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.

Amino acid starvation sensitizes cancer cells to proteasome inhibition.

We explored the crosstalk between protein degradation and synthesis in cancer cells. The tumorigenic cell line, MCF7, showed enhanced proteasome activity compared to the nontumorigenic line, MCF10A. Although there was no difference in the sensitivity of MCF7 and MCF10A cells to proteasome inhibition in complete growth medium, combining proteasome inhibition with amino acid deprivation led to reduced protein synthesis and survival of MCF7 cells, with a lesser effect on MCF10A cells. Additional cancer cell lines (including CAG and A431) could be strongly sensitized to proteasome inhibition by concomitant amino acid deprivation, whereas others were completely resistant to proteasome inhibition. We hypothesize that protein catabolism contributes to the pool of free amino acids available for protein synthesis, leading to a crucial role of the proteasome in cell survival during amino acid depletion, in some tumor cell lines.

Shift from apoptotic to necrotic cell death during human papillomavirus-induced transformation of keratinocytes.

Oncogenic transformation is a complex, multistep process, which goes through several stages before complete malignant transformation occurs. To identify early processes in carcinogenesis, we used an in vitro model, based on the initiating event in cervical cancer, papillomavirus transformation of keratinocytes. We compared gene expression in primary keratinocytes (K) and papillomavirus-transformed keratinocytes from early (E) and late (L) passages and from benzo[a]pyrene-treated L cells (BP). The transformed cells exhibit similar transcriptional changes to clinical cervical carcinoma. The number of transcripts expressed progressively decreased during the evolution from K to BP cells. Bioinformatic analysis, validated by detailed biochemical analysis, revealed substantial contraction of both pro- and antiapoptotic networks during transformation. Nonetheless, L and BP cells were not resistant to apoptotic stimuli. At doses of cisplatin that led to 30-60% apoptosis of K and E cells, transformed L and BP cells underwent 80% necrotic cell death, which became the default response to genotoxic stress. Moreover, appreciable necrotic fractions were observed in the cervical carcinoma cell line, HeLa, in response to comparable doses of cisplatin. The shrinkage of biochemical networks, including the apoptotic network, may allow a cancer cell to economize on energy usage to facilitate enhanced proliferation but leaves it vulnerable to stress. This study supports the hypothesis that the process of cancer transformation may be accompanied by a shift from apoptosis to necrosis.

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.