Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation

Protein synthesis and degradation in primary and transformed keratinocytes (A) Cells were seeded in 6-well plates.

<p>After 48 h, the medium was replaced with medium containing Promix (³⁵S-Methionine/Cysteine), and cells were incubated for another 1 h. Labeled proteins were precipitated with trichloro acetic acid (TCA) and counted in a scintillation counter. ³⁵S incorporation (cpm) was normalized to protein amount (µg), measured using the bound Coumassie blue method. Error bars represent the SD of quadruplicate samples. (B) Activity of translation factors required for cap-dependent protein synthesis. Representative western blot. Protein levels and phosphorylation of translation factors were lower in L and BP cells than in K and E cells, under standard growth conditions. ERK2 served as a gel loading control. (C) Cells were seeded in 6-well plates. After 48 h, the medium was replaced with medium containing Promix (³⁵S-Methionine/Cysteine) for 1 h, washed three times, and incubated in normal growth medium for another hour. Labeled proteins were precipitated with TCA and counted in a scintillation counter. ³⁵S incorporation retained during the “chase” was normalized to ³⁵S incorporation during the “pulse”. Error bars represent the SD of quadruplicate samples.</p

Regulation of protein synthesis during cell transformation (A) Representative western analysis of K, E, L and BP cell lysates, using the indicated antibodies.

<p>ERK2 served as a gel loading control. PKB and AMPK were visualized on one blot and P-ACC on a separate blot. The activity of PKB dropped and the activity of AMPK rose in L and BP. (B) The phosphorylation of c-Src (Y416) was induced during transformation. The amount of total c-Src protein (phosphorylated and non-phosphorylated) remained unchanged. (C) Cells were treated with PP1 to inhibit c-Src. After 4 h cells were lysed, and western blot analysis was performed. ERK2 served as a gel loading control. Representative western analysis is shown.</p

IRES-dependent translation increases in transformed cells (A) Levels of proteins that are subject to both cap and IRES-mediated translation under standard growth conditions.

<p>A representative western blot is shown. ERK2 served as a gel loading control. (B) Reciprocal constructs for cap-dependent and IRES-dependent translation of firefly luciferase (FFL) and renilla luciferase (SPL) constructs are illustrated. The ratio of IRES-dependent to cap-dependent product is represented by SPL/FFL activity for construct 1 (C1, left) and by FFL/SPL activity for construct 2 (C2, right). Bars represent the SD of duplicate samples</p

Analysis of growth and energetic parameters during cell transformation (A) Cells were grown for several hours under standard growth conditions.

<p>Growth was monitored with methylene blue staining (O.D. 630 nm, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000628#s4" target="_blank">Materials and Methods</a>). The slope of each curve represents the growth rate of the particular cell type. Error bars represent the SD of quadruplicate samples. (B) Time course of glucose uptake by K, E, L and BP cells, measured using the Amplex® Red Glucose/Glucose Oxidase Assay Kit [Molecular Probes (Invitrogen)]. Glucose consumption (O.D. 560 nm) was normalized to cell mass (O.D. 630 nm), as measured by methylene blue. The slopes represent glycolysis rates. Error bars represent the SD of duplicate samples. (C) A Clark-type oxygen electrode was used to measure oxygen consumption in primary keratinocytes (K = 1) and papilloma-transformed cells. Oxygen consumption indicates respiration rate, and was expressed as nmoles of oxygen consumed per minute per total protein amount. Error bars represent the SD of three independent experiments. (D) Cellular ATP levels were measured by the CELLTITER-GLO™ Luminescent cell viability assay (Promega). Luminescence was normalized to cell mass (O.D. 630 nm), as measured using methylene blue. Error bars represent the SD of quadruplicate samples.</p

Cell size and protein amount (A) Cell size was measured using FACS.

<p>Cell size (cell diameter) as a function of FSC is presented (K = 1). Error bars represent the SD of quadruplicate samples. (B) 64 aliquots of each cell type were counted, using a counting chamber (hemocytometer). The amount of protein per 250,000 cells was measured (K = 100%), using the bound Coumassie blue method (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000628#s4" target="_blank">Materials and Methods</a>). Error bars represent the SD of quadruplicate samples.</p

AMPK is involved in the regulation of IRES-dependent translation.

<p>L (A) and BP cells (B) were treated with 150 nM rapamycin (R) or 10 µM compound C (C) or 150 nM rapamycin in the presence of 10 µM compound C (R+C) for 24 hours. (C) E cells were treated with 150 nM rapamycin (R) or 2 mM AICAR (A) or 150 nM rapamycin in the presence of 2 mM AICAR (R+A) for 24 hours. Levels of proteins subject to both cap and IRES-translation were analyzed by Western blot using the indicated antibodies. ERK2 served as a gel loading control.</p

Short description of the functions of key factors involved in cap-mediated translation.

<p>Short description of the functions of key factors involved in cap-mediated translation.</p

Up-regulation of AMP-activated Protein Kinase in Cancer Cell Lines Is Mediated through c-Src Activation*

We report that the activation level of AMP-dependent protein kinase AMPK is elevated in cancer cell lines as a hallmark of their transformed state. In OVCAR3 and A431 cells, c-Src signals through protein kinase Cα, phospholipase Cγ, and LKB1 to AMPK. AMPK controls internal ribosome entry site (IRES) dependent translation in these cells. We suggest that AMPK activation via PKC might be a general mechanism to regulate IRES-dependent translation in cancer cells