Non-transformed Human Epithelial Cells Research Articles

Abstract LB294: Targeting fatty acid oxidation to boost the anti-tumor effects of nutrient starvation in solid malignancies

Abstract Introduction Reducing glucose and growth factor provision to cancer cells through in vitro starvation (STV) or in vivo fasting/fasting-mimicking diets (FMDs) delays tumor progression and prolongs survival in tumor-bearing mice. More recently, cyclic FMD has been shown to be safe and associated with promising metabolic and immunomodulatory effects in cancer patients. However, cyclic fasting and FMD only retard tumor progression, but they do not result in tumor cure when used alone. Here, we hypothesized that tumor cells exposed to STV activate Fatty Acid Oxidation (FAO) as an alternative metabolic pathway that could be responsible for primary or acquired tumor cell resistance to STV. Methods We investigated the effects of FAO inhibition (FAOi) in combination with STV on the proliferation and survival of human or murine cell lines of Triple Negative Breast Cancer (TNBC) (n=16), HR+HER2- breast cancer (n=3), colorectal (n=1), pancreatic (n=1) and lung cancer (n=1), as well as of non-transformed human epithelial cells (n=3). STV consisted of a 50% reduction of extracellular glucose and serum in cell growth media. To inhibit FAO, we used the Carnitine Palmitoyl transferase 1 inhibitor Etomoxir or ST1326. Results STV+FAOi resulted in cooperative or highly synergistic antiproliferative effects in all cancer models tested, but not in normal cells. At longer time points, STV+FAOi enhanced tumor cell death through the activation of apoptosis. Depending on the specific tumor type, the cooperative effects of STV and FAOi were mostly mediated by glucose or fatty acid restriction in the STV medium. On the other hand, the reduction of extracellular growth factors did not affect cell proliferation upon STV+FAOi combination. Intracellular ATP levels were reduced upon cell exposure to the combined treatment at later time points. However, the supplementation of several sources of ATP, such as glutamine or TCA cycle intermediates, to tumor cells did not rescue the antitumor effects of STV+FAOi, thus excluding the strong impact of this combination on tumor cell bioenergetics. On the other hand, STV+FAOi caused an excessive intracellular antioxidant response, which in part mediated the synergistic effects of STV+FAOi. Indeed, Etomoxir alone mildly increased the NADPH/NADP+ ratio, whereas its combination with STV resulted in a remarkable increase of this ratio. In parallel, combining the antioxidant molecules n-acetylcysteine (NAC) and reduced glutathione (GSH) with FAOi caused antitumor effects that recapitulate those produced by STV+FAOi treatment. Conclusions FAO is a potential resistance mechanism to nutrient starvation, with broad antitumor activity in different models of murine and human malignancies, but not in normal cells. In cancer cells, FAOi synergizes with STV by strongly affecting intracellular redox balance. FAOi is a new strategy that should be explored in future preclinical in vivo experiments and in clinical studies to potentiate the antitumor effects of nutrient starvation in solid malignancies. Citation Format: Marzia Santamaria, Beatrice Cantarelli, Arta Ajazi, Chiara Milanese, Giulia Salvadori, Pier Giorgio Mastroberardino, Paola Antonia Corsetto, Giancarlo Pruneri, Filippo De Braud, Claudio Vernieri. Targeting fatty acid oxidation to boost the anti-tumor effects of nutrient starvation in solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB294.

Tetraploidy-Associated Genetic Heterogeneity Confers Chemo-Radiotherapy Resistance to Colorectal Cancer Cells.

Tetraploidy, or whole-genome duplication, is a common phenomenon in cancer and preludes chromosome instability, which strongly correlates with disease progression, metastasis, and treatment failure. Therefore, it is reasonable to hypothesize that tetraploidization confers multidrug resistance. Nevertheless, the contribution of whole-genome duplication to chemo-radiotherapy resistance remains unclear. Here, using isogenic diploid and near-tetraploid clones from three colorectal cancer cell lines and one non-transformed human epithelial cell line, we show a consistent growth impairment but a divergent tumorigenic potential of near-tetraploid cells. Next, we assessed the effects of first-line chemotherapeutic drugs, other commonly used agents and ionizing radiation, and found that whole-genome duplication promoted increased chemotherapy resistance and also conferred protection against irradiation. When testing the activation of apoptosis, we observed that tetraploid cells were less prone to caspase 3 activation after treatment with first-line chemotherapeutic agents. Furthermore, we found that pre-treatment with ataxia telangiectasia and Rad3 related (ATR) inhibitors, which targets response to replication stress, significantly enhanced the sensitivity of tetraploid cells to first-line chemotherapeutic agents as well as to ionizing radiation. Our findings provide further insight into how tetraploidy results in greater levels of tolerance to chemo-radiotherapeutic agents and, moreover, we show that ATR inhibitors can sensitize near-tetraploid cells to commonly used chemo-radiotherapy regimens.

Analysis of Secretome Changes Uncovers an Autocrine/Paracrine Component in the Modulation of Cell Proliferation and Motility by c-Myc

Proteins secreted by cancer cells are a major component of tumor microenvironment. However, little is known on the impact of single oncogenic lesions on the expression of secreted proteins at early stages of tumor development. Because c-Myc overexpression is among the most frequent alterations in cancer, here we investigated the effect of sustained c-Myc expression on the secretome of a nontransformed human epithelial cell line (hT-RPE). By using a quantitative proteomic approach, we have identified 125 proteins in conditioned media of hT-RPE/MycER cells upon c-Myc induction. Analysis of the 49 proteins significantly down-regulated by c-Myc revealed a marked enrichment of factors associated with growth inhibition and cellular senescence. Accordingly, media conditioned by hT-RPE cells expressing c-Myc show an increased ability to sustain hT-RPE cellular proliferation/viability. We also find a marked down-regulation of several structural and regulatory components of the extracellular matrix (ECM), which correlates with an increased chemotactic potency of the conditioned media toward fibroblasts, a major cellular component of tumor stroma. In accordance with these data, the expression of the majority of the genes encoding proteins down-regulated in hT-RPE was significantly reduced also in colorectal adenomatous polyps, early tumors in which c-Myc is invariably overexpressed. These findings help to elucidate the significance of c-Myc overexpression at early stages of tumor development and uncover a remarkable autocrine/paracrine component in the ability of c-Myc to stimulate proliferation, sustain tumor maintenance, and modulate cell migration.

Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells

Chemoprotective effects of nutritional compounds are usually studied in cell lines. Studies using primary human colon cells have been limited due to the lack of established methods regarding their culture. We therefore optimized isolation and culture of non-transformed human epithelial cells from individual donors to enrich viable cells and sufficient amounts of intact RNA. Isolated epithelial cells were seeded in different coated cell culture dishes combined with several media (2–24h). To avoid cells from anoikis, also intact colon crypts were isolated to maintain cell interactions. These crypts were incubated with gut fermentation products (24h) derived from indigestible carbohydrates. In none of the coated (fibronectin, laminin) cell culture dishes isolated epithelial cells did attach. The number of these cells remaining in suspension, decreased already after 2h to 20%. Intact colon crypts cultured as pellets showed a stable viability up to 24h (91±4%) and were suitable to gain a sufficient quantity of RNA. The use of colon crypts with an appropriate cell culture medium could double the lifespan of intestinal epithelial cells from 12 up to 24h and represents a promising approach to study early events in carcinogenesis and chemoprevention as well as other diseases of the colon.

Abstract LB-8: A novel function of Vps34 in regulation of cell proliferation

Abstract Many new insights into the biology of class III PI3Kinase have recently been reported. Most of these studies, however, used mainly transformed cells. It is unclear whether class III PI3Kinase works similarly in non-transformed mammalian cells. We explored this by depleting two non-transformed human epithelial cell lines of Vps34, the catalytic subunit of the lipid kinase using shRNA. Interestingly, in a stark contrast to transformed cells where depletion of Vps34 was associated with decreased growth, Vps34 deficiency in non-transformed cells resulted in a marked increase in cell proliferation. The deregulated autophagy function and E-cadherin-dependent cell-cell contacts found in Vps34-depleted cells appeared to be not involved in cell growth regulation as neither silence of Atg7 expression nor inactivation of E-cadherin had any effect on cell proliferation. Reporter luciferase-based ten-pathway screens suggested an altered activity of the MAPK pathway. Indeed, biochemical analysis indicated a sustained ERK activation resulting from the impediment of endosome-mediated EGFR turnover in Vps34 deficient cells. A contribution of the endosomal pathway to Vps34-mediated regulation of cell proliferation was further confirmed by using Rab5 and Rab11 mutants that perturbed early and recycling endosomes, respectively. Importantly, inactivation of Vps34 compromised the cellular barrier against oncogene-mediated transformation, as demonstrated by the increased susceptibility of Vps34 depleted cells to Ras-induced colony formation in vitro and tumor development in mice. Our data collectively uncover a novel role for the class III PI3Kinase-endosomal pathway in regulation of cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-8. doi:10.1158/1538-7445.AM2011-LB-8

Maspin (SERPINB5) Is an Obligate Intracellular Serpin

Maspin (SERPINB5) is a tumor suppressor lost in breast and prostate cancer whose molecular function is unknown. It is a non-inhibitory member of the clade B serpins suggested to play a role in a plethora of intracellular and extracellular settings, yet its normal cellular distribution has never been clarified. Here we investigate the distribution of maspin in non-transformed human epithelial cells. By indirect immunofluorescence, maspin has a nucleocytoplasmic distribution in breast (MCF10A) and prostate (RWPE-1) cells and, by immunoblotting and pulse-chase analyses, is neither glycosylated nor secreted. Cell surface biotinylation studies also show that maspin is not present at the cell surface. Differentiation of MCF10A cells into three-dimensional acini results in the redistribution of maspin from the nucleus to the cytoplasm but does not result in secretion. Addition of an efficient conventional signal peptide to maspin directs it into the secretory pathway and results in glycosylation but not secretion. We further show that maspin in the cytoplasm of MCF10A cells is a soluble monomeric protein that is not detectably associated with the cytoskeleton or other extractable components. Taken together, these results suggest that maspin is restricted to an intracellular, possibly nuclear, role in which it influences cell-matrix interactions indirectly. It is probably released only as a consequence of cell damage or necrosis.

Radiation sensitivity increases with proliferation‐associated telomere dysfunction in nontransformed human epithelial cells

Epidemiological studies have demonstrated age differences among human adults in susceptibility to radiation, with cancer cases attributable to radiation being more frequent for older individuals at time of exposure. In addition to the notion that susceptibility increases because of progressive decline in DNA monitoring and immunosurveillance, telomere function is now emerging as a new and important factor in modulating cellular and organism sensitivity to ionizing radiation. The link between telomeres and radiosensitivity is well-documented in humans, but the causal events remain elusive. In this paper, it is shown that irradiated human epithelial cells with short dysfunctional telomeres derived from normal mammary gland display elevated DNA damage. An approach identifying the specific chromosomes with critically shortened telomeres in each donor has allowed us to conclude that short dysfunctional telomeres in human epithelial cells join radiation-induced DNA broken ends, thus interfering with their efficient repair. These findings argue against telomeres participating as sensors or transducers of DNA damage, as previously suggested. Rather, our current findings give support to the idea that dysfunctional telomeres, by acting as an additional joining option, reduce the repair fidelity of DNA broken-ends induced by radiation throughout the genome. In the mammary gland, age-dependent telomere attrition due to epithelial turnover, together with the accretion of checkpoint deficiencies, might render the accumulation of short dysfunctional telomeres. This implies that the risks associated with mammography screening could be higher than previously assumed. Our results have the possibility of imprinting a temporal dimension onto radiation sensitivity, namely, that shortened telomeres in aged cells may more easily compromise normal tissue function in the elderly.

Complete Loss of Wild-Type TP53 in a Nontransformed Human Epithelial Cell Line Is Preceded by a Phase During Which a Heterozygous TP53 Mutant Effectively Outgrows the Homozygous Wild-Type Cells

HMT-3522 is a spontaneously immortalized cell line derived from a fibrocystic breast lesion. After continuous accumulation of genetic changes, the cell line was transformed from a nontumorigenic to a malignant phenotype. One of the earliest genetic aberrations is a missense mutation of codon 179 (His179Asn) in the tumor suppressor gene TP53 leading to outgrowth of a cell type expressing only the mutant form of TP53. In this report, we extend earlier investigations to reveal the genetic background for the evolution from homozygous wild type to hemizygous mutated cells. The status of the TP53 alleles was followed at different stages by fluorescence in situ hybridization (FISH) and allele-specific PCR (ASPCR) on total DNA, as well as flow-sorted chromosomes—taking advantage of a size difference between the two homologues of chromosome 17 that harbor TP53 on 17p. This further allowed us to determine on which of the two chromosomes the mutated allele was located. The results presented here show that the cells have undergone an evolution from homozygous wild type for TP53 to heterozygous (His179Asn mutation in one allele), and finally to a hemizygous mutated state (deletion of the remaining wild-type allele). The finding of a transient period in which heterozygous cells dominate the population before the eventual outgrowth of hemizygous cells strongly indicates that the His179Asn mutation results in a tp53 protein with a dominant negative effect that does not totally abrogate the function of wild type TP53 in vitro.