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.Cancer is an evolving, complex process, which goes through several stages before full malignancy. In vivo, cell immortalization is followed by the development of benign lesions, which later progress into malignant tumors, finally metastasizing to other tissues. As it evolves, a cancer cell relinquishes pathways that interfere with proliferation and escapes from some of the restrictions of multicellular organisms. This process enables the cancer cell to proliferate in a broad range of naturally occurring microenvironments but may leave it vulnerable to rare or unexpected perturbations (1) (e.g. genotoxic stress). The reactions of cancer cells, which acquire numerous molecular changes, may differ significantly from the reactions of normal cells to genotoxic stress.Apoptosis is tightly controlled by the ability of the cell to integrate many pro-and antiapoptotic signals. Thus, the decision to live or to die in response to death signals is a choice the cell makes in the face of its cellular context. Molecular changes acquired by cancer cells may influence the connectivity of the signaling pathways and disturb the apoptotic network. As a result, the cancer cell may die by alternative death modes in response to genotoxic drugs.In order to follow the changes that occur during the evolution of cancer, we use a model in which one can follow the progression from the normal phenotype all the way to the transformed phenotype, based on the natural evolution of cervica...

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.

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 C␣, phospholipase C␥, 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.The nonreceptor tyrosine kinase c-Src was the first protooncogene to be discovered in the vertebrate genome. c-Src is overexpressed and activated in many human cancers, including lung, skin, colon, breast, and ovarian malignancies. The elevated protein levels and catalytic activity have been linked to the development of cancer and progression to metastases. However, despite the fact that c-Src is one of the oldest and most investigated proto-oncogenes, the precise functions of c-Src in cancer remain unclear. In addition to increasing cell proliferation, a key role of c-Src in cancer seems to be to promote invasion and motility, functions that might contribute to tumor progression (for review, see Refs. 1-3).Previously, we had shown that AMPK 2 activation appeared to result from progressive activation of c-Src (4). Recently published data also demonstrate that AMPK activation can be induced by c-Src, independently of the AMP/ATP ratio in endothelial cells (5, 6).The AMP-activated protein kinase (AMPK) maintains the balance between ATP production and consumption in eukaryotic cells (for review, see Refs. 7,8). Mammalian AMPK is sensitive to the cellular AMP/ATP ratio and is activated by metabolic stresses such as hypoxia and glucose deprivation. AMPK is also modulated by cytokines that regulate whole body energy balance, including leptin and interleukin-6 (for review, see Refs. 7,8).AMPK is a conserved heterotrimeric protein comprised of a catalytic (␣1 or ␣2) subunit and two regulatory (␤1 or ␤2 and ␥1, ␥2, or ␥3) subunits. Following binding of AMP to the ␥ subunit, AMPK is activated by LKB1, which phosphorylates AMPK at a conserved threonine residue (Thr-172) on the ␣ subunit. This mechanism allows the system to act as a sensor of cellular energy status (for review, see Refs. 7,8).Upon activation, AMPK switches on catabolic pathways that generate ATP, such as the uptake and metabolism of glucose and fatty acids. AMPK also switches off anabolic pathways that consume ATP, but are not necessary for survival, such as the synthesis of fatty acids, glycogen, and proteins. Thus, AMPK plays a dual role in cancer. On the one hand, AMPK activates glycolysis and angiogenesis, promoting cell survival (7-10). On the other hand, active AMPK also switches off anabolic processes, including protein synthesis, through the inhibition of mTOR (11).Previously, we found that AMPK activity increases during HPV16 transformation of human keratinocytes (4). Therefore, we were intrigued to examine the status of AMPK in cancer cells. Here, we report that AMPK activity is high in severa...

SummaryWe 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. IUBMBIUBMB Life, 62(10): 757-763, 2010