Morphoproteomic evidence of constitutively activated and overexpressed mTOR pathway in cervical squamous carcinoma and high grade squamous intraepithelial lesions

Abstract

Human papilloma virus (HPV) infection of the uterine cervix is linked to the pathogenesis of cervical cancer. Preclinical in vitro and in vivo studies using HPV-containing human cervical carcinoma cell lines have shown that the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor, erlotinib, can induce growth delay of xenografts. Activation of Akt and mTOR are also observed in cervical squamous cell carcinoma and, the expression of phosphorylated mTOR was reported to serve as a marker to predict response to chemotherapy and survival of cervical cancer patients. Therefore, we investigated: a) the expression level of EGFR in cervical squamous cell carcinoma (SCC) and high-grade squamous intraepithelial lesions (HSIL) versus non-neoplastic cervical squamous epithelium; b) the state of activation of the mTOR pathway in these same tissues; and c) any impact of these signal transduction molecules on cell cycle. Formalin-fixed paraffin-embedded tissue microarray blocks containing 20 samples each of normal cervix, HSIL and invasive SCC, derived from a total of 60 cases of cervical biopsies and cervical conizations were examined. Immunohistochemistry was utilized to detect the following antigens: EGFR; mTOR pathway markers, phosphorylated (p)-mTOR (Ser2448) and p-p70S6K (Thr389); and cell cycle associated proteins, Ki-67 and S phase kinase-associated protein (Skp)2. Protein compartmentalization and expression were quantified in regard to proportion (0-100%) and intensity (0-3+). Mitotic index (MI) was also assessed. An expression index (EI) for pmTOR, p-p70S6K and EGFR, respectively was calculated by taking the product of intensity score and proportion of positively staining cells. We found that plasmalemmal EGFR expression was limited to the basal/parabasal cells (2-3+, EI = 67) in normal cervical epithelium (NL), but was diffusely positive in all HSIL (EI = 237) and SCC (EI 226). The pattern of cytoplasmic p-mTOR and nuclear p-p70S6K expression was similar to that of EGFR; all showed a significantly increased EI in HSIL/SCC versus NL (p<0.02). Nuclear translocation of p-mTOR was observed in all SCC lesions (EI = 202) and was significantly increased versus both HSIL (EI = 89) and NL (EI = 54) with p<0.015 and p<0.0001, respectively. Concomitant increases in MI and proportion of nuclear Ki-67 and Skp2 expression were noted in HSIL and SCC. In conclusion, morphoproteomic analysis reveals constitutive activation and overexpression of the mTOR pathway in HSIL and SCC as evidenced by: increased nuclear translocation of pmTOR and p-p70S6K, phosphorylated at putative sites of activation, Ser2448 and Thr389, respectively; correlative overexpression of the upstream signal transducer, EGFR, and increases in cell cycle correlates, Skp2 and mitotic indices. These results suggest that the mTOR pathway plays a key role in cervical carcinogenesis and targeted therapies may be developed for SCC as well as its precursor lesion, HSIL.

Keywords: cervical squamous carcinoma; high grade SIL; mTOR pathway; morphoproteomics.

Figure 1

Representative hematoxylin-eosin (H&E) images of uterine cervical mucosa illustrating non-neoplastic (normal) squamous epithelium (frame A), high grade squamous intraepithelial lesion (HSIL; frame B) and invasive cervical squamous cell carcinoma (SCC, frame C). Companion images depicting the level of expression of epidermal growth factor receptor (EGFR) on the plasmalemmal (cell membranous) aspect of the epithelial cells reveal: moderate to strong (2 to 3 +) DAB [brown] chromogenic signal limited to the basal and parabasal cells of normal epithelium (frame D); and overexpression of EGFR protein in HSIL and SCC evident by strong (3+) plasmalemmal expression that decorates virtually all lesional cells (frames E and F, respectively) (DAB [brown] chromogen; original magnification ×200).

Figure 2

Relative expression levels for mTOR pathway components, mTOR (phosphorylated on serine 2448) and p70S6K (phosphorylated on threonine 389) reveal: weak to moderate (1-2+) cytoplasmic and nuclear immunoreactivity (brown [DAB] chromogenic signal) for p-mTOR (Ser 2448) in the normal cervical squamous epithelium, particularly in the basal and parabasal cells (frame A) but with moderate to strong (2-3+) cytoplasmic and nuclear expression of this analyte in both HSIL and invasive squamous cell carcinoma involving virtually all of the lesional cells (frames B and C, respectively); and with diffuse and qualitatively similar nuclear expression levels for p-p70S6K (Thr 389) in normal cervical squamous epithelium, HSIL and invasive squamous cell carcinoma (frames D, E and F respectively; also see quantitative Expression Index [EI] for each of these analytes in Figure 4 and statistical differences in Table 2. Note the moderate cytoplasmic signal for p-p70S6K in supra basal zones of normal cervical epithelium (frame D) vis-à-vis that seen in HSIL and invasive squamous cell carcinoma (frames E and F). Subepithelial and interstitial inflammatory cell infiltrate present in HSIL and invasive squamous cell carcinoma shows variable nuclear signals for p-mTOR (frames B and C) and p-p70S6K (frames E and F) (original magnification ×200).

Figure 3

Analysis of nuclear expression levels for cell cycle–associated markers, Ki-67 (G1, S, G2 and M phases of cell cycle) and Skp2 (S phase kinase-associated protein) reveals limited (parabasal only) expressions for both analytes in normal cervical squamous epithelium (frames A and B, respectively) but with strong and frequent expression involving middle to full thickness epithelium in HSIL and strong and diffuse expressions in invasive squamous cell carcinoma (frames B and D, and E and F for Ki-67 and Skp2, respectively) (original magnification ×200; also see numerical data and statistical comparisons in Tables 1 and 2).

Figure 4

Graphic representation of expression index (EI) for EGFR and mTOR pathway markers, p-mTOR (Ser 2448) and p-p70S6K (Thr 389) comparing and contrasting normal (uterine cervical squamous epithelium), HSIL and invasive SCC. EI is the product of staining intensity (0-3+) and percent of positive staining cells (0-100%). C, cytoplasmic; N, nuclear; C/P, cytoplasmic/plasmalemmal (membranous).

Figure 5

A schematic composite of the complex protein circuitries identified by the authors and others utilizing immunohistochemistry in cases of HSIL and/or invasive squamous cell carcinoma of the uterine cervix [42, 43, 48] and depicting established interrelationships with both HPV-encoded oncoproteins - E5, E6, and E7 [–5, 7, 45, 46] and with the mTOR pathway. HPV infection of cervical squamous epithelium leads to the incorporation of HPVoncogenes, E5, E6 and E7 into the genome resulting in the production of the corresponding oncoproteins. E5 activates EGFR and delays its degradation resulting in increased EGFR expression and then its signaling through the PI3'-K/Akt and ras/Raf kinase/ERK pathway, thereby contributing to phosphorylative activation of Akt and ERK1/2, respectively. This accounts for the expression of p-Akt and p-ERK1/2 in HSIL and invasive squamous cell carcinoma. Downstream signaling by p-Akt results in the constitutive activation and expression of p-mTOR (Ser 2448). Interaction of E6 oncoprotein with TSC2 (tuberin) contributes to signaling through the mTOR and probably ERK pathways [43, 45, 49, 50]. The former accounts for increased expression of p-p70S6K (Thr 389) and its downstream effector p-pS6 kinase and the latter collaborates with p-mTOR and contributes to activation of eIF4E through the phosphorylative inactivation of 4E-BP1. Additionally, p-ERK1/2 could contribute to mTOR pathway signaling by also contributing to the inactivation of TSC2 [51]. Translational synthesis of proteins including E7 oncoprotein results from the activation of eIF4E. E7 oncoprotein has been linked to growth and survival (immortalization) of HPV-associated, transformed cancer cells and to the signaling via the Akt/mTOR pathway by upregulating Akt activity. Cell cycle progression associated with mTOR and ERK pathway downstream signaling and aided by destabilization of wild type p53 by E6 oncoprotein is consistent with the increased expressions of Ki-67 and S phase kinase-associated protein (Skp) 2 and mitotic indices in HSIL and invasive squamous cell carcinoma. * indicates established immunohistochemical findings with respect to protein analytes; HSIL: high grade squamous intraepithelial lesion; HPV, human papilloma virus; mTOR, mammalian target of rapamycin; EGFR, epidermal growth factor receptor; PI3’-K, phosphatidylinositol 3-kinase; ERK, extracellular signal-regulated kinase; eIF4E, eukaryotic initiation factor 4E; 4E-BP1, 4E-binding protein-1.