Lactate Dehydrogenase B Is Required for the Growth of KRAS-Dependent Lung Adenocarcinomas

Author Manuscript Published OnlineFirst on December 6, 2012; DOI: 10.1158/1078-0432.CCR-12-2638 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Lactate Dehydrogenase B is required for the growth of KRAS-dependent lung adenocarcinomas

Mark L. McCleland1*, Adam S. Adler1*, Laura Deming1*, Ely Cosino2, Leslie Lee2, Elizabeth

M. Blackwood2, Margaret Solon1, Janet Tao1, Li Li3, David Shames4, Erica Jackson5, William F.

Forrest6, and Ron Firestein1

1Department of Pathology, 2Department of Translational Oncology, 3Department of

Bioinformatics & Computational Biology, 4Department of Molecular Diagnostics & Cancer Cell

Biology, 5Department of Research Oncology, and 6Department of Biostatistics, Genentech, Inc.,

South San Francisco, California, USA

* These authors contributed equally to this work

Corresponding Author: Ron Firestein, Genentech, Inc., 1 DNA Way, South San Francisco, CA

94080; Phone: 650-225-8441; Fax: 650-467-2625; E-mail: ronf@gene.com

Running title: LDHB is a regulator of KRAS-dependent lung cancer

Key words: LDHB, KRAS, lung cancer, glycolysis, metabolism

Conflict of interest: All authors are employed by Genentech, Inc.

Number of figures: 6

Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2012 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 6, 2012; DOI: 10.1158/1078-0432.CCR-12-2638 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Translational Relevance

Specific molecular subsets of cancer have proven to be difficult to target, leaving patients with

few therapeutic options. One such example of this is lung adenocarcinomas that exhibit

aberration in the KRAS oncogene. KRAS is altered, through activating mutation and copy

number gain, in nearly 30% of lung adenocarcinomas. We employ a loss of function screen to knockdown genes within the KRAS amplicon, and find that lactate dehydrogenase B (LDHB) is upregulated and essential in KRAS driven lung adenocarcinomas. Targeting LDHB with small molecule inhibitors may provide a therapeutic option for KRAS driven lung adenocarcinomas.

ABSTRACT

Purpose: This study aims to identify genes within the KRAS genomic amplicon that are both co-

upregulated and essential for cell proliferation when KRAS is amplified in lung cancer.

Experimental Design: We utilized an integrated genomic approach to identify genes that are

co-amplified with KRAS in lung adenocarcinomas and subsequently preformed an RNAi screen

to uncover functionally relevant genes. Subsequent functional analysis of a candidate hit from

the screen, Lactate Dehydrogenase B (LDHB), was done both in vitro and in vivo by siRNA and

shRNA mediated knockdown in a panel of lung adenocarcinoma cells lines. LDHB expression

was also investigated in patient tumors using microarray and immunohistochemistry analyses.

Results: Lung adenocarcinomas that harbor KRAS genomic copy number gain and KRAS

mutations showed elevated LDHB expression. High LDHB expression was also seen in a smaller proportion of KRAS wildtype lung cancers harboring other oncogenic drivers such as EGFR or

MET. Lung adenocarcinomas that exhibited high LDHB expression were uniquely dependent on

LDHB for proliferation both in vitro and in xenografted tumors in vivo. Consistent with a role for

LDHB in glycolysis and tumor metabolism, KRAS mutant lung adenocarcinomas exhibit an elevated expression of a glycolysis gene signature and are more dependent on glucose for growth.

Lastly, high LDHB expression was a significant predictor of shorter survival in patients with lung adenocarcinomas.

Conclusion: This study identifies LDHB as a regulator of cell growth in a subset of lung adenocarcinoma and may provide a novel therapeutic approach for treating lung cancer.

INTRODUCTION

Lung cancer is one of the most prevalent cancer forms, responsible for over one million

annual deaths worldwide. In the clinical setting, lung cancer is classified according to two main

histological types, small-cell lung cancer (SCLC) and non-small cell lung carcinoma (NSCLC)

(1). Eighty-five percent of all lung cancers are attributable to non-small-cell lung cancer

(NSCLC), of which lung adenocarcinoma is the most frequent histological subtype (1). On a

molecular level, lung adenocarcinomas frequently harbor mutations in the KRAS oncogene

(25%) and the tumor suppressor protein p53 (33%) (1). The importance of these mutations is

highlighted by the development of genetically engineered mouse models that harbor KRAS and

p53 mutations and are able to drive lung adenocarcinoma initiation and progression (2). In recent

years, molecular and cancer genomic approaches have increased our understanding of the pathogenesis of NSCLC and have led to therapies that directly target the precise genetic alterations that drive tumor growth.

The molecular genotyping of NSCLC into discrete molecular classes has redefined therapeutic approaches. Oncogenic alterations in the kinases Epidermal Growth Factor Receptor

(EGFR) and Anaplastic Lymphoma Kinase (ALK) are found in 15-25% of NSCLC (3, 4).

Patients with tumors harboring the activated EGFR and ALK kinase respond to the tyrosine kinase inhibitors gefitinib and erlotinib (5, 6) and crizotinib (7), respectively. Importantly, ALK and EGFR activation is mutually exclusive of KRAS mutation, underscoring the need for identifying and characterizing therapeutic options for KRAS driven lung cancer.

KRAS is one of the most commonly mutated oncogenes in lung cancer (1), exhibiting activating alterations in about 25-30% of lung adenocarcinomas. While point mutations in codon

12 or 13 account for the majority of KRAS mutations (8), recent studies have reported that

KRAS also undergoes copy number gain at chromosome 12p12 in 11-15% of lung tumors (9, 10).

RAS genes are members of the small GTPase super-family (11) and function to propagate growth factor signaling through activation of c-RAF and PI 3-kinase pathways. In spite of concerted efforts, KRAS mutation defines a genetic subtype of lung cancer that is currently not amenable to therapeutic intervention (12). Recent studies have utilized high throughput chemical and genetic screens to identify synthetic lethal phenotype in the context of KRAS mutations (13-

16). These screens have yielded promising kinases and other cellular machinery. Significant work is required to determine whether these interactions will translate into therapies for KRAS driven tumors.

While previous studies have mostly focused on identifying synthetic interactions in

KRAS mutant cell lines, here we set out to identify genes within the KRAS genomic amplicon that are both co-upregulated and critical for cell proliferation when KRAS is amplified. We characterized the 12p11/12 amplicon where KRAS resides, and report the presence of 18 additional genes that are co-amplified and overexpressed in lung adenocarcinomas. Using a loss of function RNAi screen on these 18 genes, we identified Lactate Dehydrogenase B (LDHB) as being specifically required in KRAS amplified lung cancer for cell proliferation. We find that

LDHB expression significantly correlates to both KRAS copy number gain and KRAS mutation in lung adenocarcinoma cell lines and tumors. LDHB knockdown reduced cell growth in KRAS mutant lung cancer both in vitro and in vivo. Lastly, LDHB was a prognostic indicator of poor outcome in lung adenocarcinomas. Our work identifies LDHB and more broadly, lactate metabolism, as a potential therapeutic target for KRAS driven lung cancer.

MATERIALS AND METHODS

Cell lines and antibodies

The following lung cell lines were used: adenocarcinoma (ALK fusion): NCI-H2228; adenocarcinoma (EGFR mutant): NCI-H1650, NCI-H1975, HCC827, NCI-H820, HCC2279,

HCC2935, HCC4011, HCC4006; adenocarcinoma (KRAS amplified): NCI-H322T, NCI-H838; adenocarcinoma (KRAS mutant): HOP-62, NCI-H2122, NCI-H1573, A427, NCI-H1734, NCI-

H2030, NCI-H2009, A549, NCI-H2126; bronchioalveolar adenocarcinoma (KRAS mutant):

NCI-H358; adenocarcinoma (ROS1 fusion, KRAS mutant): NCI-H23; non-small cell (KRAS mutant): SW1573; adenocarcinoma (Met amplification): NCI-H1993; adenocarcinoma: NCI-

H1793, RERF-LC-OK, NCI-H1568, VMRC-LCD, ABC-1, RERF-LC-KJ, NCI-H2126; bronchioalveolar adenocarcinoma: NCI-H1666; adenosquamous carcinoma: NCI-H596; large cell carcinoma: HOP-92; squamous cell carcinoma (Met amplification): EBC1. The following colon cell lines were used: SW48, SW1417, RKO, KM-12, HT55, HCA-7, C2BBe1, SW948,

SW837, SW620, SW1463, SK-CO-1, LS1034, HCT-15, DLD-1, COLO 678, CL-40. Cell lines were cultured in RPMI-1640 or DMEM (high glucose) medium, 10% Fetal Bovine Serum, and

1% Penicillin-Streptomycin (Invitrogen).

The following antibodies were used for immunoblot analysis: LDHA (Santa Cruz

Biotechnology; sc-133123), LDHB (Epitomics; 2090-1), TUBULIN (Sigma; T6074), and HRP- conjugated anti-mouse and anti-rabbit secondary antibodies (Jackson ImmunoResearch).

siRNA screen

An RNAi screen for genes that regulate lung cancer growth was carried out in four lung adenocarcinoma cell lines, two with chromosome 12p KRAS amplification (NCI-H838, NCI-

H322T) and two without KRAS amplification (RERF-LC-KJ, NCI-H1568). Genes were selected based on two main criteria: (1) they reside within the KRAS amplicon on chromosome 12p

(analysis completed with Tumorscape; http://www.broadinstitute.org/tumorscape) and (2) their gene expression and copy number were significantly upregulated in ≥10% of lung adenocarcinoma tumors and were correlated (p<0.05; analysis completed with The Cancer

Genome Atlas Data Portal; http://tcga-portal.nci.nih.gov). The heat map of 735 NSCLC tumors and cell lines was made with Integrative Genomics Viewer using data from Tumorscape.

Dharmacon siGENOME siRNAs were individually reverse transfected using

DharmaFECT 4 (Dharmacon) in 96-well format in triplicate. Four independent siRNAs were utilized for each gene. Cell number was determined with CellTiter-Glo (Promega) six days after transfection. Values were normalized to a non-targeting control siRNA, and then Z scores were calculated using the following formula: (gene value – plate average) / plate standard deviation.

To reduce the Z scores for each gene to a single comparable value, a ∆Z score was calculated: average of the Z scores from the two 12p amplified cell lines minus average Z scores of the non- amplified cell lines. Genes with a ∆Z score <-1 were considered hits in the 12p amplified cell lines. Only genes with two or more siRNA oligos with a ∆Z score <-1 were considered further.

Two independent siRNAs from the siGENOME pool were utilized for subsequent LDHB knockdown analysis (Dharmacon D-009779-01 and D-009779-02).

Gene expression analysis

For cell lines, RNA was harvested from 96 well plates three or four days after siRNA transfection using the TaqMan Gene Expression Cells-to-CT kit (Applied Biosystems). For mouse tissues/tumors, RNA was harvested using Qiagen RNeasy kit. Quantitative RT-PCR was

carried out with the Taqman One-Step RT-PCR Master Mix Reagents kit using Taqman Gene

Expression Assays according to manufacturer protocol (Applied Biosystems). All samples were

normalized to a GAPDH control.

Xenograft tumors

NCI-H2122 cells with doxycycline inducible LDHB knockdown were constructed using

the lentivirus pHush-shRNA system (17). Two independent shRNAs sequences were cloned for

LDHB knockdown (shLDHB-1 target sequence: GGATATACCAACTGGGCTA and shLDHB-

2 target sequence: GTACAGTCCTGATTGCATC). Hairpins were cloned into the pSHUTTLE-

H1 vector and then Gateway (Invitrogen) recombined into a puromycin-selectable lentiviral

vector (17). A non-targeting shRNA (pHush-shNTC) was used for control experiments and was

obtained from David Davis (Genentech). Lentivirus production and infections were carried out

as previously described (18). Cells were selected for stable integration with 2 ug/ml puromycin.

For each shRNA cell line, 5 x 106 cells were injected subcutaneously into the backs of

female NCr nude mice (Taconic) to initiate tumor growth. After tumors reached 200-300 mm3 in

size, the animals from each cell line were split into two groups and fed either 5% sucrose or 5%

sucrose + 1 mg/ml doxycycline to induce hairpin expression. Tumors with a starting volume

<200 mm3 at the time of treatment were excluded from further analysis. After seven days, three

mice from each group were euthanized and the tumors were harvested for LDHB knockdown analysis. Tumor measurements were carried out on the remaining mice every 3-4 days until Day

18 post-doxycycline administration. Mouse body weight was also recorded and showed no major changes (data not shown). Tumor growth inhibition values (TGI) were determined with the

following calculation: 100 * 1 – [(tumor volumefinal – tumor volumeinitial for doxycycline treated

group) / (tumor volumefinal – tumor volumeinitial for sucrose treated group)].

Data analysis of lung adenocarcinoma microarray data

Previously published microarray data of lung adenocarcinomas was utilized (Gene

Expression Omnibus GSE31210; (19)). For Figures 2D, 5B, and 5D, we used the previously

defined signature of RAS activation (20). Tumors (n = 226) were separated based on high or low

expression of LDHB, separated by mean LDHB expression across all tumors. The expression

level of all RAS signature genes was averaged in each group of tumors. For Figures 5A, we used

a defined glycolytic gene signature that was adapted from the Broad Institute Molecular

Signatures Database (MSigDB) gene sets for Glucose_Metabolic_Process (21). The expression

level of all glycolysis signature genes was averaged in each tumor and either separated by KRAS

gene mutation status or directly compared to the average expression of the RAS signature genes

in the same tumor.

Survival analysis

A lung adenocarcinoma tumor microarray (TMA) was obtained from the Leeds collection.

Patient samples were collected with approval from the Yorkshire and Humber East ethics committee (approval number: NRES 05/Q1206/147). In total, 170 formalin-fixed, paraffin

embedded (FFPE) lung adenocarcinomas were stained by immunohistochemistry (IHC) for

LDHB and correlated to clinical outcome. Kaplan-Meier survival curves were plotted for

survival until death or censored from the study, and for 5-year survival or censored. P-values

were calculated by a log-rank test. Fitting the data with a Cox Proportional Hazards model lead

to nearly identical P-values (data not shown). For survival analysis of LDHB gene expression,

LDHB expression and survival data were previously published (Gene Expression Omnibus

GSE11969; (22)). LDHB expression was mean-centered across all tumors then separated by

above or below the mean. All patients were censored from the study after >5 years of follow-up, thus the 5-year survival is shown. P-value was calculated by a log-rank test.

Immunohistochemistry

IHC was performed on 4 µm thick FFPE tissue sections mounted on glass slides. All IHC steps were carried out on the Ventana Discovery XT autostainer (Ventana Medical Systems).

Pretreatment was done with Cell Conditioner 1 with the standard time. Primary antibodies were used at the following concentrations: MCT1 (Santa Cruz Biotechnology; sc-365501) at 1 µg/ml,

MCT4 (Santa Cruz Biotechnology; sc-50329) at 0.1 µg/ml, LDHA (Cell Signaling Technologies;

3582) at 0.081 µg/ml, and LDHB (Epitomics; 2090-1) at 0.04 µg/ml. Slides were incubated with primary antibody for 60 minutes at 37ºC. Ventana Mouse or Rabbit OmniMap was used as the detection system. Ventana DAB and Hematoxylin II were used for chromogenic detection and counterstain. IHC analyses were performed by a board certified pathologist (R.F.).

RESULTS

Oncogenic activation of KRAS is typically achieved through single residue point

mutations that generate a constitutively active form of the protein. The KRAS locus is amplified

in 10-15% of lung adenocarcinoma tumors, suggesting an alternative mechanism of KRAS

activation in this tumor type (Supplementary Table 1; (9)). To further characterize the KRAS

locus in lung adenocarcinoma, we performed a GISTIC analysis on copy number data derived

from 735 NSCLC tumors and cell lines. While KRAS is the most significantly amplified gene

(Q-value = 10-22), the region of copy number gain is relatively broad and harbors 18 additional

genes that are both amplified and overexpressed (Figure 1A, B).

To functionally interrogate the candidate genes residing in the 12p12 region of copy

number gain, we performed a loss of function RNAi screen in lung adenocarcinoma cell lines

that harbored either 12p amplification (NCI-H838, NCI-H322T) or were disomic at this region

(RERF-LC-KJ and NCI-H1568). Gene expression and knockdown for all genes in the screen was

confirmed by quantitative RT-PCR in NCI-H838 cells (Supplementary Figure 1). By comparing

the Z scores between the amplified and disomic cell lines, genes with a differential cell

proliferation effect were identified (Figure 1C). In addition to KRAS, two additional genes,

LDHB and MED21, met our significance criteria of at least two independent siRNAs scoring at a

∆Z score of -1 or below (Z score chromosome 12p amplified lines minus Z score disomic lines).

MED21 (hSrb7) is a member of the Mediator complex and is a general regulator of transcription

(23). Lactate dehydrogenase B (LDHB) is a metabolic enzyme that catalyzes the inter-

conversion of lactate and pyruvate (24, 25).

Since LDHB and MED21 are frequently co-amplified with KRAS, we first examined

whether expression of these genes also correlate to KRAS mutation status. We assessed LDHB

and MED21 gene expression in both KRAS wild-type and mutant lung cancer cell lines and

found that both genes are upregulated in KRAS mutant cell lines (Figure 2A, B). The correlation

was significantly stronger for LDHB, which prompted us to look further into LDHB expression and function in the context of KRAS activity. LDHB protein and RNA is overexpressed in

KRAS mutant human lung tumors as well as in two mouse lung adenocarcinoma models driven

by mutant KRAS (2) (Figure 2C, D, E). Consistent with this finding, we also observed that

LDHB was upregulated in lung cancers that contain a ‘RAS activated’ gene expression signature

(20) (Figure 2F). To determine if the relationship between KRAS activation and LDHB extended

to other tumor types, LDHB expression was also examined in colon cancer, which frequently

harbors KRAS mutations (26). LDHB was not differentially expressed in colon cancer cell lines

harboring KRAS mutation, suggesting that the correlation between LDHB and KRAS may be

specific to lung adenocarcinomas (Supplementary Figure 2). We conclude that LDHB is

upregulated in lung cancer cell lines and tumors that are characterized by RAS pathway activation.

To determine if LDHB is required for the proliferation of KRAS mutant cancer cells, we

examined a panel of 24 lung adenocarcinoma cell lines for LDHB expression. Consistent with

our expression data in lung tumors, LDHB was specifically upregulated in both KRAS amplified

and KRAS mutant lung cancer cell lines when compared to KRAS wild-type cell lines (Figure

3A and Supplementary Table 2). In contrast, LDHA, the main LDH isoform, was ubiquitously

expressed in all lung cell lines examined. To explore the functional consequences of LDHB

expression in KRAS aberrant cell lines, we used two independent siRNAs to knockdown LDHB

in KRAS amplified, mutant, or wild-type lung adenocarcinoma cell lines. Both KRAS mutant

and KRAS amplified cell lines were more sensitive to LDHB knockdown (Figure 3B). To ensure

that these effects were not due to technical differences in knockdown levels, loss of LDHB was confirmed by immunoblot in each cell line tested. Moreover, no changes in LDHA protein levels were observed upon LDHB knockdown (Figure 3B). Since KRAS-mutation does not always confer dependence on KRAS for growth (27), we examined the sensitivity of each cell line to

KRAS RNAi-mediated knockdown (Figure 3B and Supplementary Figure 3). With minor exception, KRAS mutant cell lines were dependent on KRAS for growth. These results show that

KRAS dependent cell lines express elevated LDHB levels and are highly dependent on its expression for proliferation.

To determine whether LDHB itself is a downstream KRAS target, we examined LDHB gene expression after KRAS knockdown. We observed that LDHB gene expression did not directly change after KRAS knockdown (Supplementary Figure 3B), implying that LDHB is required in KRAS mutant cells, but is not directly regulated by KRAS activity.

Since LDHB expression does not appear to be directly regulated by KRAS, we considered whether LDHB is upregulated in lung adenocarcinomas that contain mutations in other oncogenic drivers (ie. EGFR mutation, c-Met amplification, ALK fusion, and ROS1 fusion). Therefore, we immunoblotted a panel of cell lines harboring these different oncogenic drivers (see Methods section for complete list of cell lines used). We find that LDHB can be upregulated in other molecular subtypes of lung adenocarcinoma, in particular those driven by c-

MET amplification and a subset of those containing EGFR mutation. The level of overexpression was similar to that observed in KRAS mutant cancers (Supplementary Figure

4A). To functionally test whether LDHB overexpression reflected a cellular dependence on

LDHB, we knocked down LDHB with two independent siRNA oligos in these additional cell lines. Overall, we find that cell lines with high levels of LDHB are significantly more sensitive to

loss of LDHB (P = 0.00005) than LDHB low expressing lines (Figure 3C, Supplemental Figure

4B, C).). Taken together these data suggest that targeting LDHB may provide a broad therapeutic option for lung cancer patients that specifically overexpress the LDHB protein.

The metabolic and growth requirements of a three-dimensional tumor can be starkly different from cancer cells grown in vitro. To test whether LDHB knockdown affects tumor growth in vivo, we utilized an inducible short hairpin (shRNA) lentiviral system to acutely deplete endogenous LDHB in fully formed tumors. Two independent shRNAs to LDHB

(shLDHB) and a non-targeting control (shNTC) were introduced into a human KRAS mutant lung cancer cell line (NCI-H2122) and subsequently grown as xenografted tumors in mice.

Doxycycline-induced LDHB knockdown in fully formed tumors led to significant tumor growth inhibition when compared to the uninduced shLDHB tumors or shNTC controls (Figure 4A).

LDHB knockdown in the tumors was confirmed by immunohistochemistry and immunoblot at day 7 after doxycycline administration (Figure 4B, C). These observations indicate that LDHB is required for growth of KRAS mutant lung tumors in vivo.

KRAS mutant lung cell lines and adenocarcinomas are dependent on glycolysis.

The ‘Warburg effect’ is named after the observation that many cancer cells generate energy using a high rate of glycolysis, instead of oxidative phosphorylation (28). The high rate of glycolysis occurs regardless of oxygen supply and produces an excess of pyruvate, which gets converted to lactate by LDHA or LDHB and subsequently exported from the cell. We hypothesized that LDHB upregulation may be associated with a more general shift in cellular metabolism toward glycolytic dependence. Since LDHB is overexpressed in KRAS mutant tumors, we analyzed and compared the expression of a defined glycolytic gene signature (see

Methods) between KRAS wild-type and KRAS mutant lung tumors. Consistent with our hypothesis, KRAS mutant tumors exhibited an elevated expression of the glycolytic gene signature compared to KRAS wild-type tumors (Figure 5A). Moreover, lung tumors that have increased expression of the RAS gene signature also show increased expression of the same glycolysis genes (Figure 5A). This effect appears to be specific to lung cancer, as a correlation between RAS signature expression and glycolytic signature expression was not observed in colon tumors (Supplementary Figure 2C). This suggests that lung tumors with KRAS mutation and RAS pathway activation are associated with a dependence on glycolysis.

To explore the functional significance of these gene expression changes, we tested the sensitivity of seven KRAS wild-type (low LDHB expressing) and six KRAS mutant (high

LDHB expressing) cell lines to the glycolysis inhibitor 2-deoxyglucose (2DG). KRAS mutant cell lines were significantly more sensitive to 2DG inhibition than KRAS wild-type cells (Figure

5B), suggesting that KRAS mutant cells are more addicted to glycolysis. Taken together these data imply that KRAS mutant tumors are associated with a shift in tumor metabolism that is characterized by increased dependence on glycolysis for energy demands.

The dependence of KRAS-mutated lung tumors on glycolysis and LDHB implies that these tumors are sensitive to perturbations in the end-stage of glycolysis, namely lactate production. In addition to LDH isoforms, which generate lactate, the monocarboxylate transporters (MCT1-4) are key transmembrane proteins that regulate the import/export of lactate

(29). While MCT1-4 all have similar structure and function, MCT1 and MCT4 are differentially upregulated in various tumor types and have garnered the most attention in cancer (30).

Therefore, we examined the expression of MCT1 and MCT4 in lung adenocarcinomas. IHC analysis revealed that MCT4, but not MCT1, was overexpressed in lung adenocarcinomas

(Figure 5C). Similar to LDHB, MCT4 expression was associated with RAS pathway activation

(Figure 5D). We suggest that KRAS mutant adenocarcinomas have upregulated components of

the lactate machinery to accommodate an increased dependence on glycolysis.

LDHB expression in lung adenocarcinomas correlates with poor clinical outcome

We next examined whether the expression of LDHB in lung cancer correlated with

clinical outcome. We scored and analyzed LDHB protein levels by IHC on a cohort of 383 lung

adenocarcinomas (Figure 6A, B), of which nearly half (n = 170) had associated survival data

(Supplementary Table 3). In Kaplan-Meyer analysis, five-year patient survival was significantly

lower in LDHB high (2 or 3 staining) as compared to LDHB low (0 or 1) tumors (33% versus

48%, log-rank p = 0.005). When we assessed the independent prognostic effect of LDHB using a

multivariate Cox model that adjusted for additional predictors of survival, such as sex, age and

tumor grade, high LDHB expression was associated with reduced five-year survival (Hazard

ratio = 1.67, 95% confidence interval (1.06, 2.62), p = 0.027) (Figure 6C, D and Supplementary

Figure 5). Since we observed that LDHB is upregulated in a subset of lung adenocarcinomas

containing that are KRAS wildtype but harbor different oncogenic drivers, we also examined if

LDHB expression is predictive of lifespan in KRAS wild-type tumors using a microarray dataset that contained associated outcome data. Indeed, KRAS wild-type lung tumors with high

expression of LDHB mRNA showed a trend with poor clinical outcome (Supplementary Figure

5C). These data indicate that LDHB is upregulated in a significant fraction of lung

adenocarcinomas and is associated with poor prognosis.

DISCUSSION

In this study, we characterized the KRAS amplicon at 12p12 and found 18 genes that are co-amplified with KRAS in NSCLC tumors. Using a loss of function RNAi screen, we subsequently knocked down all 18 genes and identified LDHB as an essential regulator of lung cancer. LDHB was upregulated and required for the growth of KRAS mutant and amplified lung cancers both in vitro and in vivo. Gene expression and functional analyses indicate that KRAS mutant lung tumors, compared to KRAS wild-type tumors, have a greater predilection for using glycolysis for their energy demands. Consistent with such a role, we find that in addition to

LDHB, the lactate transporter MCT4 is also upregulated in KRAS mutant lung cancer, suggesting a more global shift in metabolic requirements upon KRAS mutation. Finally, we show that LDHB overexpression in patients with lung adenocarcinoma have a poorer prognosis.

Carcinogenesis is a complicated, multi-step process that involves combinatorial alterations in oncogenes, tumor suppressors, and metabolic pathways. It is important to note that these alterations in intracellular signaling pathways are a type of evolution that conforms to the changing tumor micro-environment and bioenergetic requirements that allows uncontrolled cell growth. Since Warburg’s observation over 50 years ago, in which tumor cells shift from oxidative phosphorylation to aerobic glycolysis, we are only now beginning to understand the molecular details of this transformation.

In the last decade there has been accumulating evidence that oncogenes, such as PI 3- kinase (PI3K), AKT, mTOR, EGFR, KRAS, MYC, along with hypoxia-inducible factor 1

(HIF1) stimulate the transcription of key genes that mediate glycolysis and related metabolic pathways (31). For example, PI3K/AKT activation facilitates cellular glucose uptake by upregulating glucose transporters and boosting glycolysis through increasing hexokinase levels

and stimulating phosphofructokinase activity (32). Likewise, genes involved in the metabolism

of glutamine, which tumor cells need for the production of various metabolic intermediates, are

downstream targets of the MYC oncogene (33, 34). Direct regulatory inputs on metabolism are

not restricted to oncogenes, as deletion of tumor suppressors, such as p53 and PTEN, also

contribute to the metabolic shift (31). Taken together, the concerted upregulation of oncogenes and downregulation of tumor suppressors directly alters the transcriptional controls of the metabolic circuitry.

The direct role of KRAS in regulating tumor metabolism is less well defined. Tumors with oncogenic RAS correlate with numerous metabolic aberrations, including increased consumption of glucose and glutamine, increased production of lactic acid, altered expression of mitochondrial genes, and reduced mitochondrial activity (35-38). Our findings that a glycolysis gene signature is specifically upregulated in KRAS mutant lung tumors and that KRAS mutant cells are more sensitive to the glycolysis inhibitor 2DG, are consistent with such changes in tumor metabolism. Moreover, a recent report demonstrated that KRAS can also regulate glycolysis and glucose metabolism in a pancreatic cancer mouse model (39). Our observations, however, show that in other cancer types, such as colon cancer, KRAS mutant tumors do not display elevated dependence on glycolysis. Cumulatively, these studies suggest that examining the effect of KRAS on cellular metabolism should be conducted in a tumor-specific context.

Lactate dehydrogenase enzymes have important roles in aerobic glycolysis, as they are

required to convert the surplus of pyruvate generated by a high rate of glycolysis into lactate (25).

The LDHA isoform has received the most attention because it is ubiquitously expressed in

tumors and is a downstream target of HIF1. While an essential function of LDHA in tumor

metabolism has been described (40), the role of LDHB is less clear. One report has shown that

the mTOR pathway controls LDHB expression, while another report has shown that LDHB

transcription is shut off by promoter methylation in bladder, colon, and prostate cancers (41-44).

In the context of lung adenocarcinoma, we observed that LDHB is upregulated in KRAS aberrant tumors and in a subset of cell lines containing other oncogenic drivers. Irrespective of

the mutation status, cell lines that express high levels of LDHB are sensitive to its inhibition,

suggesting that expression of LDHB itself may predict response to an LDHB inhibitor . While

our data does not support that LDHB transcription is downstream of KRAS signaling, we

hypothesize that a pathway functioning in parallel with KRAS activity might drive LDHB expression. In normal tissues, LDHB is expressed in liver, red blood cells, kidney, and heart raising concern that its inhibition may have deleterious consequences (45, 46). Intriguingly, however, patients with complete loss of LDHB expression due to a hereditary recessive trait have no significant phenotypic impairment (47, 48). This suggests that an LDHB small molecule inhibitor may have minimal off-target affects in normal tissue. Future studies are necessary to identify signaling pathways that act upstream of LDHB.

Our data showing that LDHB inhibition reduced cell growth, even in the presence of

LDHA, raises an important question regarding the biochemical activity of the LDHB isoform.

Since the LDH enzymes function as both homo- and hetero-tetramers (46), it is possible that reducing LDHB somehow affects LDHA activity and the forward reaction. However, an alternative explanation is that LDHB has an exclusive function from LDHA. While biochemical data indicates that LDHA and LDHB can run in both the forward and reverse direction, LDHB has a biochemical predilection for the reverse direction (45). Perhaps LDHB functions in certain contexts to oppose LDHA or act as a governor to LDHA function. In this scenario LDHB would

at the very least slow down the conversion of pyruvate to lactate. Future work is necessary to

test these hypotheses, but is beyond the scope of this work.

In conclusion, our work identifies LDHB as a promising new therapy target for lung adenocarcinomas that overexpress LDHB and supports the notion that KRAS driven lung

cancers develop a dependence on glycolysis for their metabolic demands.

ACKOWLEDGEMENTS

We would like to thank Laura Corson, Tom Hunsaker, Anna Hitz, David Shames, Marie

Evangelista, and Georgia Hatzivassiliou for helpful comments and discussion, David Davis for reagents, and Pete Haverty for assistance in creating gene signatures.

FIGURE LEGENDS

Figure 1: RNAi screen for genes co-amplified with KRAS that regulate lung cancer cell

growth. (A) Heat map of 735 NSCLC tumors and cell lines showing copy number of a 12 Mb

region of chromosome 12 surrounding the KRAS gene. (B) Shown are Q-values (significance of

gene amplification in NSCLC data from A) of eighteen genes co-amplified with KRAS. These

genes had increased copy number and expression in primary lung tumors. (C) RNAi screen of

genes co-amplified with KRAS. Black and red bars represent effects on cell growth for each

individual siRNA oligos, as measured by ∆Z score (Z score KRAS amplified cell lines minus Z

score disomic cell lines). Red bars indicate individual siRNA oligos that had a stronger growth

inhibition in KRAS-amplified lung cancer cell lines compared to non-amplified lung cancer cell

lines. Genes with two or more individual siRNAs specifically inhibiting growth in KRAS

amplified lines are highlighted in red.

Figure 2: LDHB is upregulated in KRAS mutant lung adenocarcinomas. (A, B) Box plot of

relative MED21 (A) and LDHB (B) log2 mRNA expression in KRAS wild-type or KRAS mutant

NSCLC cell lines. P-values are a Student’s t-test. (C) Average LDHB IHC score +/- S.D. in

KRAS wild-type (n = 31) or KRAS mutant (n = 24) lung adenocarcinoma tumors. P-value is a

Student’s t-test. (D) Relative Ldhb mRNA expression is shown for normal mouse lung or

transgenic mouse models of lung cancer with KRASG12D activating mutation with or without

p53∆lox/∆lox deletion. P-values between normal and each tumor type are Student’s t-tests. (E)

Representative images of LDHB IHC protein staining in normal lung or KRASG12D; p53∆lox/∆lox lung cancer mouse model. (F) Average log2 expression RAS signature genes (20) +/- S.E.M. in

lung adenocarcimoma tumors with low (n = 97) or high (n = 129) LDHB expression (below or above mean LDHB expression across all tumors, respectively). P-value is a Student’s t-test.

Figure 3. LDHB is required for lung cancer cell line proliferation when highly expressed.

(A) Immunoblot analysis of LDHB and LDHA in lung adenocarcinoma cell lines that are KRAS wild-type, mutant, or amplified. (B) Relative cell proliferation (using CellTiter-Glo assay) of lung adenocarcinoma cell lines using two independent LDHB siRNA oligos compared to siNTC control. Immunoblot analysis of LDHB and LDHA are shown below each cell line. KRAS status refers to the mutation or amplification status of KRAS. KRAS dependent cells (denoted as +) have reduced cell proliferation after KRAS RNAi (see Supplementary Figure 3). (C) Cell lines were categorized as either high or low LDHB expressers following mean centered normalization

(cell lines above the mean were considered high and below the mean considered low). Plot shows relative cell growth (using CellTiter-Glo assay) of each cell line following LDHB knockdown (values were normalized to an siNTC control for each cell line and represent the average of two independent siLDHB oligos). Each dot or square denotes an individual cell line and error bars represent standard deviation. Colored circles and boxes highlight oncogenic mutation associated with each particular cell line. P-value indicates a statistically significant difference in cell growth between the two groups, Student’s t-test.

Figure 4: LDHB is required for lung tumor growth in vivo. (A) Tumor volumes from xenografted NCI-H2122 cells were measured over time (n = 5-6 mice per group). shRNA expression was induced with doxycycline (sucrose was used as control). Tumor growth inhibition (TGI) values were determined for each shRNA (shLDHB-1 TGI = 46%; shLDHB-2

TGI = 77%). Error bars represent S.E.M. (B) IHC staining for LDHB in representative shLDHB

xenograft tumors after 7 days of sucrose or doxycycline treatment. (C) Immunoblot of LDHB

and LDHA in xenograft tumors at day 7 post-doxycycline treatment.

Figure 5: Activated RAS is associated with increased glycolysis in lung adenocarcinomas.

(A) Average log2 expression glycolysis signature genes +/- S.E.M. is shown for lung

adenocarcimoma tumors (left panel) that are KRAS wild-type (n = 206) or KRAS mutant (n =

20). P-value is a Student’s t-test. Correlation analysis between the average log2 expression of

glycolysis signature genes and RAS signature genes in individual lung adenocarcinomas (right

panel). P-value for Pearson correlation is a one-tailed t-test. (B) Sensitivity of seven KRAS wild-

type and six KRAS mutant cell lines to 2-deoxyglucose (2DG). Each dot or square denotes an

individual cell line and error bars represent standard deviation. P-value indicates a statistically

significant difference in cell growth between the two groups, Student’s t-test. (C) Average IHC

scores +/- S.D. for LDHB, MCT1, and MCT4 are shown for normal lung tissue or

adenocarcinomas. P-values are Student’s t-tests. (D) Average log2 expression RAS signature

genes (20) +/- S.E.M. in lung adenocarcimoma tumors with low (n = 118) or high (n = 108)

MCT4 expression (below or above mean MCT4 expression across all tumors, respectively). P-

value is a Student’s t-test.

Figure 6: Patients with lung tumors expressing high LDHB protein have a poor clinical

outcome. (A) Representative IHC photomicrographs show LDHB protein expression in lung adenocarcinoma tumors. IHC score is indicated in parenthesis. (B) IHC score breakdown for a panel of normal lung tissue (n = 126) and lung adenocarcinoma tumors (n = 383). (C, D) Kaplan-

Meier survival data for lung adenocarcinoma patients, separated by LDHB IHC score. Patients with high LDHB expression (n = 99; IHC score 2 or 3) had a worse overall survival compared to patients with low LDHB expression (n = 71; IHC score 0 or 1) for a 5-year survival (C) or overall survival, as measured by death or being censored from the study (D).

REFERENCES

1. Herbst RS, Heymach JV, Lippman SM. Lung cancer. The New England journal of medicine. 2008;359:1367-80. 2. Singh M, Lima A, Molina R, Hamilton P, Clermont AC, Devasthali V, et al. Assessing therapeutic responses in Kras mutant cancers using genetically engineered mouse models. Nature biotechnology. 2010;28:585-93. 3. Camidge DR, Doebele RC. Treating ALK-positive lung cancer-early successes and future challenges. Nature reviews Clinical oncology. 2012. 4. Carter CA, Giaccone G. Treatment of nonsmall cell lung cancer: overcoming the resistance to epidermal growth factor receptor inhibitors. Current opinion in oncology. 2012;24:123-9. 5. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. The New England journal of medicine. 2004;350:2129-39. 6. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304:1497-500. 7. Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. The New England journal of medicine. 2010;363:1693-703. 8. Riely GJ, Kris MG, Rosenbaum D, Marks J, Li A, Chitale DA, et al. Frequency and distinctive spectrum of KRAS mutations in never smokers with lung adenocarcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14:5731-4. 9. Sasaki H, Hikosaka Y, Kawano O, Moriyama S, Yano M, Fujii Y. Evaluation of Kras gene mutation and copy number gain in non-small cell lung cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2011;6:15-20. 10. Weir BA, Woo MS, Getz G, Perner S, Ding L, Beroukhim R, et al. Characterizing the cancer genome in lung adenocarcinoma. Nature. 2007;450:893-8. 11. Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nature reviews Molecular cell biology. 2008;9:517-31. 12. Downward J. Targeting RAS signalling pathways in cancer therapy. Nature reviews Cancer. 2003;3:11-22. 13. Barbie DA, Tamayo P, Boehm JS, Kim SY, Moody SE, Dunn IF, et al. Systematic RNA interference reveals that oncogenic KRAS-driven cancers require TBK1. Nature. 2009;462:108-12. 14. Dolma S, Lessnick SL, Hahn WC, Stockwell BR. Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells. Cancer cell. 2003;3:285-96. 15. Luo J, Emanuele MJ, Li D, Creighton CJ, Schlabach MR, Westbrook TF, et al. A genome-wide RNAi screen identifies multiple synthetic lethal interactions with the Ras oncogene. Cell. 2009;137:835-48. 16. Torrance CJ, Agrawal V, Vogelstein B, Kinzler KW. Use of isogenic human cancer cells for high-throughput screening and drug discovery. Nature biotechnology. 2001;19:940-5.

17. Gray DC, Hoeflich KP, Peng L, Gu Z, Gogineni A, Murray LJ, et al. pHUSH: a single vector system for conditional gene expression. BMC biotechnology. 2007;7:61. 18. Adler AS, McCleland ML, Truong T, Lau S, Modrusan Z, Soukup TM, et al. CDK8 maintains tumor de-differentiation and embryonic stem cell pluripotency. Cancer research. 2012. 19. Okayama H, Kohno T, Ishii Y, Shimada Y, Shiraishi K, Iwakawa R, et al. Identification of genes upregulated in ALK-positive and EGFR/KRAS/ALK-negative lung adenocarcinomas. Cancer research. 2012;72:100-11. 20. Hoeflich KP, O'Brien C, Boyd Z, Cavet G, Guerrero S, Jung K, et al. In vivo antitumor activity of MEK and phosphatidylinositol 3-kinase inhibitors in basal-like breast cancer models. Clinical cancer research : an official journal of the American Association for Cancer Research. 2009;15:4649-64. 21. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC- 1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature genetics. 2003;34:267-73. 22. Takeuchi T, Tomida S, Yatabe Y, Kosaka T, Osada H, Yanagisawa K, et al. Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2006;24:1679-88. 23. Taatjes DJ. The human Mediator complex: a versatile, genome-wide regulator of transcription. Trends in biochemical sciences. 2010;35:315-22. 24. Bayley JP, Devilee P. The Warburg effect in 2012. Current opinion in oncology. 2012;24:62-7. 25. Porporato PE, Dhup S, Dadhich RK, Copetti T, Sonveaux P. Anticancer targets in the glycolytic metabolism of tumors: a comprehensive review. Frontiers in pharmacology. 2011;2:49. 26. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nature medicine. 2004;10:789-99. 27. Singh A, Greninger P, Rhodes D, Koopman L, Violette S, Bardeesy N, et al. A gene expression signature associated with "K-Ras addiction" reveals regulators of EMT and tumor cell survival. Cancer cell. 2009;15:489-500. 28. Warburg O. On the origin of cancer cells. Science. 1956;123:309-14. 29. Halestrap AP. The monocarboxylate transporter family--Structure and functional characterization. IUBMB life. 2012;64:1-9. 30. Pinheiro C, Longatto-Filho A, Azevedo-Silva J, Casal M, Schmitt FC, Baltazar F. Role of monocarboxylate transporters in human cancers: state of the art. Journal of bioenergetics and biomembranes. 2012. 31. Levine AJ, Puzio-Kuter AM. The control of the metabolic switch in cancers by oncogenes and tumor suppressor genes. Science. 2010;330:1340-4. 32. Deberardinis RJ, Sayed N, Ditsworth D, Thompson CB. Brick by brick: metabolism and tumor cell growth. Current opinion in genetics & development. 2008;18:54-61. 33. Gao P, Tchernyshyov I, Chang TC, Lee YS, Kita K, Ochi T, et al. c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature. 2009;458:762-5. 34. Wise DR, DeBerardinis RJ, Mancuso A, Sayed N, Zhang XY, Pfeiffer HK, et al. Myc regulates a transcriptional program that stimulates mitochondrial glutaminolysis and leads

to glutamine addiction. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:18782-7. 35. Chiaradonna F, Gaglio D, Vanoni M, Alberghina L. Expression of transforming K-Ras oncogene affects mitochondrial function and morphology in mouse fibroblasts. Biochimica et biophysica acta. 2006;1757:1338-56. 36. Chiaradonna F, Sacco E, Manzoni R, Giorgio M, Vanoni M, Alberghina L. Ras- dependent carbon metabolism and transformation in mouse fibroblasts. Oncogene. 2006;25:5391-404. 37. Gaglio D, Metallo CM, Gameiro PA, Hiller K, Danna LS, Balestrieri C, et al. Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth. Molecular systems biology. 2011;7:523. 38. Vizan P, Boros LG, Figueras A, Capella G, Mangues R, Bassilian S, et al. K-ras codon- specific mutations produce distinctive metabolic phenotypes in NIH3T3 mice [corrected] fibroblasts. Cancer research. 2005;65:5512-5. 39. Ying H, Kimmelman AC, Lyssiotis CA, Hua S, Chu GC, Fletcher-Sananikone E, et al. Oncogenic Kras Maintains Pancreatic Tumors through Regulation of Anabolic Glucose Metabolism. Cell. 2012;149:656-70. 40. Fantin VR, St-Pierre J, Leder P. Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer cell. 2006;9:425-34. 41. Leiblich A, Cross SS, Catto JW, Phillips JT, Leung HY, Hamdy FC, et al. Lactate dehydrogenase-B is silenced by promoter hypermethylation in human prostate cancer. Oncogene. 2006;25:2953-60. 42. Liao AC, Li CF, Shen KH, Chien LH, Huang HY, Wu TF. Loss of lactate dehydrogenase B subunit expression is correlated with tumour progression and independently predicts inferior disease-specific survival in urinary bladder urothelial carcinoma. Pathology. 2011;43:707-12. 43. Thangaraju M, Carswell KN, Prasad PD, Ganapathy V. Colon cancer cells maintain low levels of pyruvate to avoid cell death caused by inhibition of HDAC1/HDAC3. The Biochemical journal. 2009;417:379-89. 44. Zha X, Wang F, Wang Y, He S, Jing Y, Wu X, et al. Lactate dehydrogenase B is critical for hyperactive mTOR-mediated tumorigenesis. Cancer research. 2011;71:13-8. 45. Dawson DM, Goodfriend TL, Kaplan NO. Lactic Dehydrogenases: Functions of the Two Types Rates of Synthesis of the Two Major Forms Can Be Correlated with Metabolic Differentiation. Science. 1964;143:929-33. 46. Markert CL, Shaklee JB, Whitt GS. Evolution of a gene. Multiple genes for LDH isozymes provide a model of the evolution of gene structure, function and regulation. Science. 1975;189:102-14. 47. Kanno T, Sudo K, Takeuchi I, Kanda S, Honda N, Nishimura Y, et al. Hereditary deficiency of lactate dehydrogenase M-subunit. Clinica chimica acta; international journal of clinical chemistry. 1980;108:267-76. 48. Joukyuu R, Mizuno S, Amakawa T, Tsukada T, Nishina T, Kitamura M. Hereditary complete deficiency of lactate dehydrogenase H-subunit. Clinical chemistry. 1989;35:687- 90.

Lactate Dehydrogenase B is required for the growth of KRAS-dependent lung adenocarcinomas

Mark L. McCleland, Adam S. Adler, Laura Deming, et al.

Clin Cancer Res Published OnlineFirst December 6, 2012.

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