An individual can develop cancer if cells in their body gain genetic mutations that enable the cells to divide more rapidly and move—or metastasize—to other tissues and organs. These mutations can alter the chemistry of the cell; for example, the inside of a cancer cell is much more alkaline (has a higher pH) than the inside of a normal cell. This helps the cancer cells to grow and divide rapidly, and move to other parts of the body, but it is not clear how important this change in pH within the cell is for the development of cancer.

Previous studies have shown that many cancer cells increase the amounts or activities of the proteins that remove hydrogen ions from the cell, and so make the inside of a cell more basic. Here, Grillo-Hill et al. increased the amount of one of these proteins, NHE1, in fruit flies to study its effects on healthy cells.

The experiments showed that the larval cells that produced more NHE1 were more alkaline than normal cells, and this caused the cells to become less organized and grow more rapidly. Grillo-Hill et al. also found that NHE1 can work together with the oncogene RasV12 that promotes cancer development to enhance migration within the developing wings of the larvae.

Next, Grillo-Hill et al. tested whether the change in pH is necessary for cells to become cancerous. Flies missing the gene that makes NHE1 appeared normal. However, if these cells also have the oncogene Raf—which, like RasV12, promotes cancer—the inside of the cells gradually became so acidic that they died. Grillo-Hill et al. also found that blocking the activity of NHE1 in human cancer cells could lead to increased death but does not increase the death of normal cells.

Grillo-Hill et al.'s findings show that increasing the internal pH of healthy cells leads to characteristics similar to those found in cancer cells. Furthermore, cancer cells can die if they become more acidic. This suggests that targeting pH levels could guide the development of treatments for cancer that selectively kill cancer cells while leaving normal cells unharmed.

Dysregulated pH is a common characteristic of cancer cells, which have a lower extracellular pH (pHe) and higher intracellular pH (pHi) than normal cells. The lower pHe of tumors, confirmed by pH-sensitive PET radiotracers, MR spectroscopy and MRI (Zhang et al., 2010), contributes to local metastatic invasion (Cardone et al., 2005; Rofstad et al., 2006; Stock et al., 2008; Stock and Schwab, 2009; Busco et al., 2010; Estrella et al., 2013). Increased pHi enables a number of cancer cell behaviors, including promoting cell proliferation (Pouysségur et al., 1982; Kapus et al., 1994; Putney and Barber, 2003), glycolytic metabolism (Reshkin et al., 2000; Dietl et al., 2010; Webb et al., 2011), migration (Denker and Barber, 2002; Patel and Barber, 2005; Frantz et al., 2008; Stock and Schwab, 2009), and invasion (Reshkin et al., 2000; Hinton et al., 2009), as well as limiting apoptosis (Matsuyama et al., 2000; Lagadic-Gossmann et al., 2004). The higher pHi of cancer cells is paradoxical because increased production of metabolic acids generated by aerobic glycolysis would be predicted to lower pHi. However, many cancers have elevated expression or activity of proteins that facilitate increased pHi, including carbonic anhydrase 9 (Swietach et al., 2007), H⁺-ATPases (Martinez-Zaguilan et al., 1993; Sennoune et al., 2004; Hinton et al., 2009), the ubiquitously expressed Na⁺-H⁺ exchanger NHE1 (McLean et al., 2000; Miraglia et al., 2005; Chiang et al., 2008; Yang et al., 2011) and the monocarboxylate transporter family members MCT1 and MCT4 (Pinheiro et al., 2010; Halestrap, 2013). Therapeutic targeting of these proteins to reduce H⁺ efflux and lower pHi has been suggested for limiting cancer progression (Webb et al., 2011; Harguindey et al., 2013), based primarily on findings with xenograft models and isolated cells. Xenograft tumor growth is suppressed by inhibiting NHE1 (Lagarde et al., 1988; Yang et al., 2011) or MCT1 (Sonveaux et al., 2008; Colen et al., 2011) activity. In cell models, inhibiting NHE1 activity reduces viability of breast cancer (Reshkin et al., 2003) and leukemic cells (Rich et al., 2000; Reshkin et al., 2003), and silencing MCT4 expression with RNA interference decreases survival of renal carcinoma cells (Gerlinger et al., 2012). Although previous studies suggest important roles for pH dynamics in regulating cancer cell behaviors, whether increased pHi is sufficient or necessary for cancer progression in vivo remains unresolved.

To better understand how H⁺ efflux and increased pHi affect cancer cell behaviors we asked two distinct but related questions. First, is increased pHi by NHE1 over-expression in the absence of other transforming signals sufficient to induce dysplasia? Second, is H⁺ efflux by NHE1 necessary for oncogene-induced dysplasia? We found that over-expression of Drosophila melanogaster Dnhe2, an ortholog of mammalian NHE1, causes dysplasia, increases proliferation and facilitates oncogene-induced cell invasion in vivo. Additionally, we found that reducing H⁺ efflux genetically or pharmacologically limits oncogene-induced increases in proliferation and has synthetic lethality with oncogenic Raf in the Drosophila retina as well as in human mammary epithelial cells expressing oncogenic RasV12.

To address our first question we overexpressed Dnhe2 in Drosophila eye imaginal discs. Three NHE isoforms have been identified in Drosophila (Dnhe1-3), and sequence analysis of the C-terminal cytoplasmic domains suggests DmNhe2 is most homologous to the mammalian plasma membrane NHE1, one of nine mammalian isoforms (Figure 1A). Conserved features include a lysine/arginine-rich region (KR motif) in the juxtamembrane segment that binds phosphatidylinositol 4,5-bisphosphate in the plasma membrane (Putney and Barber, 2003), a conserved binding motif for the calcineurin homologous protein CHP (Lin and Barber, 1996; Pang et al., 2001), a glutamic acid residue in the transmembrane domain that is essential for H⁺ efflux (E358 in DmNhe2 and E266 in human NHE1) and consensus sites for phosphorylation by Akt and ATM/ATR kinases (Figure 1A, and Figure 1—figure supplements 1, 2). The only previous report on DmNhe2 function indicates a role in charge-dependent plasma membrane recruitment of Dishevelled to establish planar cell polarity (Simons et al., 2009).

Figure 1 with 3 supplements see all

Download asset Open asset

We used the GMR-GAL4 driver to over-express Dnhe2 (GMR > Dnhe2) in wandering third larval instar (wL3) eye imaginal discs, a developmental stage with proliferating cells that marks the onset of retinal pattern formation. GMR > Dnhe2 expression induced an externally rough adult eye with phenotypes consistent with dysplasia in the underlying epithelium (Figure 1B). In wild type Drosophila, the adult eye is a precisely patterned epithelial structure with hexagonal unit eyes, termed ommatidia, uniformly arrayed across the surface (Figure 1B,C, left). Wild type ommatidia contain eight photoreceptor neurons (R cells), forming a polarized, asymmetrical trapezoid (Figure 1D, tracing in Figure 1E, left). In GMR > Dnhe2 flies, ommatidial organization was disrupted and individual facets were irregular in shape and size (Figure 1B,C, middle). In section, GMR > Dnhe2 retinae had misoriented ommatidia, abnormal cell fates and missing R cells (Figure 1D,E, middle). This phenotype is similar to disrupted tissue organization with expression of a short, unregulated isoform of Dnhe2 in a subset of retinal cells using sevGAL4 (Simons et al., 2009). We also used eyelessGAL4 to express Dnhe2 earlier in eye development, which caused a very mild rough eye phenotype. To resolve whether increased H⁺ efflux by Dnhe2 is necessary for disrupted tissue architecture we generated transgenic flies expressing a mutant Dnhe2^E358I, analogous to the NHE1^E266I mutation that abrogates H⁺ efflux (Denker and Barber, 2002). Externally, GMR > Dnhe2^E358I retinae had subtle defects in patterning, including rare bristle placement defects (Figure 1B,C, right). In section, no patterning defects were evident (Figure 1D,E, right), suggesting that dysplasia in GMR > Dnhe2 eyes is dependent on ion transport.

To determine whether Dnhe2 over-expression increased pHi we generated transgenic flies expressing a genetically encoded, ratiometric mCherry-pHluorin pH sensor that was previously used to measure pHi in cultured cells (Koivusalo et al., 2010; Choi et al., 2013). After ∼24 hr of transgene expression there was no detectable difference in pHi between wild type and GMR > Dnhe2 in wL3 eye imaginal discs (data not shown). However, in pupal eyes at 42 hr after puparium formation (42H apf), pHi in GMR > Dnhe2 was significantly higher than wild type in both apical non-neural cells (Figure 1F,G) and in photoreceptor neurons (Figure 1—figure supplement 3). In contrast, pHi in pupal eyes expressing the mutant GMR > Dnhe2^E358I was not different than wild type (Figure 1F,G), indicating that this mutant lacks H⁺ efflux. The delay between onset of transgene expression and increased pHi could be due to accumulation of functionally processed DNhe2 protein. These data suggest that patterning defects in GMR > Dnhe2 retinae reflect constitutively increased pHi, although we cannot rule out possible effects of decreased pHe or altered sodium levels.

To determine the underlying cause of the adult rough eye phenotype, we examined GMR > Dnhe2 retinae at earlier time points in development. In wild-type pupal eyes, precise retinal organization is apparent on the apical surface with immunolabeling of the adherens junction protein beta-catenin (Figure 2A, schematic drawing on right). Four central cone cells (blue) display stereotyped contacts, and are enwrapped by two semi-circular primary pigment cells (orange) to form the ommatidial core. Individual ommatidia are separated by a single layer of secondary pigment cells (yellow), with tertiary pigment cells (green) and bristles (purple) at alternating vertices to form the hexagonal shape of ommatidia; these cells are collectively referred to as lattice cells. GMR > Dnhe2 pupal eyes had severely disrupted cell shapes and tissue organization, and ommatidia overall were smaller than in wild type. Ommatidia had aberrant numbers of cone and primary pigment cells (pink). Some ommatidia were fused (red), reflecting fused lenses seen in adult eyes (Figure 1B). Filamentous actin organization (labeled by rhodamine-conjugated phalloidin, purple, Figure 2A) in wild type eyes showed increased labeling in cone cells and lattice cells compared to the primary pigment cells. In GMR > Dnhe2 pupal retinae, overall phalloidin levels were lower, and the differences between cell types were less pronounced.

Figure 2

Download asset Open asset

Disorganization was present through the depth of the pupal retinal epithelium. At the level of cone cell nuclei (Figure 2B, labeled ‘c’), 4 nuclei per ommatidium are present in wild type, and phalloidin labels the center of each ommatidium. The number and arrangement of nuclei were aberrant with GMR > Dnhe2 expression, although phalloidin labeling appeared similar to wild type. Optical sections ∼3 µm deeper in the epithelium reveal R cell nuclei arranged in a characteristic pattern revealed by the neuronal antigen Elav, where four R cells (R1/3/6/7) are visible in wild type. We saw strong phalloidin labeling in the center of each ommatidium, marking the nascent rhabdomeres. In GMR > Dnhe2 pupal eyes, the number and arrangement of R cells in this section were disrupted, and we did not see the phalloidin signal in the center of the ommatidia. Optical sections ∼3 µm deeper in wild type pupal eyes show the stereotyped arrangement of the other four R cells (R2/5/7/8). In GMR > Dnhe2 retinae, this organization was disrupted, and there are more Elav-negative nuclei present than in wild type. On the basal side of the WT pupal eyes, phalloidin-labeled asters mark the centers of ommatidia, and Elav-negative nuclei of lattice cells were seen. In GMR > Dnhe2 eyes, phalloidin-labeling was decreased and did not show the same pattern as in wild type. Additionally, Elav-positive nuclei persist in this layer, suggesting severe disorganization of R cells. There were also disorganized Elav-negative nuclei, which may be lattice cells. From these data, increasing pHi in the developing eye causes profound defects in tissue architecture that affect all cell types in the developing Drosophila eye, and shows severe disruption of patterning throughout this complex, multilayered epithelium. These phenotypes are consistent with dysplasia, and are similar to effects of expressing activated, oncogenic Raf, as shown below.

Consistent with increased pHi being a conserved proliferative signal from yeasts (Orij et al., 2012) to mammals (Pouysségur et al., 1982; Kapus et al., 1994; Putney and Barber, 2003), cell proliferation was substantially increased with GMR > Dnhe2 expression. A regulated band of proliferation in wL3 eye imaginal discs termed the second mitotic wave (SMW) is present posterior to the morphogenetic furrow, a physical indentation in the epithelium that serves as a spatial and temporal benchmark of eye development. Posterior to the SMW, cells divide stochastically. With GMR > Dnhe2 expression, we found a significant increase in the number of proliferating phospho-histone H3 (phosH3) positive cells within this posterior region (Figure 2C,D). We also expressed Dnhe2 in the posterior compartment of the developing wing imaginal disc using an engrailed driver (enGAL4). In en > GFP, Dnhe2 expressing wing discs, there was overgrowth and dysplasia only in the expressing compartment, while non-expressing tissue resembled wild type (Figure 2E). In wild type wing discs, the number of phosH3 positive cells was relatively similar in the posterior and anterior compartments (Figure 2F,G). With en > Dnhe2 expression, however, this distribution was markedly different with significantly more proliferating cells in the posterior compared with anterior compartment (Figure 2F,G). Together, these data suggest that Dnhe2 acts in a cell-autonomous manner to promote proliferation and dysplasia.

NHE1 activity promotes directed cell migration (Denker and Barber, 2002; Patel and Barber, 2005; Stock and Schwab, 2009) and invasion (Stock and Schwab, 2009; Busco et al., 2010) in cultured cells; however effects on these behaviors in vivo remain unresolved. We tested for synergy between Dnhe2 and oncogenic RasV12 using the ptc-GAL4 driver to express transgenes in a stripe of cells along the anterior/posterior compartment boundary in wL3 wing discs; this assay induces mosaic transgene expression to distinguish autonomous and non-autonomous effects, and can reveal effects on tissue organization, proliferation and invasive/migratory cell behaviors (Vidal et al., 2006; Petzoldt et al., 2013) (Figure 3). Expression of RasV12 induced a wider ptc > GFP stripe compared to control (∼1.6-fold increase), while expression of Dnhe2 alone did not (Figure 3A–C). However, co-expression of RasV12 and Dnhe2 markedly increased the ptc > GFP stripe width greater than twofold (Figure 3C). We also observed a significant increase in the overall size of the wing disc with ptc > RasV12, Dnhe2 compared with either transgene alone or wild type (Figure 3A,D). To determine invasive phenotypes, we identified single GFP-positive cells in stacks of confocal images. In control and Dnhe2-expressing discs we found only a single instance of a few (Rofstad et al., 2006; Stock and Schwab, 2009; Zhang et al., 2010) single GFP-positive cells, and no examples in RasV12-expressing discs (Figure 3E). However, in ptc > RasV12, Dnhe2 discs, we found examples of invasive cells in all discs examined. Further, we found several different types of invasive behaviors, including basal expansion of the ptc stripe, single invasive cells and streams of cells migrating out of the stripe into neighboring tissue in the posterior compartment (Figure 3E). These data show cell autonomous effects of Dnhe2 expression on growth and migration behaviors, as well as non-autonomous effects on tissue growth.

Figure 3

Download asset Open asset

To address our second question on whether H⁺ efflux is necessary for oncogene-induced dysplasia, we generated precise deletions of the Dnhe2 using FLP-mediated recombination of FRT-bearing transposon insertions that flank the Dnhe2 coding region (Figure 4A,B). This strategy avoided excision of a neighboring gene CG9257 that caused larval lethality in the existing Dnhe2¹ allele (Simons et al., 2009). Deletions were screened by PCR by using primer pairs to test for the presence of coding sequences as well as for the recombinant P-element generated by recombination (Figure 4B). Five independent excision lines were obtained, and all showed identical phenotypes. Homozygous Dnhe2^null flies had ∼20% survival to eclosion (Figure 4C), and surviving adult flies appeared morphologically normal. The lethality of Dnhe2^null flies was rescued by expression of Dnhe2 using the muscle-specific mef2-GAL4 driver, but not using the neuronal elav-GAL4 driver (Figure 4C).

Figure 4

Download asset Open asset

To test the effects of deleting Dnhe2 with expression of an activated oncogene we initially used GMR-GAL4 to drive activated RasV12; however GMR > RasV12 caused pupal lethality, which precluded phenotypic analysis in adult flies. We therefore induced expression of constitutively active, oncogenic Raf (Brand and Perrimon, 1994) (GMR > Raf). Retinal architecture in homozygous Dnhe2^null flies was organized and regular, resembling wild type (Figure 5A,B). The external surface of GMR > Raf eyes was disorganized with indistinct facets and a rough exterior retinal surface (Figure 5A,B). Some ommatidia showed thin or absent lenses. In section, GMR > Raf had ommatidial defects with mis-orientation and occasional missing rhabdomeres (Figure 5C, tracing in Figure 5D). Additionally, GMR > Raf rhabdomeres were morphologically distinct from wild type, appearing more rectangular, especially in R1, R3, R5 and R6. Externally, Dnhe2^null; GMR > Raf appeared identical to GMR > Raf (Figure 5A,B), except that the eye color was noticeably lighter and yellowed. Tangential retinal sections revealed a complete loss of tissue architecture and cell structure, with histological features of coagulative necrosis, including loss of rhabdomere morphology, absence of distinct cell membranes and diffusion of pigment granules (Figure 5C). Live, dissected whole-mount adult eyes revealed a significant reduction of the cohesive, red-pigmented retinal epithelial layer, such that the remaining Dnhe2^null; GMR > Raf tissue was thin and transparent in appearance (data not shown). These observations support a synthetic lethal interaction between loss of Dnhe2 and expression of oncogenic Raf.

Figure 5

Download asset Open asset

Synthetic lethality of Dnhe2^null; GMR > Raf was progressive, as determined by examining earlier developmental stages. In wL3 eye discs, Dnhe2^null tissue morphology was indistinguishable from wild type (Figure 6A); however there were fewer proliferating cells than in wild type as indicated by phosH3 staining (Figure 6A,B). Expression of GMR > Raf led to disrupted tissue morphology, with irregular ommatidial spacing and folding of the eye disc, presumably due to increased proliferation (Figure 6A,B). Compared with GMR > Raf the morphology of Dnhe2^null; GMR > Raf appeared less disrupted (Figure 6A) and hyperproliferation was suppressed (Figure 6B). Pupal eyes were assayed 42H apf for expression of the neuronal antigen Elav, and we found that cells in all genotypes retain their fates as differentiated neurons (Figure 6C). Beta-catenin immunolabeling showed distinct differences in the morphology of cell contacts (Figure 6D, line drawing in Figure 6E). Dnhe2^null pupal eyes appeared wild type: individual ommatidia had 4 cone cells and 2 primary pigment cells visible on the apical surface, and a single row of secondary pigment cells separating neighboring ommatidia. However, cell morphologies were slightly abnormal (Figure 6D,E). Secondary pigment cells were thicker than in wild type, and cell shapes and cell–cell contacts were irregular. With expression of GMR > Raf, cell morphologies were profoundly aberrant, with extra and mis-shapen cone cells and ommatidia mis-aligned along the D/V axis, as seen in adults. Additionally, immature cone cell contacts were maintained and secondary pigment cells were absent between some pairs of neighboring ommatidia. These defects were enhanced in Dnhe2^null; GMR > Raf pupal eyes, where all cell types appeared smaller and more cells had abnormal morphologies. Additionally, there were more ommatidia with extra cone cells. Immunolabeling pupal eyes for cleaved caspase 3 (CC3), a marker of apoptotic cells, showed no staining in wild type, Dnhe2^null or GMR > Raf eyes. However, in Dnhe2^null; GMR > Raf eyes there were a few CC3–positive cells in all retina examined, even though the normal phase of cell death concludes ∼12 hr before this time point (data not shown). CC3 staining was not abundant enough to indicate widespread cell death at 42H apf. Our data suggest that Dnhe2 deletion causes progressive defects in Raf-expressing cells, which culminates in synthetic lethality prior to eclosion.

Figure 6

Download asset Open asset

Changes in pHi correlated with the progressive morphological defects described above. We measured pHi using the mCherry-pHluorin biosensor in live wL3 eye imaginal discs, approximately 24 hr after the onset of oncogenic Raf expression (Figure 7A). Compared with wild type, the pHi in Dnhe2^null eye discs was significantly lower and GMR > Raf was significantly higher (Figure 7B). In contrast, pHi in Dnhe2^null; GMR > Raf discs was not different than wild type (p > 0.05). This suggests that the increased pHi with expression of oncogenic Raf requires Dnhe2 activity. We also measured pHi in live 42H apf pupal eyes, and found that pHi in GMR > Raf was still significantly higher than wild type pupal eyes (Figure 7C,D). In contrast to larval eye discs, however, the pHi of Dnhe2^null; GMR > Raf was significantly lower than wild type and lower than GMR > Raf alone (Figure 7C,D). These data suggest that pHi progressively decreases through pupal morphogenesis in Dnhe2^null; GMR > Raf retinae, and this could contribute to the synthetic lethality observed in adult eyes.

Figure 7

Download asset Open asset

We also found synthetic lethality in clonal cells with loss of H⁺ efflux and oncogene expression. In clonal human mammary MCF10A cells we found that loss of NHE1 activity lowers pHi and increases cell death selectively in cells expressing oncogenic RasV12. We first used an estrogen receptor-induced H-RasV12 (ER-RasV12) and suppressed NHE1 expression with shRNA. Tamoxifen-induced Ras expression markedly increased rate of pHi recovery from an acid load (Figure 8A, Figure 8—figure supplement 1). Tamoxifen increased pHi in ER-Ras cells and with NT shRNA but not in vector control cells (Figure 8B). However, with NHE1 shRNA pHi was significantly lower (Figure 8B), indicating NHE1 is necessary for increased pHi with oncogenic Ras. Tamoxifen treatment increased NHE1 expression (Figure 8C), while NHE1 shRNA treatment significantly reduced NHE1 protein expression (Figure 8C; two different NHE1 shRNA constructs showed similar results). A similar NHE1-dependent increase in pHi was shown using MCF10A cells stably expressing H-RasV12 (sRasV12) and 5(N-ethyl-N-isopropyl) amiloride (EIPA) to pharmacologically inhibit NHE1 activity (Figure 8E,F). As a control, we show that EIPA treatment does not effect Ras expression (Figure 8G). Although pHi was higher in MCF10A cells with RasV12 and inhibited NHE1 activity than in Drosophila Dnhe2^null; GMR > Raf pupal eyes, measuring pHi of cultured cells requires washes that could remove less adherent, dying cells with a lower pHi. Quantifying dying cells by flow cytometry using fluorescently labeled AnnexinV showed NHE1 shRNA or EIPA significantly increased cell death with RasV12 (p < 0.05) (Figure 8D,H) but not in control cells. These results combined with our in vivo findings in Drosophila indicate that limiting H⁺ efflux causes synthetic lethality with oncogene expression.

Figure 8 with 1 supplement see all

Download asset Open asset

Decreasing H⁺ efflux also increased lethality of invasive tumor cells having a complex mutational landscape compared with only H-RasV12 expression as described above. We used SW620 metastatic colorectal cells, which have an activating mutation in K-Ras (G12V), mutations in the tumor suppressors SMAD4 (frameshift), p53 (R273H, P309S), and adenomatous polyposis coli (APC) (Q1338*), as well as MDA-MB-231 invasive breast metastatic cells, which have a different activating mutation in K-ras (G13D), a mutant B-Raf (G464V) and mutations in the tumor suppressors cyclin-dependent kinase inhibitor 2A (deletion), and p53 (R280K). In both cell lines, EIPA treatment for 72 hr significantly decreased pHi (Figure 9A) and increased cell death compared with controls (Figure 9B). These data indicate that loss of H⁺ efflux limits survival of cells containing multiple oncogenic lesions and suggest a broad applicability of limiting H⁺ efflux to increase cell death across cancer types.

Figure 9

Download asset Open asset

With the advent of low-cost whole genome sequencing, personalized chemotherapeutic treatments currently dominate discussion of new cancer therapies. However, the balkanization of cancers by causative mutation or tissue of origin ignores the shared disease physiology in most cancers as highlighted by the ‘hallmarks of cancer’ (Hanahan and Weinberg, 2011). Dysregulated pHi is an emerging hallmark of cancer that is associated with traditional hallmarks, including resisting cell death, sustaining proliferative signaling, activating invasion and metastasis, deregulating cellular energetics and tumor-promoting inflammation. Therefore, targeting pHi-regulatory proteins in cancer cells could address distinct pathological characteristics in parallel.

Our data provide an in vivo functional corollary of the established increased pHi and upregulation of H⁺ efflux mechanisms in cancer cells. Building on previous reports that NHE1 activity and expression is increased in human cancers (McLean et al., 2000; Miraglia et al., 2005; Chiang et al., 2008; Yang et al., 2011), we show that engineered over-expression of an NHE1 ortholog in vivo increased pHi by ∼0.3 pH units, which is comparable to the increase seen with oncogene transformation. Moreover, over-expression of Dnhe2 was sufficient to induce dysplasia and hyper-proliferation phenotypes, and it enhanced oncogene-mediated invasion. Further, co-expressing RasV12 and Dnhe2 had a strong, synergistic effect on growth and invasion. A similar synergistic effect between expression of RasV12 and the C subunit of the V-ATPase, Vha44 was recently reported (Petzoldt et al., 2013), which supports the ability of increased H⁺ efflux to enhance RasV12-induced phenotypes. However, Vha44 enhances invasion with RasV12 but not activated Src or Abl, suggesting an oncogene-specific synergy. Important to determine in future studies are the relative effects of increased pHi on growth and invasion, and whether decreased pHe contributes to the non-autonomous effects we observed in imaginal discs.

Our data also indicate that reducing H⁺ efflux in cancer cells suppresses multiple pathological characteristics in parallel. Previous studies identified distinct responses to reduced H⁺ efflux, such as tumor growth in xenograft models (Lagarde et al., 1988; Sonveaux et al., 2008; Colen et al., 2011; Yang et al., 2011) or viability of isolated cancer cells (Rich et al., 2000; Reshkin et al., 2003). We now show progressive effects of limiting H⁺ efflux with oncogene expression, including initially suppressing hyper-proliferation and dysplasia, followed by decreasing pHi, and culminating in a synthetic lethal interaction with histological features of coagulative necrosis. Coagulative necrosis is characteristically found within the central region of solid tumors (Searle et al., 1975; Lagarde et al., 1988), and is thought to be induced by extreme physiological cellular stresses. Removing Dnhe2 in an otherwise wild type genetic background decreased pHi but had no effect on larval tissue morphology. Dnhe2^null retinal pHi in pupae was slightly higher than in larvae but not significantly different compared with wild type. These data suggest that in the absence of Dnhe2, pHi homeostasis during metamorphosis can be maintained by alternative ion transport mechanisms, changes in cellular buffering capacity and/or metabolic changes. While our Drosophila studies evaluate synthetic lethality in the ~20% Dnhe2 homozygous mutant ‘escaper’ flies that survive to eclosion, our studies in several transformed cell lines also show a synthetic lethality interaction, suggesting a conserved mechanism. The synthetic lethal interaction we observed suggests H⁺ efflux is necessary for dissipating oncogene-generated acids, most likely from increased glycolytic metabolism. Our studies further show that these lethal interactions occur in two genetically complex, physiologically adapted invasive tumor cell lines.

Our data build on the long-standing observation of increased pHi in cancer cells and suggest several new directions for future investigation. First, our findings indicate that the biosensor pHluorin can be used to resolve pHi dynamics in vivo during tumor formation and metastasis and whether there are spatially distinct pHi dynamics within a tumor that might inform us about tumor properties and plasticity. Second, the rough eye phenotype induced with GMR > Dnhe2 provides a new model for genetic screens to identify pH-sensitive pathways and previously unrecognized pH sensors regulating cell growth and tissue architecture. Dysplasia with increased pHi in the absence of oncogenes is likely in part dependent on cancer-promoting pH sensors (Schönichen et al., 2013), including focal adhesion kinase (FAK) and the actin regulatory protein cofilin that support tumor growth and metastasis (Wang et al., 2007; Cance et al., 2013) and have increased activity at higher pHi (Frantz et al., 2008; Choi et al., 2013). Third, the synthetic lethality we show suggests that therapeutic strategies targeting H⁺ efflux transporters or pH sensors with selective roles in oncogene-induced responses could limit metastatic progression with minimal off-target effects.

1. 1. Brand AH
   2. Perrimon N

   (1994) Raf acts downstream of the EGF receptor to determine dorsoventral polarity during Drosophila oogenesis

   Genes & Development 8:629–639.

   https://doi.org/10.1101/gad.8.5.629

   • Google Scholar
2. 1. Busco G
   2. Cardone RA
   3. Greco MR
   4. Bellizzi A
   5. Colella M
   6. Antelmi E
   7. Mancini MT
   8. Dell'Aquila ME
   9. Casavola V
   10. Paradiso A
   11. Reshkin SJ

   (2010) NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space

   The FASEB Journal 24:3903–3915.

   https://doi.org/10.1096/fj.09-149518

   • Google Scholar
3. 1. Cance WG
   2. Kurenova E
   3. Marlowe T
   4. Golubovskaya V

   (2013) Disrupting the scaffold to improve focal adhesion kinase-targeted cancer therapeutics

   Science Signaling 6:pe10.

   https://doi.org/10.1126/scisignal.2004021

   • Google Scholar
4. 1. Cardone RA
   2. Casavola V
   3. Reshkin SJ

   (2005) The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis

   Nature Reviews Cancer 5:786–795.

   https://doi.org/10.1038/nrc1713

   • Google Scholar
5. 1. Chiang Y
   2. Chou CY
   3. Hsu KF
   4. Huang YF
   5. Shen MR

   (2008) EGF upregulates Na+/H+ exchanger NHE1 by post-translational regulation that is important for cervical cancer cell invasiveness

   Journal of Cellular Physiology 214:810–819.

   https://doi.org/10.1002/jcp.21277

   • Google Scholar
6. 1. Choi C-H
   2. Webb BA
   3. Chimenti MS
   4. Jacobson MP
   5. Barber DL

   (2013) pH sensing by FAK-His58 regulates focal adhesion remodeling

   The Journal of Cell Biology 202:849–859.

   https://doi.org/10.1083/jcb.201302131

   • Google Scholar
7. 1. Colen CB
   2. Shen Y
   3. Ghoddoussi F
   4. Yu P
   5. Francis TB
   6. Koch BJ
   7. Monterey MD
   8. Galloway MP
   9. Sloan AE
   10. Mathupala SP

   (2011)

   Metabolic targeting of lactate efflux by malignant glioma inhibits invasiveness and induces necrosis: an in vivo study

   Neoplasia 13:620–632.

   • Google Scholar
8. 1. Debnath J
   2. Muthuswamy SK
   3. Brugge JS

   (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures

   Methods 30:256–268.

   https://doi.org/10.1016/S1046-2023(03)00032-X

   • Google Scholar
9. 1. Denker SP
   2. Barber DL

   (2002) Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1

   The Journal of Cell Biology 159:1087–1096.

   https://doi.org/10.1083/jcb.200208050

   • Google Scholar
10. 1. Denker SP
    2. Huang DC
    3. Orlowski J
    4. Furthmayr H
    5. Barber DL

    (2000) Direct binding of the Na–H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation

    Molecular Cell 6:1425–1436.

    https://doi.org/10.1016/S1097-2765(00)00139-8

    • Google Scholar
11. 1. Dietl K
    2. Renner K
    3. Dettmer K
    4. Timischl B
    5. Eberhart K
    6. Dorn C
    7. Hellerbrand C
    8. Kastenberger M
    9. Kunz-Schughart LA
    10. Oefner PJ
    11. Andreesen R
    12. Gottfried E
    13. Kreutz MP

    (2010) Lactic acid and acidification inhibit TNF secretion and glycolysis of human monocytes

    The Journal of Immunology 184:1200–1209.

    https://doi.org/10.4049/jimmunol.0902584

    • Google Scholar
12. 1. Estrella V
    2. Chen T
    3. Lloyd M
    4. Wojtkowiak J
    5. Cornnell HH
    6. Ibrahim-Hashim A
    7. Bailey K
    8. Balagurunathan Y
    9. Rothberg JM
    10. Sloane BF
    11. Johnson J
    12. Gatenby RA
    13. Gillies RJ

    (2013) Acidity generated by the tumor microenvironment drives local invasion

    Cancer Research 73:1524–1535.

    https://doi.org/10.1158/0008-5472.CAN-12-2796

    • Google Scholar
13. 1. Frantz C
    2. Barreiro G
    3. Dominguez L
    4. Chen X
    5. Eddy R
    6. Condeelis J
    7. Kelly MJ
    8. Jacobson MP
    9. Barber DL

    (2008) Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding

    The Journal of Cell Biology 183:865–879.

    https://doi.org/10.1083/jcb.200804161

    • Google Scholar
14. 1. Gerlinger M
    2. Santos CR
    3. Spencer-Dene B
    4. Martinez P
    5. Endesfelder D
    6. Burrell RA
    7. Vetter M
    8. Jiang M
    9. Saunders RE
    10. Kelly G
    11. Dykema K
    12. Rioux-Leclercq N
    13. Stamp G
    14. Patard JJ
    15. Larkin J
    16. Howell M
    17. Swanton C

    (2012) Genome-wide RNA interference analysis of renal carcinoma survival regulators identifies MCT4 as a Warburg effect metabolic target

    The Journal of Pathology 227:146–156.

    https://doi.org/10.1002/path.4006

    • Google Scholar
15. 1. Grillo-Hill BK
    2. Webb BA
    3. Barber DL

    (2014) Ratiometric imaging of pH probes

    Methods in Cell Biology 123:429–448.

    https://doi.org/10.1016/B978-0-12-420138-5.00023-9

    • Google Scholar
16. 1. Halestrap AP

    (2013) The SLC16 gene family-structure, role and regulation in health and disease

    Molecular Aspects of Medicine 34:337–349.

    https://doi.org/10.1016/j.mam.2012.05.003

    • Google Scholar
17. 1. Hanahan D
    2. Weinberg RA

    (2011) Hallmarks of cancer: the next generation

    Cell 144:646–674.

    https://doi.org/10.1016/j.cell.2011.02.013

    • Google Scholar
18. 1. Harguindey S
    2. Arranz JL
    3. Polo Orozco JD
    4. Rauch C
    5. Fais S
    6. Cardone RA
    7. Reshkin SJ

    (2013) Cariporide and other new and powerful NHE1 inhibitors as potentially selective anticancer drugs – an integral molecular/biochemical/metabolic/clinical approach after one hundred years of cancer research

    Journal of Translational Medicine 11:282.

    https://doi.org/10.1186/1479-5876-11-282

    • Google Scholar
19. 1. Hinton A
    2. Sennoune SR
    3. Bond S
    4. Fang M
    5. Reuveni M
    6. Sahagian GG
    7. Jay D
    8. Martinez-Zaguilan R
    9. Forgac M

    (2009) Function of a subunit isoforms of the V-ATPase in pH homeostasis and in vitro invasion of MDA-MB231 human breast cancer cells

    The Journal of Biological Chemistry 284:16400–16408.

    https://doi.org/10.1074/jbc.M901201200

    • Google Scholar
20. 1. Kapus A
    2. Grinstein S
    3. Wasan S
    4. Kandasamy R
    5. Orlowski J

    (1994)

    Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation

    The Journal of Biological Chemistry 269:23544–23552.

    • Google Scholar
21. 1. Koivusalo M
    2. Welch C
    3. Hayashi H
    4. Scott CC
    5. Kim M
    6. Alexander T
    7. Touret N
    8. Hahn KM
    9. Grinstein S

    (2010) Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling

    The Journal of Cell Biology 188:547–563.

    https://doi.org/10.1083/jcb.200908086

    • Google Scholar
22. 1. Lagadic-Gossmann D
    2. Huc L
    3. Lecureur V

    (2004) Alterations of intracellular pH homeostasis in apoptosis: origins and roles

    Cell Death and Differentiation 11:953–961.

    https://doi.org/10.1038/sj.cdd.4401466

    • Google Scholar
23. 1. Lagarde A
    2. Franchi A
    3. Paris S
    4. Pousségur J

    (1988) Effect of mutations affecting Na+:H+ antiport activity on Tumorigenic potential of hamster lung fibroblasts

    Journal of Cellular Biochemistry 36:249–260.

    https://doi.org/10.1002/jcb.240360306

    • Google Scholar
24. 1. Lin X
    2. Barber DL

    (1996) A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange

    Proceedings of the National Academy of Sciences of USA 93:12631–12636.

    https://doi.org/10.1073/pnas.93.22.12631

    • Google Scholar
25. 1. Martinez-Zaguilan R
    2. Lynch RM
    3. Martinez GM
    4. Gillies RJ

    (1993)

    Vacuolar-type H(+)-ATPases are functionally expressed in plasma membranes of human tumor cells

    The American Journal of Physiology 265:C1015–C1029.

    • Google Scholar
26. 1. Matsuyama S
    2. Llopis J
    3. Deveraux QL
    4. Tsien RY
    5. Reed JC

    (2000) Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis

    Nature Cell Biology 2:318–325.

    https://doi.org/10.1038/35014006

    • Google Scholar
27. 1. McLean LA
    2. Roscoe J
    3. Jorgensen NK
    4. Gorin FA
    5. Cala PM

    (2000)

    Malignant gliomas display altered pH regulation by NHE1 compared with nontransformed astrocytes

    American Journal of Physiology Cell Physiology 278:C676–C688.

    • Google Scholar
28. 1. Miraglia E
    2. Viarisio D
    3. Riganti C
    4. Costamagna C
    5. Ghigo D
    6. Bosia A

    (2005) Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin

    International Journal of Cancer Journal International Du Cancer 115:924–929.

    https://doi.org/10.1002/ijc.20959

    • Google Scholar
29. 1. Orij R
    2. Urbanus ML
    3. Vizeacoumar FJ
    4. Giaever G
    5. Boone C
    6. Nislow C
    7. Brul S
    8. Smits GJ

    (2012) Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pH(c) in Saccharomyces cerevisiae

    Genome Biology 13:R80.

    https://doi.org/10.1186/gb-2012-13-9-r80

    • Google Scholar
30. 1. Pang T
    2. Su X
    3. Wakabayashi S
    4. Shigekawa M

    (2001) Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers

    The Journal of Biological Chemistry 276:17367–17372.

    https://doi.org/10.1074/jbc.M100296200

    • Google Scholar
31. 1. Parks AL
    2. Cook KR
    3. Belvin M
    4. Dompe NA
    5. Fawcett R
    6. Huppert K
    7. Tan LR
    8. Winter CG
    9. Bogart KP
    10. Deal JE
    11. Deal-Herr ME
    12. Grant D
    13. Marcinko M
    14. Miyazaki WY
    15. Robertson S
    16. Shaw KJ
    17. Tabios M
    18. Vysotskaia V
    19. Zhao L
    20. Andrade RS
    21. Edgar KA
    22. Howie E
    23. Killpack K
    24. Milash B
    25. Norton A
    26. Thao D
    27. Whittaker K
    28. Winner MA
    29. Friedman L
    30. Margolis J
    31. Singer MA
    32. Kopczynski C
    33. Curtis D
    34. Kaufman TC
    35. Plowman GD
    36. Duyk G
    37. Francis-Lang HL

    (2004) Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome

    Nature Genetics 36:288–292.

    https://doi.org/10.1038/ng1312

    • Google Scholar
32. 1. Patel H
    2. Barber DL

    (2005) A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

    The Journal of Cell Biology 169:321–329.

    https://doi.org/10.1083/jcb.200412145

    • Google Scholar
33. 1. Petzoldt AG
    2. Gleixner EM
    3. Fumagalli A
    4. Vaccari T
    5. Simons M

    (2013) Elevated expression of the V-ATPase C subunit triggers JNK-dependent cell invasion and overgrowth in a Drosophila epithelium

    Disease Models & Mechanisms 6:689–700.

    https://doi.org/10.1242/dmm.010660

    • Google Scholar
34. 1. Pinheiro C
    2. Reis RM
    3. Ricardo S
    4. Longatto-Filho A
    5. Schmitt F
    6. Baltazar F

    (2010) Expression of monocarboxylate transporters 1, 2, and 4 in human tumours and their association with CD147 and CD44

    Journal of Biomedicine & Biotechnology 2010:427694.

    https://doi.org/10.1155/2010/427694

    • Google Scholar
35. 1. Pouysségur J
    2. Chambard JC
    3. Franchi A
    4. Paris S
    5. Van Obberghen-Schilling E

    (1982) Growth factor activation of an amiloride-sensitive Na+/H+ exchange system in quiescent fibroblasts: coupling to ribosomal protein S6 phosphorylation

    Proceedings of the National Academy of Sciences of USA 79:3935–3939.

    https://doi.org/10.1073/pnas.79.13.3935

    • Google Scholar
36. 1. Pouysségur J
    2. Sardet C
    3. Franchi A
    4. L'Allemain G
    5. Paris S

    (1984) A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH

    Proceedings of the National Academy of Sciences of USA 81:4833–4837.

    https://doi.org/10.1073/pnas.81.15.4833

    • Google Scholar
37. 1. Putney LK
    2. Barber DL

    (2003) Na-H exchange-dependent increase in intracellular pH times G2/M entry and transition

    The Journal of Biological Chemistry 278:44645–44649.

    https://doi.org/10.1074/jbc.M308099200

    • Google Scholar
38. 1. Reshkin SJ
    2. Bellizzi A
    3. Caldeira S
    4. Albarani V
    5. Malanchi I
    6. Poignee M
    7. Alunni-Fabbroni M
    8. Casavola V
    9. Tommasino M

    (2000) Na+/H+ exchanger-dependent intracellular alkalinization is an early event in malignant transformation and plays an essential role in the development of subsequent transformation-associated phenotypes

    FASEB Journal 14:2185–2197.

    https://doi.org/10.1096/fj.00-0029com

    • Google Scholar
39. 1. Reshkin SJ
    2. Bellizzi A
    3. Cardone RA
    4. Tommasino M
    5. Casavola V
    6. Paradiso A

    (2003)

    Paclitaxel induces apoptosis via protein kinase A- and p38 mitogen-activated protein-dependent inhibition of the Na+/H+ exchanger (NHE) NHE isoform 1 in human breast cancer cells

    Clinical Cancer Research 9:2366–2373.

    • Google Scholar
40. 1. Rich IN
    2. Worthington-White D
    3. Garden OA
    4. Musk P

    (2000)

    Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na(+)/H(+) exchanger

    Blood 95:1427–1434.

    • Google Scholar
41. 1. Rofstad EK
    2. Mathiesen B
    3. Kindem K
    4. Galappathi K

    (2006) Acidic extracellular pH promotes experimental metastasis of human melanoma cells in athymic nude mice

    Cancer Research 66:6699–6707.

    https://doi.org/10.1158/0008-5472.CAN-06-0983

    • Google Scholar
42. 1. Schönichen A
    2. Webb BA
    3. Jacobson MP
    4. Barber DL

    (2013) Considering protonation as a posttranslational modification regulating protein structure and function

    Annual Review of Biophysics 42:289–314.

    https://doi.org/10.1146/annurev-biophys-050511-102349

    • Google Scholar
43. 1. Searle J
    2. Lawson TA
    3. Abbott PJ
    4. Harmon B
    5. Kerr JF

    (1975) An electron-microscope study of the mode of cell death induced by cancer-chemotherapeutic agents in populations of proliferating normal and neoplastic cells

    The Journal of Pathology 116:129–138.

    https://doi.org/10.1002/path.1711160302

    • Google Scholar
44. 1. Sennoune SR
    2. Bakunts K
    3. Martínez GM
    4. Chua-Tuan JL
    5. Kebir Y
    6. Attaya MN
    7. Martínez-Zaguilán R

    (2004) Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity

    American Journal of Physiology Cell Physiology 286:C1443–C1452.

    https://doi.org/10.1152/ajpcell.00407.2003

    • Google Scholar
45. 1. Simons M
    2. Gault WJ
    3. Gotthardt D
    4. Rohatgi R
    5. Klein TJ
    6. Shao Y
    7. Lee HJ
    8. Wu AL
    9. Fang Y
    10. Satlin LM
    11. Dow JT
    12. Chen J
    13. Zheng J
    14. Boutros M
    15. Mlodzik M

    (2009) Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization

    Nature Cell Biology 11:286–294.

    https://doi.org/10.1038/ncb1836

    • Google Scholar
46. 1. Sonveaux P
    2. Végran F
    3. Schroeder T
    4. Wergin MC
    5. Verrax J
    6. Rabbani ZN
    7. De Saedeleer CJ
    8. Kennedy KM
    9. Diepart C
    10. Jordan BF
    11. Kelley MJ
    12. Gallez B
    13. Wahl ML
    14. Feron O
    15. Dewhirst MW

    (2008) Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice

    The Journal of Clinical Investigation 118:3930–3942.

    https://doi.org/10.1172/JCI36843

    • Google Scholar
47. 1. Stehbens S
    2. Pemble H
    3. Murrow L
    4. Wittmann T

    (2012) Imaging intracellular protein dynamics by spinning disk confocal microscopy

    Methods in Enzymology 504:293–313.

    https://doi.org/10.1016/B978-0-12-391857-4.00015-X

    • Google Scholar
48. 1. Stock C
    2. Cardone RA
    3. Busco G
    4. Krähling H
    5. Schwab A
    6. Reshkin SJ

    (2008) Protons extruded by NHE1: digestive or glue?

    European Journal of Cell Biology 87:591–599.

    https://doi.org/10.1016/j.ejcb.2008.01.007

    • Google Scholar
49. 1. Stock C
    2. Schwab A

    (2009) Protons make tumor cells move like clockwork

    Pflügers Archiv 458:981–992.

    https://doi.org/10.1007/s00424-009-0677-8

    • Google Scholar
50. 1. Swietach P
    2. Vaughan-Jones RD
    3. Harris AL

    (2007) Regulation of tumor pH and the role of carbonic anhydrase 9

    Cancer Metastasis Reviews 26:299–310.

    https://doi.org/10.1007/s10555-007-9064-0

    • Google Scholar
51. 1. Vidal M
    2. Larson DE
    3. Cagan RL

    (2006) Csk-deficient boundary cells are eliminated from normal Drosophila epithelia by exclusion, migration, and apoptosis

    Developmental Cell 10:33–44.

    https://doi.org/10.1016/j.devcel.2005.11.007

    • Google Scholar
52. 1. Wang W
    2. Eddy R
    3. Condeelis J

    (2007) The cofilin pathway in breast cancer invasion and metastasis

    Nature Reviews Cancer 7:429–440.

    https://doi.org/10.1038/nrc2148

    • Google Scholar
53. 1. Webb BA
    2. Chimenti M
    3. Jacobson MP
    4. Barber DL

    (2011) Dysregulated pH: a perfect storm for cancer progression

    Nature Reviews Cancer 11:671–677.

    https://doi.org/10.1038/nrc3110

    • Google Scholar
54. 1. Wolff T

    (2000)

    Histological techniques for the Drosophila eye

    Drosophila Protocols.

    • Google Scholar
55. 1. Yang X
    2. Wang D
    3. Dong W
    4. Song Z
    5. Dou K

    (2011) Expression and modulation of Na(+) /H(+) exchanger 1 gene in hepatocellular carcinoma: a potential therapeutic target

    Journal of Gastroenterology and Hepatology 26:364–370.

    https://doi.org/10.1111/j.1440-1746.2010.06382.x

    • Google Scholar
56. 1. Zhang X
    2. Lin Y
    3. Gillies RJ

    (2010) Tumor pH and its measurement

    Journal of Nuclear Medicine 51:1167–1170.

    https://doi.org/10.2967/jnumed.109.068981

    • Google Scholar

Author details

1. Bree K Grillo-Hill

   Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   BKG-H, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Changhoon Choi

   Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   CC, Acquisition of data, Analysis and interpretation of data

   Competing interests

   The authors declare that no competing interests exist.

3. Maite Jimenez-Vidal

   Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   MJ-V, Generated Dnhe2^null Drosophila lines, Contributed unpublished essential data or reagents

   Competing interests

   The authors declare that no competing interests exist.

4. Diane L Barber

   Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, United States

   Contribution

   DLB, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   diane.barber@ucsf.edu

   Competing interests

   The authors declare that no competing interests exist.

• 2,924

  views

• 686

  downloads

• 67

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.