces apoptosis in both rapamycin-sensitive and rapamycin-resistant CRCs, suggesting that selective targeting of mTORC2 may represent a novel therapeutic strategy for treatment of CRC.

Figure 1. Expression of mTORC1 and mTORC2 in CRC tissues and cell lines

A and B. Immunohistochemical analysis of mTOR, Raptor, Rictor, and pAkt in representative colorectal adenocarcinomas and adjacent normal mucosa (tissue microarray, 10X magnification; n=45 cases; 90 tumor cores, 8 non-neoplastic cores). C. Expression and activation of mTOR signaling pathway components in HCT116, KM20, SW480 and Caco-2 CRC cells treated with 20nM rapamycin for 24 h.

Figure 2. HCT116 and KM20 cells are rapamycin-sensitive whereas Caco-2 and SW480 cells are rapamycin-resistant

Assessment of cell proliferation by counting cell numbers directly (right) after rapamycin treatment for 48h in A. HCT116; B. KM20; C. Caco-2; D. SW480 cells (* p<0.05 vs. control). Western blot analysis (left) demonstrating expression patterns of pAkt^Ser473 and p-p70S6K^Thr389 after rapamycin treatment.

Figure 3. Rictor siRNA decreases the proliferation of rapamycin-sensitive and rapamycin-resistant CRC cells

Assessment of cell proliferation by counting cell numbers directly (middle) or MTS cell proliferation assay (right) in A. HCT116; B. KM20; C. Caco-2; D. SW480 cells transfected with Raptor, Rictor or NTC siRNA and assessed by western blotting (left) at 72h after transfection, (* p<0.05 vs. NTC siRNA).

Figure 4. Functional effects of shRNA-mediated stable inhibition of mTORC1 and mTORC2 in rapamycin-sensitive HCT116 CRC cells

A. Assessment of specific protein knockdown using Western blot analysis; B. Cell proliferation analysis by counting cell numbers directly (* p<0.05 vs. control shRNA); C. Apoptosis assessment using an ELISA detecting nucleosomes in the cytoplasm (* p<0.05 vs. control shRNA; † p<0.05 vs. -serum), D. Cell cycle progression analysis using flow cytometric analysis.

Figure 5. Functional effects of shRNA-mediated stable inhibition of mTORC1 and mTORC2 in rapamycin-resistant SW480 CRC cells

A. Assessment of specific protein knockdown using Western blot analysis; B. Cell proliferation analysis by counting cell numbers directly (* p<0.05 vs. control shRNA); C. Apoptosis assessment using an ELISA detecting nucleosomes in the cytoplasm (* p<0.05 vs. control shRNA; † p<0.05 vs. -serum), D. Cell cycle progression analysis using flow cytometric analysis.

Figure 6. Inhibition of mTORC1 and mTORC2 reduces the tumorigenic potential of rapamycin-sensitive and rapamycin-resistant CRC cells in vivo

Athymic nude mice were inoculated subcutaneously with A. HCT116 shNTC, HCT116 shmTOR, HCT116 shRaptor and HCT116 shRictor cells B. SW480 shNTC, SW480 shmTOR, SW480 shRaptor and SW480 shRictor cells. The size of the tumors was measured after 25 days. Five mice were used in each group, and the cells were inoculated at one site in each mouse. Bar graphs showing tumor volume (left) and tumor weight (middle) are shown along with representative mice with tumors from each group (right) (* p<0.05 vs. control shRNA).