Chernet BT and Levin M (2014), Transmembrane voltage potential of somatic cell...

XB-ART-49065

Oncotarget 2014 May 30;510:3287-306. doi: 10.18632/oncotarget.1935.

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Transmembrane voltage potential of somatic cells controls oncogene-mediated tumorigenesis at long-range.

Chernet BT , Levin M .

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The microenvironment is increasingly recognized as a crucial aspect of cancer. In contrast and complement to the field's focus on biochemical factors and extracellular matrix, we characterize a novel aspect of host:tumor interaction - endogenous bioelectric signals among non-excitable somatic cells. Extending prior work focused on the bioelectric state of cancer cells themselves, we show for the first time that the resting potentials of distant cells are critical for oncogene-dependent tumorigenesis. In the Xenopus laevis tadpole model, we used human oncogenes such as mutant KRAS to drive formation of tumor-like structures that exhibited overproliferation, increased nuclear size, hypoxia, acidity, and leukocyte attraction. Remarkably, misexpression of hyperpolarizing ion channels at distant sites within the tadpole significantly reduced the incidence of these tumors. The suppression of tumorigenesis could also be achieved by hyperpolarization using native CLIC1 chloride channels, suggesting a treatment modality not requiring gene therapy. Using a dominant negative approach, we implicate HDAC1 as the mechanism by which resting potential changes affect downstream cell behaviors. Based on published data on the voltage-mediated changes of butyrate flux through the SLC5A8 transporter, we present a model linking resting potentials of host cells to the ability of oncogenes to initiate tumorigenesis. Antibiotic data suggest that the relevant butyrate is generated by a native bacterial species, identifying a novel link between the microbiome and cancer that is mediated by alterations in bioelectric signaling.

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Species referenced: Xenopus laevis
Genes referenced: cacnb3 clic1 gli1 grap2 hdac1 hdac3 kcna5 kras mag nppb rel slc5a8 slc6a4l tp53
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	Figure 1. Induced tumor-like structures (ITLS) exhibit characteristics reminiscent of human tumors(A) Rates of proliferation were analyzed in vivo in fluorescent cell cycle indicators (FUCCI pair: mKO2-zCdt1 and mAG-zGeminin) injected, ITLS-bearing embryos at stage 34. ITLS regions (black bars) have >65% more cells that are in G2/S/M phase (green insert, mAG-zGeminin) than there are in G1 phase (red insert, mKO2-zCdt1). Unperturbed regions (grey bars) showed no difference between the number of cells in G1 (red insert) and G2/S/M (green insert) phases. N=8 for all four categories. P<0.001, one-way ANOVA, tukey's post hoc analysis; different letters indicate statistically significant difference; scale bar = 250 µm.(B) Immunoperoxidase analysis of hypoxia using detection of pimonidazole protein adducts (black arrowhead) in cells reveals a 12 fold increase for immunoperoxidase staining for hypoxia per unit area in ITLS (yellow traces) than in surrounding healthy tissue. N=7; P<0.0001 Student's t-test; scale bar = 500 µm.(C) H&E staining of KRASG12D ITLS sections (yellow circular trace, 40X) for evidence of features of neoplasia reveals disorganized growth patterns, including misplaced mesodermal and endodermal cells, and mesodermal cells that are larger than those present in unperturbed regions. (D) Automated analysis of the size of Hoechst Blue stained nuclei reveals a 10% increase in the size of tumor nuclei compared to nuclei from unaffected cells. A change in morpholology of the nuclei that includes a progressive increase in nucleus size has been documented in human breast cancer cells [82]. N=16 (2371 nuclei) for both ITLS and unaffected regions.(E) Intracellular pH measurements in ITLS and Control regions were made using the fluorescent pH reporter dye BCECF. (I) A decreased fluorescence is observed in ITLS cells (T) compared to a normal region (N). (II) Upon quantification, levels of fluorescence correspond to pH values of 6.6 in ITLS and 7.41 in control regions. N=8 for both treatments; P<0.05 Student's t-test; scale bar = 1mm in tail fragment; 150 µm in magnified inserts.(F) The response of innate immunity to ITLS formation was investigated using anti-XL2 immunohistochemistry to mark the presence of leukocytes: leukocytes are primarily present around ITLS (white circular trace) and 1-3 grid (300 µM) away from ITLSs. N=12; P<0.01, one-way ANOVA, Tukey's post hoc analysis; different letters indicate statistically significant difference; scale bar = 500 µm. Error bars indicate ± 1 s.e.m in A, B, E, F.
	Figure 2. Long-range hyperpolarization suppresses the formation ITLSs(A) To visualize and track ITLSs, mRNA encoding a fusion construct of Xrel3 and tdTomato was injected into a single blastomere of 16-cell stage embryos. (I, II) ITLSs resulting from Xrel3-2A-tdTomato injections were highly fluorescent when visualized under a TRITC filter set. (III) Overlay of bright field (BF) and TRITC images shows the co-localization of ITLS and red fluorescent signal, confirming that foci of oncogene expression are the cells that make up ITLS. Scale bar = 1 mm.(B) To test whether long-range hyperpolarization suppresses ITLS formation, a potassium based hyperpolarizing channel (Kv1.5-GFP3) and Xrel3-2A-tdTomato were injected in distantly separated blastomeres of a 16-cell stage embryo. (I) While morphologically-apparent ITLSs were often missing from these embryos, the oncogenic protein was present as evidenced by the tdTomato fluorescent signal (II, II). Robust expression of the hyperpolarizing channel protein (Kv1.5-GFP3) was observed in the head and gut regions (IV, V), and away from Xrel3-2A-tdTomato expressing cells (VI). Scale bar = 1 mm.(C) Fold change in ITLS formation for oncogene injected embryos with hyperpolarized treatments compared to oncogene-only injected embryos. Oncogene-only injected embryos with ITLS have a ratio of 1, and ratio values below and above one represent fewer and more embryos with ITLS, respectively. To show the effect of ITLS suppression due to change in Vmem, three different oncogenes and hyperpolarizing reagents (based on Cl− and K+) were used. When Kv1.5 (K+ hyperpolarizing channel) was introduced non-locally in Xrel3 and Gli1 injected embryos, 39.4% and 29.4% decreases, respectively, in ITLS formation were observed. A 37.5% decrease in ITLS formation was also achieved using KRASG12D as an oncogene and 70mM Cl− as a hyperpolarizing reagent. N=225-349 embryos for each treatment; *P<0.01 Student's t-test. Error bars indicate ± 1 s.e.m.
	Figure 3. A suppression screen for chloride channels is employed to identify a channel responsible for long-range ITLS suppression(A) Oncogene injected embryos were placed in a high chloride concentration (70mM) solution, followed immediately by separate treatments with very well-known chloride transport blockers: Anthracene-9-Carboxylic Acid (ACA), indanyloxyacetic acid (IAA) and 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). (B) High chloride treatment of oncogene-injected embryos results in ~31%% decrease in KRASG12D ITLS incidence. While blocking chloride channels using ACA and NPPB did not affect the rate of this suppression, IAA treatments in the presence of high Cl− restored ITLS incidence back to oncogene-only levels. N=162, 164, 186 for oncogene only, oncogene + high Cl−, and oncogene + high Cl− + IAA, respectively; *P<0.05, χ2 compared to tumor incidence in KRASG12D only injected embryos.
	Figure 4. Hyperpolarization by the influx of Cl- through CLIC1 mediates long-range ITLS suppression(A) To visualize and track ITLSs, KRASG12D mRNA was co-injected with GFP3 (I) and tdTomato (II) into a single blastomere of 16-cell stage embryos. In both cases, ITLS co-localize with the respective fluorescent signals, confirming that oncogene-expressing cells make up ITLS. Scale bar = 1 mm.(B) When mRNA encoding Xenopus wildtype CLIC1-GFP3 was injected at a distance from KRASG12D -tdTomato, morphologically-apparent ITLS formation was often suppressed despite the presence of oncogenic protein (III). Whereas overexpression of a CLIC1 dominant negative (CLIC1C24S-tdTomato) in non-oncogene expressing cells prevents ITLS suppression despite the presence of high Cl− in the media (IV). Scale bar = 1 mm. (C) Changes in ITLS formation incidence for oncogene and CLIC1 (mutant or wildtype) injected embryos in 70mM Cl− media. ITLS incidence from oncogene-only injection was normalized to 1 so that it can be used to measure against the effects of other treatments. Embryos injected with KRASG12D followed by a high Cl− treatment showed a 31.5% decrease in ITLS incidence. Overexpression of the wildtype CLIC1 and high Cl− bath also lowered the number of embryos with ITLS by 44.3% while resulting in smaller ITLS in escapees. Overexpression of the mutant CLIC1, which encodes for defective multimer channels, appeared to block ITLS suppression by a high Cl- treatment, implicating native CLIC1 channels in Vmem mediated, long-range suppression of ITLS. N= 336-364 embryos for each treatment; P=0.01, one-way ANOVA, Tukey's post hoc analysis; different letters indicate statistically significant difference. Error bars indicate ± 1 s.e.m.
	Figure 5. Long-range suppression of ITLS by hyperpolarization is HDAC1-dependent(A)Hyperpolarization can be transduced into transcriptional pathways by processes that include Vmem-dependent transport of signaling molecules (e.g. serotonin transport through SERT), electrophoresis of morphogenes through gap junctions (GJC), calcium signaling via voltage-gated Ca++ channels (VGCC), and voltage dependent butyrate transport that results in HDAC inhibition. To identify the responsible transduction mechanism, each process was independently blocked: 10 µM fluoxetine shuts down SERT; H7, a dominant connexin disrupts the transport function of endogenous gap junctions; 0.1 mM cadmium chloride effectively blocks VGCCs; and a dominant negative 0.5 ng HDAC1 injection inactivates endogenous HDAC1. (B) In each case, a treatment used to probe a given transduction mechanism was targeted non-local to KRASG12D injected cells. KRASG12D injections and treatments were administered at the 16-cell stage, and embryos were then raised to stage 34 before scoring the presence of ITLS.(C) When analyzing ITLS incidences compared to oncogene only injected embryos – DN-HDAC1blocks the effects of CLIC1 mediated hyperpolarization, thus brining ITLS formation to the level of oncogene-only injection. Introducing DN-HDAC1 at a distance from KRASG12D without hyperpolarization increases ITLS incidence by 35.6%, implicating a role for HDAC1 in long-range ITLS suppression. N=164-251 embryos; *P<0.05, χ2 compared to tumor incidence in KrasG12D only injected embryos.
	Figure 6. Targeted killing of butyrate producing Bacteria increases ITLS incidence To determine the source of butyrate, which is responsible for ITLS suppression through HDAC inhibition, variety of antibiotics were used to target butyrate-producing BacteriaCompared to KRASG12D-only injected embryos, novobiocin and antibiotic cocktail (gentamicin, metrodinazole, vancomycin, and clindamycin) treatments significantly increased ITLS formation by 32.1% and 26.8%, respectively. Adding nalidixic acid, which works by inhibiting DNA gyrase as novobiocin does, did not significantly alter ITLS formation, showing only an 8.2% increase in ITLS incidence. N=537, 318, 431, 117 for KRASG12D, KRASG12D + Nalidiixic Acid, KRASG12D + Novobiocin, KRASG12D + antibiotics cocktail; P<0.05, *P<0.01, χ2 compared to ITLS incidence in KRASG12D-only injected embryos.
	Figure 7. A model for V – mediated, HDAC1-dependent, long-range control of cell dynamics during neoplastic transformation It is known that HDAC1 inhibition leads to the altered transcriptional of regulation of several ion channel genes including NaV 1.2 [197] – a voltage gated sodium channel encoding geneOne model consistent with our data is that the resulting Na+ gradient (more positive charge) would attract butyrate producing bacteria away from oncogene expressing cells (illustrated using Escherichia coli in Supp. Fig 1). Gram-positive bacteria are necessary for the production of butyrate, which gets imported into cells via SLC5A8 to reduce proliferation rate by inducing cell cycle arrest in tumor cells. Driving these bacteria away from ITLS cells would reduce the number of butyrate molecules available near oncogene cells, thus blocking ITLS suppression.

References [+] :

Abdalla, Correlation of nuclear morphometry of breast cancer in histological sections with clinicopathological features and prognosis. 2009, Pubmed