Simultaneously Targeting Tissue Transglutaminase and Kidney Type Glutaminase Sensitizes Cancer Cells to Acid Toxicity and Offers New Opportunities for Therapeutic Intervention

Most cancer cells undergo characteristic metabolic changes that are commonly referred to as the Warburg effect, with one of the hallmarks being a dramatic increase in the rate of lactic acid fermentation. This leads to the production of protons, which in turn acidifies the microenvironment surrounding tumors. Cancer cells have acquired resistance to acid toxicity, allowing them to survive and grow under these detrimental conditions. Kidney type glutaminase (GLS1), which is responsible for the conversion of glutamine to glutamate, produces ammonia as part of its catalytic activities and has been shown to modulate cellular acidity. In this study, we show that tissue, or type 2, transglutaminase (TG2), a γ-glutamyl transferase that is highly expressed in metastatic cancers and produces ammonia as a byproduct of its catalytic activity, is up-regulated by decreases in cellular pH and helps protect cells from acid-induced cell death. Since both TG2 and GLS1 can similarly function to protect cancer cells, we then proceeded to demonstrate that treatment of a variety of cancer cell types with inhibitors of each of these proteins results in synthetic lethality. The combination doses of the inhibitors induce cell death, while individual treatment with each compound shows little or no ability to kill cells. These results suggest that combination drug treatments that simultaneously target TG2 and GLS1 might provide an effective strategy for killing cancer cells

HSF1 aptamer inhibits mitogenic signaling.

<p>(<b>A</b>) HSF1 inhibition attenuates EGF receptor activation following the addition of EGF to HeLa cells. (<b>B</b>) HSF1 inhibition by iaRNA^HSF1 causes a depletion of the total levels and activated forms of Erk1/2. The left most three lanes are a serial dilution of parental line extracts that provides a quantification standard curve. Ectopic expression of HSP70 or HSP90 suppresses the inhibition of mitogenic signaling in the iaRNA^HSF1 expressing cells.</p

Expression of the RNA construct targeting HSF1 inhibits its occupancy at heat shock loci <i>in vivo</i>.

<p>(A) Control RNA (RevRA1) and aptamer RNA (iaRNA^HSF1) constructs are expressed to similar levels in HeLa and IMR90 cells after 24 hrs post transfection (RNA values normalized to GAPDH, n = 3). (<b>B</b>) Disruption of HSF1's interaction with its cognate DNA elements by iaRNA ^HSF1. ChIP assays in iaRNA^HSF1 (or RevRA1) expressing HeLa cells show that iaRNA^HSF1 expression can effectively inhibit HSF1 binding to the <i>Hsp90</i> and <i>Hsp70</i> promoter loci <i>in vivo</i> (n = 3). Antibodies used in ChIP assays are specific for mammalian HSF1 or HSF2 proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096330#pone.0096330-Sarge2" target="_blank">[20]</a>. BG  =  Background.</p

Specific binding of the aptamer to human HSF1 <i>in vitro</i>.

<p>(<b>A</b>) Electrophoretic motility shift assay (EMSA) using radiolabeled iaRNA^HSF1 (1 nM) and increasing amounts of human HSF1 protein shows that the aptamer binds to its target avidly. (<b>B</b>) Quantification of independent EMSA reveals the apparent affinity of the iaRNA^HSF1 for HSF1 as Kd∼25 nM (n = 5).</p

Effective targeting of HSF1 activity reduces the levels of molecular chaperone proteins.

<p>(<b>A</b>) Western blot analysis showing the depletion of specific molecular chaperone proteins in aptamer or control expressing cells. Hsp90 co-expression rescues specific molecular chaperones observed in HSF1 inhibited aptamer expressing cells. The asterisk indicates PARP degradation product, a marker of apoptosis. The left most three lanes are a serial dilution of parental line extracts that provides a quantification standard curve. (<b>B</b>) Quantification of the results observed in panel A (n = 4, error indicates %SEM).</p

Inhibiting Heat Shock Factor 1 in Human Cancer Cells with a Potent RNA Aptamer

<div><p>Heat shock factor 1 (HSF1) is a master regulator that coordinates chaperone protein expression to enhance cellular survival in the face of heat stress. In cancer cells, HSF1 drives a transcriptional program distinct from heat shock to promote metastasis and cell survival. Its strong association with the malignant phenotype implies that HSF1 antagonists may have general and effective utilities in cancer therapy. For this purpose, we had identified an avid RNA aptamer for HSF1 that is portable among different model organisms. Extending our previous work in yeast and Drosophila, here we report the activity of this aptamer in human cancer cell lines. When delivered into cells using a synthetic gene and strong promoter, this aptamer was able to prevent HSF1 from binding to its DNA regulation elements. At the cellular level, expression of this aptamer induced apoptosis and abolished the colony-forming capability of cancer cells. At the molecular level, it reduced chaperones and attenuated the activation of the MAPK signaling pathway. Collectively, these data demonstrate the advantage of aptamers in drug target validation and support the hypothesis that HSF1 DNA binding activity is a potential target for controlling oncogenic transformation and neoplastic growth.</p></div

iaRNA^HSF1 expression attenuates transformed growth.

<p>HSF1 inhibition by iaRNA^HSF1 inhibits transformed growth in soft agar. Soft agar analysis of non-transfected HeLa cells (top left) or control RNA over-expressing HeLa (bottom left), shows that iaRNA^HSF1 over-expression (bottom right) inhibits cellular transformation (colony formation) in a similar manner as treatment of HeLa cells with 150 nM 17-AAG (top right) (Day 14).</p

MALS and AUC based analysis of solution oligomeric state.

<p><b>a)</b> MALS data for GACwt displaying mass fractions of oligomeric species in the protein solution in the protein concentration range 1.4 µM to 9.7 µM as detected on MALS with estimated R_z values (RMS radius) given on the x-axis. Elution concentration of the dominant peak is given in the legends. The error bars represent calculated fitting errors. <b>b)</b> MALS data for GACwt displaying phosphate dependent changes in protein oligomeric state. Mass fractions of species as detected on MALS with estimated R_z values given on the x-axis. Protein concentration was kept constant at 31 µM for all samples. The error bars represent calculated fitting errors. <b>c)</b> AUC sedimentation velocity data for GACwt construct showing a protein concentration dependent distribution of oligomeric states. The obtained continuous size distributions are plotted against the S values, corrected for buffer density and viscosity at 20°C (S_w20), for the different protein concentrations as shown in the figure. <b>d)</b> Inorganic phosphate dependence of the activity of GACwt. The data is shown as mean +/− SD from three independent experiments. The line through the data points is drawn by inspection.</p

Solution concentrations and basic biophysical parameters derived from the SAXS data.

<p>Forward intensity scattering, the <b>I(0)</b> values, are estimated from data un-scaled for concentration. <b>Guinier Approximate MW (Guinier App</b><b>MW)</b> and <b>Guinier App R_g</b> are estimated from the forward scattering. <b>OLIGO MW</b> and <b>OLIGO R_g</b> estimates are derived from OLIGOMER program analysis. <b>EOM MW</b> and <b>EOM R_g</b> are estimates generated from EOM analysis. Theoretical MW for glutaminase C wild type construct is 234.20 kDa for tetramer and 117.10 kDa for dimer. Theoretical R_g is 57.50 Å for tetramer and 41.64 Å for dimer. The theoretical radius of Gyration for an octamer and a 16mer growing in an elongated direction is 94.7 Å and 186.5 Å respectively. Theoretical MW for glutaminase C construct with truncated C-terminal is 103.24 kDa for dimeric and 318.72 kDa for hexameric protein. R_g for GACwt and GACΔC are estimated to be very similar within the accuracy of SAXS data.</p

SAXS based analysis of solution systems flexibility and oligomeric states.

<p>a) Bar plot depicting the EOM estimated oligomer distribution (marked with E in legend), the SASRFMX distribution of dimers and tetramers (marked with S) and the OLIGOMER analysis estimated distribution (marked with O) of GACwt at the analyzed protein concentrations given on the x-axis in µM units. <b>b)</b> Bar plot depicting the EOM estimated oligomer distribution (marked with E) and the OLIGOMER analysis estimated distribution (marked with O) of GACΔC at the analyzed protein concentrations given on the x-axis in µM units. For a) and b) the EOM-derived distribution was estimated by taking the structures giving the best fit to the experimental curve. <b>c)</b> Bar plot showing the derived oligomer distribution given by OLIGOMER analysis as volume fractions for GACwt phosphate titration screen. <b>d)</b> Bar plot showing the OLIGOMER distribution as volume fractions for GACΔC phosphate titration screen. <b>e)</b> EOM analysis of concentrations screen of GACwt. R_g distribution of GACwt corresponding to the pool of structures (given pool of dimers, tetramers and octamers is shown in green). <b>f)</b> EOM analysis of concentrations screen of GACΔC. R_g distribution of GACΔC corresponding to the pool of structures (given pool of dimers, tetramers and octamers is shown in green).</p