Selection of Metastatic Breast Cancer Cells Based on Adaptability of Their Metabolic State

A small subpopulation of highly adaptable breast cancer cells within a vastly heterogeneous population drives cancer metastasis. Here we describe a function-based strategy for selecting rare cancer cells that are highly adaptable and drive malignancy. Although cancer cells are dependent on certain nutrients, e.g., glucose and glutamine, we hypothesized that the adaptable cancer cells that drive malignancy must possess an adaptable metabolic state and that such cells could be identified using a robust selection strategy. As expected, more than 99.99% of cells died upon glutamine withdrawal from the aggressive breast cancer cell line SUM149. The rare cells that survived and proliferated without glutamine were highly adaptable, as judged by additional robust adaptability assays involving prolonged cell culture without glucose or serum. We were successful in isolating rare metabolically plastic glutamine-independent (Gln-ind) variants from several aggressive breast cancer cell lines that we tested. The Gln-ind cells overexpressed cyclooxygenase-2, an indicator of tumor aggressiveness, and they were able to adjust their glutaminase level to suit glutamine availability. The Gln-ind cells were anchorage-independent, resistant to chemotherapeutic drugs doxorubicin and paclitaxel, and resistant to a high concentration of a COX-2 inhibitor celecoxib. The number of cells being able to adapt to non-availability of glutamine increased upon prior selection of cells for resistance to chemotherapy drugs or resistance to celecoxib, further supporting a linkage between cellular adaptability and therapeutic resistance. Gln-ind cells showed indications of oxidative stress, and they produced cadherin11 and vimentin, indicators of mesenchymal phenotype. Gln-ind cells were more tumorigenic and more metastatic in nude mice than the parental cell line as judged by incidence and time of occurrence. As we decreased the number of cancer cells in xenografts, lung metastasis and then primary tumor growth was impaired in mice injected with parental cell line, but not in mice injected with Gln-ind cells

Elucidating the Reactivity of Oxygenates on Single-Atom Alloy Catalysts.

Publication status: PublishedDoping isolated transition metal atoms into the surface of coinage-metal hosts to form single-atom alloys (SAAs) can significantly improve the catalytic activity and selectivity of their monometallic counterparts. These atomically dispersed dopant metals on the SAA surface act as highly active sites for various bond coupling and activation reactions. In this study, we investigate the catalytic properties of SAAs with different bimetallic combinations [Ni-, Pd-, Pt-, and Rh-doped Cu(111), Ag(111), and Au(111)] for chemistries involving oxygenates relevant to biomass reforming. Density functional theory is employed to calculate and compare the formation energies of species such as methoxy (CH3O), methanol (CH3OH), and hydroxymethyl (CH2OH), thereby understanding the stability of these adsorbates on SAAs. Activation energies and reaction energies of C-O coupling, C-H activation, and O-H activation on these oxygenates are then computed. Analysis of the data in terms of thermochemical linear scaling and Bro̷nsted-Evans-Polanyi relationship shows that some SAAs have the potential to combine weak binding with low activation energies, thereby exhibiting enhanced catalytic behavior over their monometallic counterparts for key elementary steps of oxygenate conversion. This work contributes to the discovery and development of SAA catalysts toward greener technologies, having potential applications in the transition from fossil to renewable fuels and chemicals

Elucidating the Reactivity of Oxygenates on Single-Atom Alloy Catalysts

Doping isolated transition metal atoms into the surface of coinage-metal hosts to form single-atom alloys (SAAs) can significantly improve the catalytic activity and selectivity of their monometallic counterparts. These atomically dispersed dopant metals on the SAA surface act as highly active sites for various bond coupling and activation reactions. In this study, we investigate the catalytic properties of SAAs with different bimetallic combinations [Ni-, Pd-, Pt-, and Rh-doped Cu(111), Ag(111), and Au(111)] for chemistries involving oxygenates relevant to biomass reforming. Density functional theory is employed to calculate and compare the formation energies of species such as methoxy (CH3O), methanol (CH3OH), and hydroxymethyl (CH2OH), thereby understanding the stability of these adsorbates on SAAs. Activation energies and reaction energies of C–O coupling, C–H activation, and O–H activation on these oxygenates are then computed. Analysis of the data in terms of thermochemical linear scaling and Bro̷nsted–Evans–Polanyi relationship shows that some SAAs have the potential to combine weak binding with low activation energies, thereby exhibiting enhanced catalytic behavior over their monometallic counterparts for key elementary steps of oxygenate conversion. This work contributes to the discovery and development of SAA catalysts toward greener technologies, having potential applications in the transition from fossil to renewable fuels and chemicals

Highly adaptable triple-negative breast cancer cells as a functional model for testing anticancer agents.

A major obstacle in developing effective therapies against solid tumors stems from an inability to adequately model the rare subpopulation of panresistant cancer cells that may often drive the disease. We describe a strategy for optimally modeling highly abnormal and highly adaptable human triple-negative breast cancer cells, and evaluating therapies for their ability to eradicate such cells. To overcome the shortcomings often associated with cell culture models, we incorporated several features in our model including a selection of highly adaptable cancer cells based on their ability to survive a metabolic challenge. We have previously shown that metabolically adaptable cancer cells efficiently metastasize to multiple organs in nude mice. Here we show that the cancer cells modeled in our system feature an embryo-like gene expression and amplification of the fat mass and obesity associated gene FTO. We also provide evidence of upregulation of ZEB1 and downregulation of GRHL2 indicating increased epithelial to mesenchymal transition in metabolically adaptable cancer cells. Our results obtained with a variety of anticancer agents support the validity of the model of realistic panresistance and suggest that it could be used for developing anticancer agents that would overcome panresistance

Contrasting Function of Structured N-Terminal and Unstructured C-Terminal Segments of Mycobacterium tuberculosis PPE37 Protein

Pathogens frequently employ eukaryotic linear motif (ELM)-rich intrinsically disordered proteins (IDPs) to perturb and hijack host cell networks for a productive infection. Mycobacterium tuberculosis has a relatively high percentage of IDPs in its proteome, the significance of which is not known. The Mycobacterium-specific PE-PPE protein family has several members with unusually high levels of structural disorder and disorder-promoting Ala/Gly residues. PPE37 protein, a member of this family, carries an N-terminal PPE domain capable of iron binding, two transmembrane domains, and a disordered C-terminal segment harboring ELMs and a eukaryotic nuclear localization signal (NLS). PPE37, expressed as a function of low iron stress, was cleaved by M. tuberculosis protease into N- and C-terminal segments. A recombinant N-terminal segment (P37N) caused proliferation and differentiation of monocytic THP-1 cells, into CD11c, DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin)-positive semimature dendritic cells exhibiting high interleukin-10 (IL-10) but negligible IL-12 and also low tumor necrosis factor alpha (TNF-α) secretion—an environment suitable for maintaining tolerogenic immune cells. The C-terminal segment entered the macrophage nucleus and induced caspase-3-dependent apoptosis of host cells. Mice immunized with recombinant PPE37FL and PPE37N evoked strong anti-inflammatory response, validating the in vitro immunostimulatory effect. Analysis of the IgG response of PPE37FL and PPE37N revealed significant immunoreactivities in different categories of TB patients, viz. pulmonary TB (PTB) and extrapulmonary TB (EPTB), vis-a-vis healthy controls. These results support the role of IDPs in performing contrasting activities to modulate the host processes, possibly through molecular mimicry and cross talk in two spatially distinct host environments which may likely aid M. tuberculosis survival and pathogenesis

A Strategy for Testing Anticancer Agents.

<p>The strategy described in this paper is depicted as a flow diagram. See text for further details.</p

Evidence of a High EMT and a Drug-Tolerant State in MA Cells.

<p>(A) Overlap in gene expression changes between MA1 and MA2 variants. The Venn diagram depicts the number of gene probes that detected significantly higher or lower RNA levels in MA1 and MA2 cells than in the parental cell line. The common 1735 gene probes that detected the shared alterations between MA1 and MA2 cells were identified in Microsoft Excel by combining the spreadsheets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s002" target="_blank">S2</a>) and then searching for duplicate primary sequence names. (B) We analyzed by Western blotting ZEB1 and GRHL2 proteins in MA2 variants and the parental cell line SUM149-Luc (labeled SUM149). After selection in Gln-free medium, MA2 variants were cultured in a medium without or with Gln as indicated at the bottom. p1 and p7 represent passages in Gln-free medium. The parental cell line was cultured in a medium with dialyzed FBS for six passages prior to preparation of lysates for Western blots to match the MA cells. For the rightmost lane, MA2 cells were cultured in Gln-containing medium for four passages prior to preparation of the lysate. We blotted β-actin and vinculin as controls; β-actin is reduced in MA cells grown without Gln, as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487-Singh2" target="_blank">[20]</a>. (C) We analyzed by western blotting trimethylation at lysine 4 of histone H3 and acetylation at lysine 14 of histone H3 in MA1 cells and the parental cell line SUM149-Luc (labeled SUM149). We blotted total histone H3 as a control. The star marks a background band that serves as an additional gel loading control in western blots. We cultured MA cells continuously in Gln- medium for 11 passages after selection (Gln-, middle lane), or we cultured MA cells in Gln- medium for 9 passages and then switched them back to Gln+ medium for 4 passages (Gln+, right lane). We measured the relative intensities of histone H3 bands detected as K4me3 and K14ac modified forms and normalized them by dividing with the intensity of total H3 band from the corresponding samples. The normalized data are shown in a graphical form on the right. (D) A model of therapy resistance in MA cells. According to our data, the balance of epithelial versus mesenchymal phenotypes, which is determined by the GRHL2/ZEB1 ratio, is shifted toward mesenchymal phenotype in MA variants. The sizes of ovals and rectangles and the widths of arrows indicate relative levels/strengths. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s001" target="_blank">Tables S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s002" target="_blank">S2</a>.</p

Amplification of the <i>FTO</i> Gene in MA Cells.

<p>(A) CGH array analysis was performed with MA cells compared to the SUM149-Luc cell line. Chromosomal gains (indicated in red) and losses (indicated in green) in MA1 and MA2 variants are presented as a composite graphic penetrance summary after removal of pseudoautosomal regions from the X and Y chromosomes. Chromosome numbers and the specific loci involved are indicated. Further details are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s003" target="_blank">Tables S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s004" target="_blank">S4</a>. (B) FTO protein overexpression in MA cells. The cell lysates used in the analysis shown in Fig. 2 were used for these Western blots. The lysates were from the parental cell line, MA2 variants in Gln-free medium at passages 1 and 7, and MA2 variants after 4 passages in Gln-containing medium. We blotted vimentin and COX-2 as controls; we had previously detected their overexpression in MA1/Gln-independent variants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487-Singh2" target="_blank">[20]</a>. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s004" target="_blank">Tables S4</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109487#pone.0109487.s006" target="_blank">S6</a>.</p

Evidence of an Elevated Panresistance in MA Cells.

<p>MA cells and the SUM149-Luc parental cell line were simultaneously treated with various anticancer agents as indicated to determine their relative level of resistance under the conditions that would inhibit most of the proliferating cells. (A) Increased resistance to EGFR and MEK inhibition in MA cells. The cells were treated with erlotinib or AZD6244 for 11 days (d) as indicated (each treatment killed most of the cells) and were allowed to recover in a drug-free medium before the colonies were stained. Combination treatment with the two drugs lasted 10 days, since it killed cells sooner than did the single-drug treatments; then, the cells were allowed to recover for 4 days. (B) Resistance to crizotinib in MA cells. (C) Resistance to PI3K/mTOR inhibition in MA cells. The cells were treated with BEZ235 for 14 days (d), passaged, treated with doxorubicin for 7 days (the treatment killed most of the cells), and allowed to recover and form colonies in a drug-free medium for before the colonies were stained (top). The bottom panel shows a similar experiment in which cells were treated with paclitaxel instead of doxorubicin. (D) Resistance to salinomycin in MA cells. The top panel shows bright-field photographs of representative cells (10X magnification) after 6 days of treatment with salinomycin. The cells were treated with salinomycin for 7 days (which killed most of the cells) and were allowed to recover and form colonies in a drug-free medium before the colonies were stained (bottom panel). (E) Resistance to thioridazine in MA cells. (F) Resistance to PEITC in MA cells. (G) Resistance to itraconazole in MA cells. The parental SUM149-Luc cell line and MA1 cells were treated in parallel with 1 µM itraconazole for 9 days (which killed most of the cells in the parental cell line) and were allowed to recover and grow in a drug-free medium for 5 days before being stained. Since itraconazole was ineffective in killing MA1 cells, the cells grew into a continuous monolayer rather than colonies. (H) Resistance to FAC combination chemotherapy agents in MA cells. (I) Resistance to FEC combination chemotherapy agents in MA cells.</p

Selection of Rare Cancer Cells That Survive Lack of Glutamine and Glucose.

<p>(A) Half a million SUM149-Luc cells were plated in a 10-cm dish. The next day, the medium was changed to a medium containing dialyzed FBS and lacking glutamine. After growth for 34 days in the Gln-deficient medium, we stained the colonies with crystal violet. (B) Half a million SUM149-FP76 cells were plated in a 10-cm dish. The next day, the medium was changed to a medium containing dialyzed FBS lacking glucose (left dish) or lacking both glucose and glutamine (right dish). After 15 days, the media were replaced with complete medium. We stained the colonies with crystal violet after 18 days in complete medium.</p