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

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

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

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

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

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