PerspectiveCancer

Akt Demoted in Glioblastoma

Sci. Signal.  21 Apr 2009:
Vol. 2, Issue 67, pp. pe26
DOI: 10.1126/scisignal.267pe26

Abstract

In glioblastomas, an Akt-independent, PTEN (phosphatase and tensin homolog deleted on chromosome ten)–regulated signaling pathway links EGFR (epidermal growth factor receptor) to the phosphorylation of TOR (target of rapamycin) and of the ribosomal protein S6 and to the control of cell replication. Although PKCα (protein kinase Cα) has been identified as an essential component, the detailed wiring of this previously unexplored noncanonical pathway remains to be worked out.

The canonical signaling pathway from EGFR (epidermal growth factor receptor) to TOR (target of rapamycin) proceeds through PI3K (phosphoinositide 3-kinase) and Akt (cellular homolog of the akt retroviral oncoprotein). Akt, a serine-threonine protein kinase, has multiple targets and functions; activated Akt strongly promotes cell replication and survival. Akt commonly shows a gain of function in cancer and is considered a promising cancer target. However, the surprising outcome of a recent study on glioblastoma is that, at least in this tumor, Akt is irrelevant for the proliferative phenotype of the transformed cell.

Glioblastomas frequently show enhanced EGFR activity as a result of gene amplification or mutation (14). However, only a minority of these tumors responds to EGFR inhibitors. This clinical response is correlated with PTEN (phosphatase and tensin homolog deleted on chromosome ten) status. Only tumors that are wild-type for PTEN are sensitive to EGFR inhibition; PTEN tumors are resistant (5). A recent study by Fan and collaborators (6) now reports the discovery of a noncanonical PTEN-dependent signaling pathway that connects EGFR with its downstream targets, the serine-threonine kinase TOR, and the ribosomal protein S6.

The essential experimental findings of Fan et al. can be summarized as follows (Fig. 1). The EGFR inhibitor Erlotinib interfered with the proliferation of PTEN+ glioblastoma cell lines but did not affect the replication of PTEN glioblastoma cells. The effect of Erlotinib on cell proliferation correlated with reduced phosphorylation of TOR and of the ribosomal protein S6, making interference with the phosphorylation of these two proteins a reliable indicator for the cytostatic activity of Erlotinib in glioblastoma. In contrast, inhibition of Akt, as judged by its reduced phosphorylation at serine residue 473 (S473), did not correlate with the phosphorylation of TOR or S6 or with Erlotinib-induced cessation of glioblastoma cell proliferation. Even in PTEN+ glioblastoma cells, all three isoforms of Akt could be down-regulated with pharmacological inhibitors and short interfering RNA (siRNA) without affecting the phosphorylation status of S6K or cellular replication as reflected by the distribution of cell cycle phases.

Fig. 1

Signaling events in glioblastoma point to PKCα as a critical intermediary between EGFR and TOR. (A to C) Akt phosphorylation is disconnected from the phosphorylation of TOR and S6 and from inhibition of cell growth. (A) In PTEN+ glioblastoma cells, Erlotinib decreases the phosphorylation of Akt, TOR, and S6. It also inhibits cell proliferation. (B) In PTEN glioblastoma cells, Erlotinib still reduces Akt phosphorylation but has no effect on TOR and S6 phosphorylation. It also fails to inhibit cell growth. (C) Decreasing the activity of all three isoforms of Akt in PTEN+ with a combination of inhibitors and siRNA does not affect the phosphorylation status of TOR or that of S6. (D to F) Phosphorylation of PKC and S6 are correlated and tied to the inhibition of cell growth. (D) In PTEN+ glioblastoma cells, Erlotinib decreases the phosphorylation of PKC and S6. (E) In PTEN glioblastoma cells, Erlotinib is ineffective at down-regulating PKC and S6 phosphorylation. (F) An inhibitor of PKC decreases phosphorylation of PKC and S6 and interferes with cell growth regardless of the PTEN status of the cells. Erl, Erlotinib; BIM 1, bisindolylmaleimide; ∞ represents cell replication, red ↓ indicates decreased; = indicates no significant change.

The mitogen-activated protein kinase (MAPK) pathway, which is also stimulated by EGFR, can also lead to S6 phosphorylation. However, the abundance of phosphorylated S6 was unresponsive to treatment with the MAPK kinase (MEK) inhibitor PD98059. This observation ruled out the MAPK signaling pathway as an alternative link between EGFR and TOR in glioblastoma. With Akt and MAPK signaling eliminated, protein kinase C (PKC) emerged as another potential link between EGFR and TOR. The new experimental evidence reported by Fan et al. supports this possibility.

In PTEN glioblastoma cells, the amounts of total and of phosphorylated PKC were increased, suggesting that there was increased expression and constitutive activation of PKC in the absence of PTEN. In contrast, PTEN+ glioblastoma cells showed a phosphorylated form of PKC only after treatment with EGF, regardless of any amplification of EGFR.

Erlotinib blocked the EGF-induced phosphorylation of PKC. Although inhibition of PTEN with bisperoxovanadium did not itself change the amount of total or phosphorylated PKC, it reduced the ability of Erlotinib to block the EGF-induced phosphorylation of PKC and of S6. A combination of immunological and knock-down techniques identified PKCα as the relevant PKC isoform linking EGFR and TOR.

Additional evidence for the role of PKC was provided by experiments that revealed the effects of PKC gain of function. Activation of PKC by phorbol esters, which elicited appearance of the phosphorylated forms of this kinase, led to S6 phosphorylation, a process that was insensitive to Erlotinib. Expression of a constitutively active form of PKCα in PTEN+ glioblastoma cells tempered the effect of Erlotinib on S6 phosphorylation and on cell replication. However, mere overexpression of PKCα did not interfere with the ability of Erlotinib to inhibit the phosphorylation of S6. Finally, an inhibitor of PKC, BIM I, reduced S6 phosphorylation in PTEN+ and PTEN glioblastoma cell lines and interfered with their proliferation. In primary glioblastomas, the amounts of phosphorylated S6 and PKCα were correlated but were unrelated to the amount of phosphorylated Akt. These observations on growth signals in glioblastoma show that the critical connection between EGFR and TOR is not routed through Akt, but involves PKCα. PKCα appears to be negatively regulated by PTEN; in the absence of this regulation, inhibitors of EGFR fail to affect TOR activity and cellular replication.

The identification of a noncanonical, alternative signaling pathway from EGFR to TOR is important, but it raises many questions. PKCα has emerged from these studies as a potential cancer target in glioblastoma. The possibility that the noncanonical EGFR-TOR pathway includes other components—perhaps some that have not yet been implicated in this pathway—presents a challenge and an opportunity to identify and validate additional targets for therapy of glioblastoma. The unusual signaling in this tumor may eventually reveal weak spots that could make glioblastoma uniquely vulnerable to targeted therapy.

As yet, we know little about the EGFR-PKCα-TOR axis. The current evidence is compatible with the conclusion that PKCα operates downstream of PTEN; however, its position vis-à-vis TOR is not clearly defined. This issue could be resolved with inhibitors of PKCα and with new, ATP-competitive inhibitors of TOR (7). Sensitivity of TOR phosphorylation to inhibitors of PKC would place PKC upstream of TOR; sensitivity of PKC phosphorylation to such a nonrapamycin TOR inhibitor would place PKC downstream of TOR.

TOR has numerous cellular functions. These are carried out by two multiprotein complexes that show distinct target specificities. TORC1 (TOR complex 1) consists of TOR, raptor, and LST8. TORC2 (TOR complex 2) is composed of TOR, LST8, and rictor (810). Knockout studies have illuminated the functional differences between TORC1 and TORC2 (11, 12). TORC1 is essential for very early embryonic development; TORC2 is only required in mid-gestation. Downstream targets of TORC1 are p70S6 kinase and S6; the specific targets of TORC2 include the hydrophobic phosphorylation motif of Akt at S473 and the turn and hydrophobic phosphorylation motifs of PKCα. This last could place PKCα downstream of TORC2, but would not rule out the possibility that PKCα is upstream of TORC1. The disconnect between activation of PKCα and phosphorylation of Akt at S473 is puzzling, because both phosphorylations result from the activity of TORC2. This discrepancy suggests that other, as-yet-unidentified players affect these signaling events.

Erlotinib-sensitive and -resistant glioblastomas were also recently studied by expression profiling (13). This study showed that expression of two genes correlated with resistance to Erlotinib: IGF (encoding insulin-like growth factor) and PIK3C2b. Both encode important growth signaling molecules, but PIK3C2b, encoding the β isoform of class II PI3K (phosphoinositide-3-kinase) is of particular interest here. Unlike the isoforms of class I PI3K, some of which play well-documented roles in cancer, connections between class II PI3Ks and cancer are tentative (14). The β isoform of class II PI3K can be recruited to the EGFR and can activate Akt, initiating a signal chain that probably extends to TOR (15, 16). The products of class II PI3Ks, phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-phosphate, are substrates of PTEN, and PTEN can therefore also regulate the activities of class II PI3Ks (17). Whether the β isoform of class II PI3K is linked to the alternative EGFR-TOR signaling pathway that includes PKCα remains to be determined. The studies on Erlotinib resistance in glioblastoma add new dimensions to EGFR signaling and point out notable gaps in our understanding. They also offer the promise of new therapeutic approaches that will emerge from studies of the EGFR-PKCα-TOR connection.

Acknowledgments

18.Work by the authors is supported by grants from the National Cancer Institute. This is manuscript number 20077 of The Scripps Research Institute.

References and Notes

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