HIF-2Α Deletion Promotes Kras-Driven Lung Tumor Development

HIF-2α deletion promotes Kras-driven lung tumor development

Jolly Mazumdara,b, Michele M. Hickeya,c,1, Dhruv K. Panta,d, Amy C. Durhame, Alejandro Sweet-Corderof, Anil Vachanig, Tyler Jacksh, Lewis A. Chodosha,d, Joseph L. Kissili, M. Celeste Simona,b,j,2, and Brian Keitha,d

aAbramson Family Cancer Research Institute, cCell and Molecular Biology Graduate Group, dDepartment of Cancer Biology, and jDepartment of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; bHoward Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104; eDepartment of Pathobiology, University of Pennsylvania School of Veterinary Medicine, Philidelphia, PA 19104; fDepartment of Pediatrics, Stanford University, Stanford, CA 94305; gAbramson Research Center, Philadelphia, PA 19104; hKoch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139; and iThe Wistar Institute, Philadelphia, PA 19104

Edited by Steven L. McKnight, University of Texas Southwestern, Dallas, TX, and approved June 21, 2010 (received for review February 1, 2010) Non-small cell lung cancer (NSCLC) is the leading cause of cancer In a recent report, ectopic expression of a nondegradable HIF- deaths worldwide. The oxygen-sensitive hypoxia inducible factor 2α promoted growth of KrasG12D-driven murine lung tumors (13). (HIF) transcriptional regulators HIF-1α and HIF-2α are overexpressed Relative to KrasG12D controls, KrasG12D lung tumors expressing in many human NSCLCs, and constitutive HIF-2α activity can pro- stabilized HIF-2α were larger, displayed evidence of enhanced mote murine lung tumor progression, suggesting that HIF proteins angiogenesis and epithelial-mesenchymal transition (EMT), and may be effective NSCLC therapeutic targets. To investigate the con- resulted in decreased survival (13). These phentoypes were cor- sequences of inhibiting HIF activity in lung cancers, we deleted related with increased expression of multiple HIF-2α target genes Hif-1α or Hif-2α in an established KrasG12D-driven murine NSCLC including VEGFR2, Snail, and others (13). Collectively, these model. Deletion of Hif-1α had no obvious effect on tumor growth, results demonstrated that increased HIF-2α activity can drive whereas Hif-2α deletion resulted in an unexpected increase in tumor NSCLC growth and underscore the potential utility of developing burden that correlated with reduced expression of the candidate targeted therapies to inhibit HIF proteins in general, and HIF-2α tumor suppressor gene Scgb3a1 (HIN-1). Here, we identify Scgb3a1 in particular, for NSCLC and renal cell carcinomas (RCC). α α α α as a direct HIF-2 target gene and demonstrate that HIF-2 regulates To determine the effects of eliminating HIF-1 and HIF-2 CELL BIOLOGY Scgb3a1 expression and tumor formation in human KrasG12D-driven expression in NSCLC, we used conditional “floxed” alleles to delete NSCLC cells. AKT pathway activity, reported to be repressed by HIF-1α or HIF-2α in lung tumors using the murine KrasG12D Scgb3a1, was enhanced in HIF-2α-deficient human NSCLC cells NSCLC model (13). Deletion of HIF-1α in these tumor cells had no and xenografts. Finally, a direct correlation between HIF-2α and detectable effect on tumor growth but, surprisingly, deletion of SCGB3a1 expression was observed in approximately 70% of human HIF-2α actually increased tumor number and size. This phenotype NSCLC samples analyzed. These data suggest that, whereas HIF-2α correlated with reduced expression of Scgb3a1 (14) also known as overexpression can contribute to NSCLC progression, therapeutic high in normal-1 (HIN-1), a candidate tumor suppressor gene im- inhibition of HIF-2α below a critical threshold may paradoxically plicated in lung, breast, pancreatic, and other cancers (15–17). We promote tumor growth by reducing expression of tumor suppressor demonstrate here that Scgb3a1 is a direct HIF-2α target gene and genes, including Scgb3a1. that HIF-2α deficient lung tumor xenografts are characterized by enhanced AKT signaling, consistent with previous observations hypoxia | inducible mouse model | non-small cell lung cancer | tumor that Scgb3a1 suppresses AKT activity in human breast cancer cells suppressor (18). We further demonstrate that ectopic expression of Scgb3a1 suppresses the growth of HIF-2α–deficient lung tumor xenografts, espite the access of normal pulmonary epithelia to atmo- concomitant with reduced AKT signaling. Finally, a direct corre- α Dspheric O2 levels, tumor hypoxia has been observed in human lation between Hif-2 and Scgb3a1 expression was observed in non-small cell lung cancer (NSCLC) and correlates with poor human NSCLC cells and primary NSCLC tumors. These data prognosis and decreased disease-free survival (1, 2). Cells can suggest that, although HIF-2α overexpression can contribute to overcome hypoxic stress through multiple mechanisms, including NSCLC progression, therapeutic inhibition of HIF-2α below the stabilization of hypoxia inducible factor (HIF) transcriptional a critical threshold may actually promote tumor growth by repres- regulators. HIFs consist of an oxygen labile α-subunit and a con- sing Scgb3a1 and other HIF-2α target genes. stitutively expressed β-subunit (also called ARNT), which to- Results gether bind hypoxia response elements (HREs) to activate a large HIF-2α Deletion Promotes KrasG12D-Induced Lung Tumor Growth. The number of target genes encoding proteins that mediate adaptive G12D responses to hypoxic stress (3, 4). Two distinct subunits, HIF-1 inducible LSL-Kras murine genetic model generates lung and HIF-2, have been demonstrated to regulate partly over- tumors that faithfully model human lung adenocarcinoma initi- lapping sets of target genes in hypoxic cells, although each protein also fulfills unique essential functions (5–7). Many HIF target genes encode proteins important for tumor Author contributions: J.M., M.M.H., A.S.-C., T.J., M.C.S., and B.K. designed research; J.M., M.M.H., A.S.-C., and B.K. performed research; A.V. provided human NSCLC samples; J.M., growth and progression, including glycolytic enzymes, VEGF, D.K.P., A.C.D., J.L.K, L.A.C., and B.K. analyzed data; and J.M. and B.K. wrote the paper. matrix metalloproteinase-2 (MMP2), transforming growth factors The authors declare no conflict of interest. α β α β and (TGF- and TGF- ), and numerous others (4, 8). Col- This article is a PNAS Direct Submission. lectively, these factors modulate cancer cell metabolism and can Freely available online through the PNAS open access option. promote angiogenesis, invasion, and metastasis, suggesting that Data deposition: The data reported in this paper have been deposited in the Gene Ex- HIF proteins may represent suitable targets for antitumor ther- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE22575). apies (9–11). It is interesting to note that both HIF-1α and HIF-2α 1Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115. proteins are overexpressed in approximately 50% of human 2To whom correspondence should be addressed. E-mail: [email protected]. NSCLCs (12), further suggesting they may contribute directly to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. NSCLC progression. 1073/pnas.1001296107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1001296107 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 fl Δ ation and progression (19). To evaluate the effect of HIF-2α loss- 2A), and a similar trend at 12 wk. Similarly, KrasG12D/Hif-2α / of-function in lung tumor progression, we crossed mice carrying animals displayed significantly increased numbers of adenomas fl Δ conditional floxed Hif-2α or null Hif-2α alleles (20) to mice (Fig. 2B) at 24 wk compared with control animals. A similar trend carrying the LSL-KrasG12D allele. The resulting experimental toward increased numbers of adenocarcinomas was observed at fl Δ Δ KrasG12D/Hif-2α / and control KrasG12D/Hif-2α+/ animals were 24 wk but did not achieve statistical significance (Fig. 2C). Col- treated with adenovirus encoding the Cre recombinase by intra- lectively, these data indicate that loss of HIF-2α accelerates tumor nasal instillation. Cre-mediated deletion of the Lox-Stop-Lox (LSL) progression in this context. Interestingly, deletion of HIF-1α in gene cassette in the LSL-KrasG12D allele produced KrasG12D-driven parallel experiments did not alter disease kinetics (Fig. S2 G–I), lung tumors as previously described (19) and in this case also underscoring the differential effects of HIF-1α and HIF-2α in fl deleted the conditional Hif-2α allele. As the control KrasG12D/ these tumors. Δ Hif-2α+/ animals harbor a wild-type Hif-2α allele, HIF-2α function The increased size of HIF-2α–deficient tumors correlated with was consequently retained in tumor tissue (Fig. S1A). Southern elevated cell proliferation (Fig. 2 D and E) and decreased apo- fl blot analysis confirmed efficient deletion of the Hif-2α allele spe- ptosis (Fig. 2 F and G), which was not observed in HIF-1α– cifically in tumors but not surrounding lung tissue (Fig. S1B). deficient tumors (Fig. S2 J and K). We also noted increased To evaluate the effect of HIF-2α deletion on lung tumor numbers of infiltrating leukocytes in HIF-2α–deleted tumors progression, animals were analyzed 12 wk and 24 wk after ade- (Fig. S3 A and B), and granulocytes in particular (Fig. S3 C and noviral Cre delivery (Fig. S1C). Based on the recent report that D), suggesting that inflammatory responses may differ between Δ Δ Δ HIF-2α gain-of-function promotes lung tumor progression and KrasG12D/Hif-2α / , and KrasG12D/Hif-2α+/ lung tumors. correlates with poor survival in this model (12, 13), we predicted that deletion of HIF-2α would reduce tumor burden and pro- Identification of Scgb3a1 as a HIF-2α Target. To investigate molec- fl Δ gression. Surprisingly, KrasG12D/Hif-2α / mutant mice displayed ular mechanisms underlying HIF-2α’s tumor suppressive effects, a significant increase in tumor number and size at 12 wk (Fig. 1 we conducted global gene expression profiling on individual fl Δ Δ A, C, and D) and 24 wk (Fig. 1 B, E, and F) postinfection, as tumors from KrasG12D/Hif-2a / or control KrasG12D/Hif-2α+/ compared with their control littermates. Notably, deletion of fl a floxed Hif-1α allele (21) in parallel experiments had no de- G12D tectable effect on tumor number or volume in Kras lung A +/ fl/ BC+/ fl/ +/ fl/ tumors (Fig. S2 A–F). 45 6 1.6 40 1.4 Hif-2α Deletion Promotes Tumor Progression. Lung tumors gener- * 5 * 35 1.2 ated in LSL-KrasG12D mice display distinct types of progressive 30 4 1 lesions including epithelial hyperplasia, adenomas, and adeno- 25 G12D 3 0.8 carcinomas (19). HIF-2α deficient Kras lung tumors dis- 20 played a significant increase in hyperplastic lesions at 24 wk (Fig. 15 2 0.6 10 0.4 1 5 Adenomas /mouse 0.2 Hyperplasias /mouse 0 0 Adenoarcinomas /mouse 0 A 12 week B 24 week 12 wk 24 wk 12 wk 24 wk 12 wk 24 wk

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. 0 4 24 wk 2 0 X F G +/ fl/ +/ fl/ 16 d C D E F 2 0 X l 12 wk 12 wk 24 wk 24 wk e 14 i * 18 ) 16 ) 6 40 2 2 16 12 14 * 36 * * er f * 5 µ m 32 ( 14 12 10

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24 / 10 8 ce 8 3 8 20 + 6 6 TUNEL 2 16 6 4 8 4 EL 4 1 N 2 4 2 Tumor no. per mouse Tumor no. per mouse Tumor area Tumor area/field ( µ m 2 0 0 0 0 TU +/ fl/ +/ fl/ +/ fl/ +/ fl/

% 0 Δ 24 wk Fig. 1. Hif-2α deletion increases tumor burden. (A–F) KrasG12D/Hif-2α+/ fl Δ (designated +/Δ), or KrasG12D/Hif-2α / (designated fl/Δ) mice were infected Fig. 2. Hif-2α deletion promotes tumor proliferation and progression. H&E Δ with Adeno-Cre virus and killed 12 (A)or24(B) wk postinfection (w.p.i.). H&E stained lung sections prepared from KrasG12D/Hif-2α+/ (+/Δ)orKrasG12D/Hif- fl Δ staining of murine lung sections indicate increased tumor burden in Hif-2α 2α / (fl/Δ) animals were graded for hyperplastic lesions (A), adenomas (B), deleted cohorts. Tumor number and area were measured using an automated and adenocarcinomas (C). (D and E) Control and mutant tumors were Axiovision system (Materials and Methods). (C–F) At 12 and 24 w.p.i, fl/Δ assessed 24 w.p.i. for proliferation by Ki67 staining (n = 6). (F and G)En- animals presented significantly increased tumor number (C and E) and size (D hanced cell death in +/Δ tumors as compared with Δ/Δ mutant tumors (n =6) and F) compared with +/Δ controls. (n =9–11 in each group). Error bars (C–F) as assessed by a TUNEL assay. Sections were imaged at 20×. Error bars rep- represent SEM. *P < 0.05. resent SEM. *P < 0.05.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1001296107 Mazumdar et al. Downloaded by guest on September 25, 2021 mice. Specifically, RNA was isolated from tumors from experi- To extend our studies to human NSCLC, we introduced mental and control animals (n = 7 for each group) 28 wk after a retroviral shRNA gene construct targeting human Hif-2α infection and analyzed independently. Comparisons between the mRNA (23) into human A549 lung adenocarcinoma cells, which genotypes identified a small set of genes (Table S1) whose ex- harbor an activating Kras mutation (24). Despite extensive pression was significantly and reproducibly reduced in HIF-2α– screening, only partial HIF-2α knockdown was observed in mul- deficient tumors, whereas expression levels of most genes were tiple cell clones, as revealed by Western blot and qRT-PCR unchanged. Subsequent quantitative RT-PCR (qRT-PCR) anal- analyses (Fig. 3 B and C). Nevertheless, pooled HIF-2α knock- ysis on the same tumor RNAs revealed dramatic down-regulation down cells (Fig. 3D), as well as independent knockdown cell of multiple transcripts, including those encoding Scgb3a1, lacto- clones HIF-2α KD C1 and HIF-2α KD C2 (Fig. 3E), exhibited transferrin, aquaporin 4, and ceruloplasmin (Fig. 3A). In contrast, a significant reduction in Scgb3a1 transcript levels (approximately transcript levels of the HIF-1α target gene aconitase (Aco1) were 2.5-fold), consistent with our previous observations. A similar unaffected by Hif-2α deletion (Fig. 3A). The reduced expression reduction was observed for transcripts encoding aquaporin 4 of Scgb3a1 was particularly intriguing as (i) Scgb3a1 is expressed (Aqp4), ceruloplasmin (CP), and VEGF (Fig. 3D and Fig. S4 A primarily in epithelial organs including lung, mammary gland, and B) but not the HIF-1α specific target PGK1 (Fig. S4B). As trachea, prostate, pancreas, and salivary gland (22); (ii) Scgb3a1 a control for functional specificity, we introduced a murine cDNA expression is silenced in a variety of human cancers including encoding HIF-2α into the human HIF-2α KD C1 knockdown cells lung, breast, pancreas, and prostate (15, 16); and (iii) down- (designated HIF-2α KD C1R, Fig. 3C), which in turn restored regulation of Scgb3a1 is a significant independent predictor of Scgb3a1 transcript levels (Fig. 3E). Interestingly, HIF-1α de- poor clinical outcome in early stage NSCLC (17). pletion in A549 cells (Fig. 3B) did not alter transcript levels of

A +/ fl/ 1.2 ** ** 1 ** ** ** ** ** * 0.8 CELL BIOLOGY 0.6 0.4

0.2

(normalized to18s) 0 4 P rp Fold change in mRNA Ltf C x36 -0.2 Aqp o1a5 o Aco1 lc11a2 Gab lc b Scgb3a1 S S Slc27a1 F B C A549 VC HIF-2 KD C1 HIF-2 KD C2 HIF-1 KD HIF-2 KD HIF-2 KD C1R 2 6 VC VC C1 C2 VC VC C1 C2 ** 5 HIF-1 1.5 * * 4 Fig. 3. HIF-2α regulates Scgb3a1 tumor suppressor 1 3 HIF-2 gene expression. (A) A qRT-PCR analysis of mRNAs from KrasG12D/Hif-2α+/Δ (+/Δ)orKrasG12D/Hif-2αﬂ/Δ (ﬂ/Δ) 2 tumors (n = 7) harvested 28 w.p.i. Probe sets were se- 0.5 actin 1 lected from microarray analysis (Table S1). (B and C) A549 human NSCLC cells transduced with retroviral

Fold change in mRNA α α α 0 0 shRNA constructs targeting HIF-1 (HIF-1 KD) or HIF-2 NDFXNDFX α hHIF-2 mHIF-2 (HIF-2 KD) or with an empty viral control (VC). (B)In- dependent clones (C1 and C2) were assessed for HIF-1α α VC HIF-2 KD VC and HIF-2 protein levels. n = normoxia (21% O2). DFX = D E F deferoxamine (100 μM) treatment to stabilize HIF-α HIF-1 KD HIF-2 KD C1 HN HIF-2 KD C2 subunits. (C). Endogenous human HIF-2α transcript lev- HIF-2 KD C1R els were reduced in HIF-2α KD cells (hHIF-2α, Left). HIF-2α α 6 9 rescue cells (HIF-2 KD C1R) were engineered through * 8 stable expression of murine HIF-2α mRNA in HIF-2α KD 8 ** α 5 * 7 C1 cells (mHIF-2 , Right). (D) Hypoxic induction of 7 Scgb3a1, Aqp4, and CP transcript levels was reduced in * 6 ** 4 * 6 pooled HIF-2α KD cells but not HIF-1α KD cells. (E) Scgb3a transcript levels were reduced in hypoxic HIF-2α 5 5 ** 3 KD cell clones C1 and C2 (0.5% O2, 16 h) but restored in ormoxic mRNA 4 4 hypoxic HIF-2α KD C1R cells. (F) Nuclear extracts (n =3) n 3 were isolated from uninfected hypoxic A549 cells (0.5% 2 c/ 3 2 2 O2, 16 h), sonicated, and immunoprecipitated with HIF- 1 oxi 2α antibodies. DNA was ampliﬁed using primers for HRE 1 1 regions 4, 5, and 6 (designated H4, H5, and H6) and 0 Hyp 0 0 normalized to isotype matched IgG antibody. Error bars Hypoxic induction of mRNA Scgb3a1 Aqp4 CP Scgb3a1 Relative promoter occupancy H4 H5 H6 (C–F) represent SD. *P < 0.05; **P < 0.005.

Mazumdar et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 Scgb3a1 or other genes identiﬁed in the microarray experiment A B VC HIF-2 KD C1 (Fig. 3D and Fig. S4C). HIF-2 KD C1R Our results indicate that HIF-2α regulates Scgb3a1 expression 350 VC 3 HIF-2 KD C1 Scgb3a1 ) ** 3 HIF-2 KD C1 in lung adenocarcinoma cells in a cell autonomous manner and 300 2.5 ** HIF-2 KD C1R that this activity is not shared by HIF-1α. We next investigated the 250 α 2 possibility that HIF-2 regulates Scgb3a1 directly. Analysis of 200 1.5 human and murine Scgb3a1 gene sequences revealed multiple 150 1 putative HREs spanning the upstream promoter and enhancer 100 regions (Fig. S4D). ChIP assays revealed HIF-2α occupation of 50 0.5 two HREs (H4 and H5) in the Scgb3a1 promoter in A549 cells, Tumor volume (mm 0 0

which increased (4- to 7-fold) under hypoxic conditions (Fig. 3F). 1 5 10 15 20 25 30 35 Fold change in tumor weight Not all HREs in the Scgb3a1 promoter appear functional, as H6 d.p.i (1x106 cells) fails to bind HIF-2α (Fig. 3F). Moreover, HIF-1α failed to bind H4 and H5 (Fig. S4E). Collectively, these data demonstrate that C 1 D Scgb3a1 is a direct HIF-2α target gene. C1 KDT1 C2 KDT2 KD C1 HIF-2 KD C Scgb3a1 α A549 HIF-2 KD C2 HIF-2 KD C1 R HIF-2 pAKT To test the effects of HIF-2 knockdown in tumor formation by S473 6 HIF-1 the A549 cells, we implanted 5 × 10 HIF-2α KD C1 or HIF-2α KD AKT HIF-2 C2 cells s.c. in immunocompromised mice to generate xeno- pGSK3 graft tumors. Consistent with our results from the autochthonous Scgb3a1 pAKT GSK HIF-2α deficient lung tumors, xenograft tumors from HIF-2α KD S473 C3 KDT3 C4 KDT4 fi AKT C1 cells displayed a modest but signi cant growth advantage over pAKT A549 control tumors and a similar trend was also observed for pGSK3 S473 AKT HIF-2α KD C2 cells (Fig. S5 A and B). As the recipient mice had to GSK pGSK3 be killed between 25 and 35 d due to the large size of these xen- 4EBP-1 × 6 α GSK ografts, the experiment was repeated using fewer (1 10 )HIF-2 actin KD C1 cells (Fig. 4A). In this experiment, HIF-2α KD C1 xenografts displayed a clear and significant growth advantage over the Fig. 4. HIF-2α suppresses A549 xenograft growth and AKT signaling. (A) vector control xenografts, and restored HIF-2α expression (HIF-2α Nude (Nu/Nu) mice were injected s.c. with 1 × 106 HIF-2α KD C1 cells. A549 fl KD C1R cells) suppressed tumor growth to levels essentially in- cells expressing the empty vector (VC) injected in contralateral ank served as controls, and individual tumor volumes were measured (mm3) at indicated distinguishable from the vector control tumors (Fig. 4 A and B). α fi α days postimplantation (d.p.i). HIF-2 KD C1 cells (n = 10) formed signi cantly Importantly, ectopic expression of human Scgb3a1 in the HIF-2 larger tumors compared with control cells (n = 10). Tumors formed by mHIF- KD C1 cells (Fig. S5B) also effectively suppressed tumor growth, 2α expressing HIF-2α KD C1R cells (n = 6) were comparable to control tumors implicating Scgb3a1 as a critical mediator of HIF-2α activity in this (n = 6). Pooled VC tumor volumes from two simultaneously conducted in- setting (Fig. 4B). dependent experiments [(VC; HIF-2α KD C1) and (VC; HIF-2α KD C1R)] were used for computation. (B) Ectopic expression of HIF-2α or Scgb3a1 in HIF-2α HIF-2α Deficiency Reduces AKT Signaling. Enforced Scgb3a1 ex- KD C1 cells inhibits tumor growth. Xenografts (n = 10) were generated as in pression was shown to inhibit AKT pathway signaling associated A and tumor weights measured at 35 d. (C) A549 wild-type cells, HIF-2α KD with decreased AKT phosphorylation at Ser473 in human breast C1, HIF-2α KD C2, HIF-2α KD C1R, and HIF-2α KD C1 cells expressing Scgb3a1 cancer cells (18). We therefore hypothesized that HIF-2α were cultured under hypoxic conditions (0.5% O2) for 20 h and whole cell extracts analyzed for AKT pathway activation by Western blot analysis. Actin knockdown in A549 cells would increase AKT pathway activity. α α α served as the loading control. (D) HIF-2 KD C1 [n = 4 (KD T1-4)] and con- Exposure of HIF-2 KD C1 and HIF-2 KD C2 cells to hypoxia tralateral A549 control [n = 4 (C1-4])] xenograft tumors were processed for fi 473 signi cantly increased levels of phopshorylated AKT at Ser whole cell protein extraction and analyzed by Western blot. Error bars (Fig. 4C). Moreover, levels of phosphorylated AKT downstream represent SEM. **P < 0.005. effectors including pGSK-3β and 4EBP-1 were higher compared with control cells (Fig. 4C). Notably, restoring either HIF-2α or Scgb3a1 expression in the HIF-2α KD C1 cells reduced AKT, Discussion GSK-3β, and 4EBP-1 phosphorylation to nearly control levels α (Fig. 4C). A similar trend was observed in the xenograft tumors, Many human cancers display a direct correlation between HIF- as Western blot analysis revealed that HIF-2α KD C1 tumors overexpression and poor prognosis (4, 8), consistent with the displayed increased pAKT and pGSK3β levels compared with idea that HIF target genes positively regulate critical features of control tumors (Fig. 4D). tumor progression, including cancer cell metabolism, survival, movement, and local angiogenesis. This correlation has spurred HIF-2α Levels Correlate with SCGB3a1 in Human NSCLC. The role of the development of HIF inhibitors as therapeutic treatments for HIF-2α in regulating Scgb3a1 led us to investigate whether HIF- cancer, as well as other diseases (11). The specific activities of HIF- 2α and SCGB3a1 transcript levels correlate in human NSCLC. 1α and HIF-2α in tumor progression are quite complex, however, The qRT-PCR analysis of 15 independent human adenocarci- and appear to vary depending on cell type and oncogenic back- noma biopsies revealed that in 73% (11/15), HIF-2α mRNA ground. For example, although HIF-1α overexpression drives levels were significantly reduced relative to normal tissue, which murine mammary tumor progression and metastasis in a murine correlated directly with reduced SCGB3a1 mRNA expression model (25), it also inhibits the growth of certain von Hippel-Lindau (Fig. 5A). In contrast, tumors that retained normal HIF-2α (VHL)-deficient RCCs (26, 27), in part by restricting the activity mRNA levels did not display statistically significant changes in of the c-Myc oncoprotein (28, 29). In contrast, HIF-2α expression SCGB3a1 mRNA levels. To further investigate the connection potentiates c-Myc signaling in RCCs and promotes tumor growth between HIF-2α and SCGB3a1 expression, we evaluated lung (29). Furthermore, a recent report demonstrated that expression cancer datasets available through the Oncomine dataset re- of a stabilized (gain-of-function) Hif-2α allele similarly promotes pository (www.oncomine.org). Filtering specifically for NSCLC tumor progression in the LSL-KrasG12D murine NSCLC model tumor datasets containing HIF-2α and SCGB3a1 probes, we (13). These and other data suggest that inhibiting either HIF-1α found significant correlation between HIF-2α and SCGB3a1 or HIF-2α (or both) may be of therapeutic value in specific expression in three of four datasets (Fig. 5B). disease settings.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1001296107 Mazumdar et al. Downloaded by guest on September 25, 2021 α A CT CT 2 and SCGB3a1 mRNA levels observed in approximately 70% of 1.6 6 human lung adenocarcinomas analyzed and in three of four * * 1.4 5 NSCLC datasets obtained from the Oncomine database. 1.2 Interestingly, our results contrast directly with those from 4 1 a recent report in which HIF-2α inhibition in A549 cells, as well as 0.8 3 in HCT116 colon carcinoma and U87MG glioblastoma cells, ac- 0.6 2 tually reduced xenograft tumor growth (31). The nature of these 0.4 disparate results is not clear, as identical shRNA sequences were 0.2 1 Fold change in mRNA Fold change in mRNA 0 0 used; however, our data are supported by two important controls. HIF-2 Scgb3a1 HIF-2 Scgb3a1 First, the phenotypes observed in our HIF-2α deficient A549 cells are reversed by enforced expression of a murine HIF-2α B HIF-2 /Scgb3a1 gene expression correlation in NSCLC datasets. mRNA that escapes shRNA inhibition, confirming the HIF-2α Dataset Pearson Correlation p-value dependent nature of these phenotypes. Second, the HIF-2α de- Bild (n=111) 0.437 1.60E-06 ficient A549 xenograft tumors phenocopy directly the behavior of autochthonous tumors generated by Hif-2α genetic deletion in the Kim (n=138) 0.377 5.10E-06 LSL-KrasG12D model. Ding (n= 68) 0.161 0.168 The tumor suppressive activity of HIF-2α we observed was Bittner (n=73) 0.503 3.02E-06 surprising, as expression of a stabilized HIF-2α in the identical LSL-KrasG12D model promoted tumor growth, and HIF-2α over- Over expression similarly promotes growth of RCCs (29). In particular, C expressed Vegf Angiogenesis, it raises the question of how HIF-2α can behave as both a tumor HIF-2 Vegfr-2 EMT transition Snail suppressor and an oncogene in the same NSCLC model. It appears Tumor that entirely different sets of HIF-2α target genes mediate these growth effects in a manner perhaps analogous to the proproliferative and proapoptotic activities of c-Myc (32). Whereas increased HIF-2α Deleted Akt signaling HIF-2 Scgb3a1 Lactotransferrin ? function elevates the expression of genes associated with angio- Ceruloplasmin ? genesis and epithelial-to-mesenchymal transition (EMT) in LSL- G12D Kras NSCLCs (13), HIF-2α deletion in the same model CELL BIOLOGY α Fig. 5. HIF-2 and SCGB3a1 levels correlate in NSCLC samples. (A and B) RNA diminishes the expression of a putative tumor suppressor Scgb3a1, was isolated from fresh frozen or formaldehyde/paraformaldehyde fixed human NSCLC tumors (designated T) (n = 15), used to generate cDNA for among other genes. quantitative RT-PCR analysis. Uninvolved adjacent lung tissue (designated C) Collectively, these results strongly suggest that inhibition of served as control. Transcipt levels were normalized to 18s RNA; 73% (11 out of overexpressed HIF-2α in NSCLCs (and perhaps other carcino- 15) NSCLC samples analyzed demonstrated significantly decreased levels of mas) might have beneficial effects but that reducing HIF-2α both HIF-2α and SCGB3a1 mRNA compared with adjacent control tissue (Left). function below a critical threshold might also drive tumor pro- Samples with normal SCGB3a1 expression (n = 4) did not display any signifi- gression by inhibiting the expression of specific tumor suppressor cant change in HIF-2α level (Right). Error bars represent SEM. *P < 0.05. (B) genes. This model (Fig. 5C) assumes that HIF-2α overexpression Significant correlation between HIF-2α and SCGB3a1 mRNA expression was either fails to increase Scgb3a1 function above normal levels or observed in three (of four) human NSCLC datasets obtained from Oncomine. that increased Scgb3a1 activity is offset by elevated expression of (C) Schematic representation of the opposing roles of HIF-2α in tumorigenesis, α α other HIF-2 targets. It is also interesting to note that the time at wherein both HIF-2 overexpression through activation of protumorigenic α target genes, and HIF-2α deletion through loss of expression of candidate which HIF-2 function affects tumor progression differs between tumor suppressor genes, including Scgb3a1, could promote tumor growth. the two models. In a previous study (13), expression of stabilized Other HIF-2α targets such as lactotransferrin and ceruloplasmin may also in- HIF-2α was initiated at the earliest stage in lung tumorigenesis fluence tumor outcome, but their role in tumor suppression is as yet unclear. (i.e., Ad-Cre infection). In contrast, HIF-2α protein stabilization in the lung tumors of our control mice probably occurs subsequent to Kras activation, either as a result of tumor hypoxia or additional The data presented here indicate that HIF-2α has an un- oncogenic events. The importance of this temporal difference in expected tumor suppressive role in KrasG12D-driven lung tumors expression is not yet clear. In general, however, our results caution and that reduced expression of the HIF-2α target Scgb3a1 con- that inhibition of HIF complexes in cancer treatment may require tributes to this effect. This interpretation is supported by the a narrower therapeutic window than initially imagined. observation that restored Scgb3a1 expression inhibited the Our results also raise a number of interesting questions for growth of HIF-2α–deficient A549 xenograft tumors. Moreover, α– fi subsequent work, including (i) to what degree does the sup- elevated AKT signaling was observed in HIF-2 de cient A549 pression of other putative HIF-2α target genes (lactotransferrin, α cells and was inhibited by restored expression of either HIF-2 ceruloplasmin, etc.) contribute to the phenotypes we observe; (ii) or Scgb3a1. These observations are consistent with previous are there noncell-autonomous effects of HIF-2α deletion that reports that enforced Scgb3a1 expression inhibits AKT activity in regulate LSL-KrasG12D NSCLC tumor progression (for example, human breast cancer cells (18). the increase in granulocyte infiltration); and (iii) is the relation fi Scgb3a1 was originally identi ed as a candidate tumor sup- between HIF-2α and Scgb3a1 observed only in the context of pressor through gene expression studies comparing human mam- activating Kras mutations or does it extend to other oncogenic mary carcinomas to normal mammary epithelial cells (14). It was backgrounds? Importantly, how does Scgb3a1 regulate AKT ac- subsequently shown that Scgb3a1 expression is reduced in human tivity and are additional signaling pathways involved in its tumor lung, prostate, pancreatic, and nasopharyngeal cancers, and cor- suppressive effects? To what degree does elevated AKT activity, responding hypermethylation of the Scgb3a1 promoter was in concert with other direct and indirect HIF-2α targets, con- reported for multiple malignancies (15–17, 30). Moreover, de- tribute to the proliferative and apoptotic phenotypes we observe creased Scgb3a1 expression was identified as a primary in- in KrasG12D-driven, HIF-2α deficient lung tumors? dependent predictor of poor clinical outcome in early stage human In summary, we used both autochthonous and xenograft ge- NSCLCs (17). Our data indicate that Scgb3a1 expression is directly netic tumors to reveal a tumor suppressive effect of HIF-2α in regulated by HIF-2α in human lung adenocarcinoma cells: this NSCLC and identify its direct target gene Scgb3a1 as a down- relationship is underscored by the direct correlation between HIF- stream effector. This may be a more general phenomenon: for

Mazumdar et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 example, a previous report demonstrated that either HIF-1α or lation kit (Ambion) and purified on RNeasy spin columns (Qiagen) as de- HIF-2α expression can limit tumor growth in genetically manip- scribed in SI Materials and Methods; cDNA was synthesized using a high ulated GS9L glioma and murine embryonic stem cells by altering capacity c-DNA transcription kit (Applied Biosystems). Transcript levels were normalized to 18S rRNA using an Applied Biosystems 7900HT analyzer and angiogenesis and tumor cell apoptosis (33). Determining the −ΔCT −ΔCT cellular conditions and molecular mechanisms that distinguish the represented as fold change 2 (normoxia)/ 2 (hypoxia); mean nor- moxic value was set to 1. tumor promoting, or tumor suppressive, activities of HIF-1α and α HIF-2 proteins will clearly require additional investigation. cDNA Microarray and Oncomine Analysis. Microarray analysis was performed Materials and Methods by the University of Pennsylvania Microarray Core facility, and Oncomine data were analyzed as described in SI Materials and Methods. Generation of and Characterization of Inducible LSL-KrasG12D, Conditional Hif- αfl Mice, and Lung Tumors. Mice carrying the previously described inducible ChIP Analysis. A549 cells were cultured either under normoxia or hypoxia, (LSL-KrasG12D) knock-in allele (19), the conditional floxed Hif-2αfl allele, and crosslinked with 1% formaldehyde for 20 min at 37 °C, and quenched with the related null Hif-2αΔ allele (20) were intercrossed to generate experi- 1 M glycine. Nuclear lysates were analyzed as described in SI Materials mental and control animals (SI Materials and Methods). Lung morphometry, and Methods. histology, and immunohistochemistry were performed as described in SI Materials and Methods. Xenograft Assays. We injected 5 × 106 or 1 × 106 (as indicated) viable HIF-2α KD C1 or HIF-2α KD C2 cells in a 1:1 mix of DMEM:growth factor reduced matrigel Cell Culture and Infection. A549 cells (ATCC) were grown in DMEM with 10% matrix (BD) s.c. into one flank of 6-wk-old nude (NU/NU) female mice (Charles FBS (Gemini), amino acids, and antibiotics (SI Materials and Methods); A549 River Laboratories). The other flank was either injected with wild-type A549 cells were infected with retroviruses encoding shRNAs targeting HIF-1α (34), cells or cells carrying the empty vector (VC). Tumor volume (mm3) was de- HIF-2α (23) (designated HIF-1α KD and HIF-2α KD, respectively), or with the retroviral backbone as the negative viral control (VC). termined using a caliper every 5 d postimplantation (d.p.i). Animals were killed 5 wk postimplantation, or earlier (3-4 wk) in case of ulcerating tumors. Western Blot Analysis. Protein extracts were prepared and analyzed as de- ’ < scribed in SI Materials and Methods. For hypoxic analysis, cells were cultured Statistics. P values were determined using the Student s t test; P 0.05 was considered significant. under 0.5% O2 and 5% CO2 for 16–20 h in an InVivo2 400 hypoxia workstation with O2 control (Ruskinn Technology Ltd.). Western blots were probed with antibodies against HIF-1α (Cayman for mouse protein, R&D for human pro- ACKNOWLEDGMENTS. We thank Shetal Patel and Ania Warczyk (University tein), HIF-2α (Novus Biologicals), Scgb3a1 (Abcam), AKT, pAKT, GSK-3β, pGSK- of Pennsylvania) for assistance with immunohistochemistry and xenograft assays, Hongwei Yu and Alan Wanicur (University of Pennsylvania) for 3-α/β, p4EBP1, and β-actin (Cell Signaling). preparation of tissue sections, and William Y. Kim (University of North Carolina) and the Simon laboratory for helpful discussions. This work was RNA Purification, Reverse Transcription, and Real-Time PCR Amplification. RNA supported by the Howard Hughes Medical Institute (M.C.S.) and National was extracted with TRIzol (Invitrogen) or RecoverAll Total nucleic Acid Iso- Institutes of Health Grant HL66130 (to B.K. and M.C.S.).

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