Constitutive Overactivation of Phospholipase D/mTORC2 Pathway in Uterine Leiomyosarcomas vs STUMPs, Leiomyomas and Myometria
Q Shen, X Duan, ME Rodriguez, S Dhingra, RE Brown. The University of Texas Medical School at Houston, Houston, TX
Background: mTOR exists in two complexes: mTORC1 and mTORC2. mTORC1 is predominantly cytoplasmic and sensitive to rapamycin, while a significant proportion of mTORC2 is nuclear and relatively resistant to rapamycin. We previously reported mTOR was constitutively activated in uterine leiomyosarcomas; this activation specifically occurred in the nucleus, suggesting a role for mTORC2, not mTORC1, in leiomyosarcoma tumorigenesis. Unlike mTORC1, which is known to be activated by PI3K/Akt, little is known about the regulation of mTORC2 activity. Recently phospholipase D (PLD) and its metabolite phosphatidic acid (PA) have emerged as an alternative regulator of the mTOR signaling-binding of PA activates both mTORC1 and mTORC2; the effect of PA is competitive with rapamycin. In this study, we investigated the expression of the two major PLD isoforms, PLD1 and PLD2, and one of the key downstream mediators of mTORC2, p-Akt (Ser473) in the leiomyosarcomas (LMS), uterine smooth muscle tumors of uncertain malignant potential (STUMPs), leiomyomas (LM) and normal myometria.
Design: A formalin-fixed, paraffin-embedded tissue microarray was constructed including 12 LMS, 8 STUMPs, 17 LM, and 11 normal myometria. Immunohistochemistry was used to detect PLD1, PLD2, and p-Akt (Ser473). Protein expression was quantified with brightfield microscopy regarding staining pattern, cellular percentage and intensity.
Results: PLD2 expression is predominantly in the nucleus. Strong PLD2 expression is seen in 83% LMS, 38% STUMPs versus 12% LM and 9% in controls. As for PLD1, nuclear expression is the main pattern in LMS, whereas faint cytoplasmic staining is the main pattern in all other groups. Moderate PLD1 expression is seen in 59% LMS but none in STUMPs, LM or controls. mTORC2 phosphorylates Akt at Ser473. In line with our prior findings on p-mTOR, p-Akt (Ser473) also resides largely in the nucleus in LMS. In contrast, it exhibits weak cytoplasmic and nuclear expression in all other groups. Strong p-Akt staining is seen in 59% LMS versus 12% STUMPs, and 0% in LM and controls.
Conclusions: We report for the first time that PLD is overexpressed in uterine leiomyosarcomas. Our data show the PLD/mTORC2 pathway is constitutively overactivated in leiomyosarcomas, evidenced by nuclear co-localization and overexpression of PLD1, PLD2, p-mTOR and p-Akt (Ser473). These findings provide a rationale for targeting PLD activity to enhance the efficacy of rapamycin in leiomyosarcomas.
Wednesday, March 24, 2010 9:30 AM
Poster Session V # 228, Wednesday Morning