Variant Ciz1 is a circulating biomarker for early-stage PNAS PLUS lung cancer

Gillian Higginsa,b, Katherine M. Ropera,b, Irene J. Watsona,b, Fiona H. Blackhallc, William N. Romd, Harvey I. Passe, Justin F. X. Ainscoughf, and Dawn Coverleya,b,1

aCizzle Biotech, University of York, Yorkshire YO10 5DD, United Kingdom; bDepartment of Biology, University of York, Yorkshire YO10 5YW, United Kingdom; cPaterson Institute for Cancer Research, University of Manchester, Lancashire M20 4BX, United Kingdom; dDivision of Pulmonary, Critical Care and Sleep Medicine, New York University School of Medicine, New York, NY 10016; eNew York University Langone Medical Center, New York, NY 10016; and fSchool of Medicine, Leeds University, Yorkshire LS2 9JT, United Kingdom

Edited by Dennis A. Carson, University of California at San Diego, La Jolla, CA, and approved September 20, 2012 (received for review June 15, 2012) There is an unmet need for circulating biomarkers that can detect CDK2, and with the cyclin-dependent kinase inhibitor p21 (7) early-stage lung cancer. Here we show that a variant form of the and also plays an indirect role in DNA replication by modulating nuclear matrix-associated DNA replication factor Ciz1 is present in the expression of genes, including cyclin D, that influence cell 34/35 lung tumors but not in adjacent tissue, giving rise to stable proliferation (8). Normally, Ciz1 is attached to the salt- and protein quantifiable by Western blot in less than a microliter of nuclease-resistant protein component of the nucleus referred plasma from lung cancer patients. In two independent sets, with to as the “nuclear matrix” and resides within foci that partially 170 and 160 samples, respectively, variant Ciz1 correctly identified colocalize with sites of DNA replication (9), implicating Ciz1 in patients who had stage 1 lung cancer with clinically useful accuracy. the spatial organization of DNA replication. Here we describe a For set 1, mean variant Ciz1 level in individuals without diagnosed Ciz1 variant that lacks part of a C-terminal domain involved in tumors established a threshold that correctly classified 98% of nuclear matrix attachment (Fig. 1A). Expression of this stable small cell lung cancers (SCLC) and non-SCLC patients [receiver variant is apparently restricted to tumor cells, making clinical operator characteristic area under the curve (AUC) 0.958]. Within exploitation as a cancer biomarker highly tractable. set 2, comparison of patients with stage 1 non-SCLC with asymp- tomatic age-matched smokers or individuals with benign lung Results nodules correctly classified 95% of patients (AUCs 0.913 and 0.905), As part of a gene-focused analysis of function, we cloned human MEDICAL SCIENCES with overall specificity of 76% and 71%, respectively. Moreover, Ciz1 from a SCLC cell line and recovered multiple variants, in- using the mean of controls in set 1, we achieved 95% sensitivity cluding a prevalent transcript in which 24 nucleotides from the 3′ among patients with stage 1 non-SCLC patients in set 2 with 74% end of exon 14 (2475_2498del) is excluded, leading to in-frame specificity, demonstrating the robustness of the classification. RNAi- deletion of eight amino acids (VEEELCKQ). Analysis of the mediated selective depletion of variant Ciz1 is sufficient to restrain sequence surrounding exons 14 and 15 revealed a second splice the growth of tumor cells that express it, identifying variant Ciz1 donor site within exon 14 (2475/6) that could support alternative as a functionally relevant driver of cell proliferation in vitro and in splicing. Location identifiers refer to Ciz1 reference sequence vivo. The data show that variant Ciz1 is a strong candidate for a NM_012127.2. We refer to the whole of predicted exon 14 as cancer-specific single marker capable of identifying early-stage lung “14a,” the shorter alternative as “14b,” and Ciz1 transcripts cancer within at-risk groups without resort to invasive procedures. harboring 14b as “b-variant.” Transcript frequencies among ESTs that map to the Ciz1 Unigene cluster Hs. 212395 suggested that ung cancer is the leading cause of cancer death worldwide. b-variant is prevalent in neuroendocrine lung tumors, and this fi LApproximately 80% of lung tumors are classified as nonsmall prevalence was con rmed by analysis of SCLC cell lines using cell lung cancer (NSCLC), including squamous cell carcinoma independent detection methods (Fig. S1 and below). and adenocarcinoma, and the remainder as small cell type As a nuclear matrix protein characterized by resistance to (SCLC). SCLCs are primarily neuroendocrine in origin, ranging harsh extraction conditions, b-variant could offer a robust bio- marker with potential to remain stable and detectable in body from low-grade typical carcinoid to high-grade neuroendocrine fluids. Consistent with this idea, an affinity-purified polyclonal tumors (HGNTs), although some HGNTs are classified as large antibody directed against the unique peptide encoded at the cell type (1, 2). The risk of lung cancer is increased dramatically junction of exon 14b/exon15 (Fig. S2) detected b-variant protein by smoking, and genetic factors appear to play a role in our by Western blot in 1 μL of plasma from patients with SCLC and ability to deal with smoking-related damage. However, clearly NSCLC but not from healthy individuals (Fig. 1B). A diffuse but heritable forms of increased lung cancer risk are not common. specific band of 50–60 kDa (Fig. S2) is reproducibly seen and Lung cancer diagnosis and staging relies heavily on imaging, remained stable even after extended periods of storage at 4 °C. suggesting that imaging may offer a route to early detection in high-risk groups. Although the impact of early detection on sur- vival has been questioned, several studies have looked at the potential benefit (3), and it recently became clear that screening fi Author contributions: J.F.X.A. and D.C. designed research; G.H., K.M.R., I.J.W., and D.C. with low-dose spiral CT can achieve a signi cant reduction in performed research; F.H.B., W.N.R., and H.I.P. contributed new patient samples; G.H. and mortality among heavy smokers (4). However, because around a D.C. analyzed data; and J.F.X.A. and D.C. wrote the paper. quarter of individuals require follow-up procedures to investigate Conflict of interest statement: The findings reported in this paper arise directly out of suspicious imaging results, the cost of this approach is extremely basic research at the University of York (York, Yorkshire, United Kingdom) on the Ciz1 high, highlighting the need for a second-line noninvasive test that protein and its role in the spatial and temporal organization of DNA replication. The fi findings are the result of an academic and commercial collaboration, funded in part by can con rm malignancy. Here we present evidence that protein- Cizzle Biotech, which is a spin-out company of the University of York. G.H. and D.C. are level detection of a variant form of the nuclear matrix protein partially funded by Cizzle Biotech. D.C. and J.F.X.A hold shares in Cizzle Biotech. Ciz1 has the potential to meet this need. Ciz1 promotes initiation This article is a PNAS Direct Submission. of mammalian DNA replication, where it helps coordinate the 1To whom correspondence should be addressed. E-mail: [email protected]. sequential functions of cyclin E- and A-dependent protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. kinases (5). It interacts directly with cyclins E and A (6), with 1073/pnas.1210107109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1210107109 PNAS Early Edition | 1of8 Downloaded by guest on September 24, 2021 Fig. 1. Variant Ciz1 protein in 170 samples in plasma set 1. (A) Ciz1 gene showing translated exons (numbered) and the alternative-splicing event at the exon 14/15 junction which gives rise to b-variant Ciz1. Exons that encode DNA replication domain (5) and nuclear matrix anchor domain (9) are indicated by dotted lines above. Exons that are commonly excluded from known Ciz1 variants (23) are shaded in dark gray. A complete representation of Ciz1 alternative splice variants was assembled previously (24), and transcript diversity was discussed recently (25, 26). (B) Western blot showing b-variant protein detected with antibody 2B in 1 μL of plasma from patients with SCLC or NSCLC plus five samples from individuals with no diagnosed disease. Endogenous Ig is used to normalize for loading (control). (C) Box-and-whisker plots showing the median, upper, and lower quartile, range, and outliers for data derived from Western blots by densitometry. Results for a total of 119 pretreatment lung cancer patients (Left) with the indicated type and stage of disease (Right), plus 51 samples from individuals with no disease or from patients with COPD, asthma, or anemia are shown. Individual sample values are given in Dataset S1. Using a threshold set at the mean of the noncancer samples, the test correctly classified 98% of all 119 lung cancer patients, with specificity of 85%. (D) ROC curve, with 95% confidence interval, generated for all 170 samples (AUC is 0.958). Student’s two-tailed t test with unequal variance returned a P value of <0.0001 for the noncancer samples compared with all sample sets from individuals with lung cancer.

Quantification of b-variant protein in 119 SCLC and NSCLC patients with early-stage lung cancer and those without lung samples (summary information in Table 1 with sample-specific cancer. However, for maximum clinical utility a blood test must information in Dataset S1) gave a mean signal intensity of 95.8 be able to identify individuals with early-stage lung cancer within densitometry units with an SD of 51.8, indicating considerable populations that are at high risk of developing the disease. heterogeneity among patients. In contrast, the mean signal in- An important potential application is as a test for lung cancer tensity for 51 individuals without diagnosed malignancy (who had in individuals with lung nodules identified by CT. A test that can chronic obstructive pulmonary disease, asthma, anemia, or no positively identify those individuals with lung cancer could help disease) was just 14.1 (SD 15.2). Unexpectedly, limited- and reduce the frequency of surgical intervention and favorably affect extensive-stage SCLC and stages 1, 2, 3, and 4 NSCLC were all both cost and outcome. To look specifically at the discriminatory significantly different from the control groups when analyzed power of b-variant Ciz1 in comparison with (i) age-matched separately (Fig. 1C), with a trend toward expression increasing smokers with ≥10 y of cigarette smoking history but without with stage for stages 1, 2, and 3 NSCLC. Receiver operating char- diagnosed cancer and (ii) individuals with nonmalignant lung acteristic (ROC) analysis for all 170 samples returned an area nodules or inflammatory lung disease, we analyzed a second, under the curve (AUC) of 0.958 (Fig. 1D), demonstrating con- independent, archived set of case and control plasma samples siderable discriminatory power in this context. To our knowledge, (set 2, summary information in Table 1 with sample-specific in- and despite considerable effort and progress with circulating formation in Dataset S2), using high-throughput Western blots biomarkers for other cancers (10, 11), no other single biomarker and just 0.5 μL of each sample. Comparison of the smokers is capable of achieving this level of discrimination between (median age 64.5 y) with 20 individuals with stage 1 squamous

2of8 | www.pnas.org/cgi/doi/10.1073/pnas.1210107109 Higgins et al. Downloaded by guest on September 24, 2021 Table 1. Summary characteristics of patients in plasma sets 1 and 2 PNAS PLUS Diagnosis/classification No. of patients Median age Sex (%F) Median pack years* Mean b-variant (±SD)

Set 1 170 Stage 1 NSCLC 52 62 40 N/d 65.7 ± 40.0 Stage 2 NSCLC 6 66.5 50 N/d 94.8 ± 29.8 Stage 3 NSCLC 16 66 38 N/d 103.6 ±− 26.3 Stage 4 NSCLC 14 69 43 N/d 85.0 ± 52.1 Limited stage SCLC 21 67 48 N/d 112.0 ± 67.1 Extensive stage SCLC 10 64 70 N/d 118.0 ± 70.1 All cancer 119 65 44 N/d 95.8 ± 51.8 COPD 5 59 20 N/d −0.3 ± 4.3 Asthma 12 42.5 58 N/d 17.6 ± 16.9 Anemia 20 47.5 80 N/d 23.7 ± 8.0 No diagnosed disease 14 36 29 N/d 5.9 ± 12.3 All noncancer 51 46 55 N/d 14.1 ±15.2 Set 2 160 Stage 1 squamous cell carcinoma 20 67 20 45 64.3 ± 32.0 Stage 1 adenocarcinoma 20 71.5 60 41 35.2 ± 14.4 Benign lung nodules 20 60 85 20 8.6 ± 22.0 Inflammatory lung disease 20 67.5 60 30 6.3 ± 0.4 Smokers 80 64.5 55 34 6.35 ± 12.2

N/d, no data. *One pack year is defined as 20 cigarettes smoked every day for a year.

cell carcinoma (median age 67 y) and 20 patients with stage were applied as a secondary screen to high-risk groups with 1 adenocarcinoma (median age 71.5 y) revealed clear discrimi- suspicious CT results (4), b-variant Ciz1 could halve the number MEDICAL SCIENCES natory power in both contexts and ROC AUC values in excess of of individuals referred for costly and invasive follow-up procedures. 0.9 (Fig. 2). Western blot images for all samples are shown in In the context of high-risk smokers without CT-based diagnosis, Fig. S3, and raw and normalized results are given in Dataset S2. the biomarker also would appear to have utility, with 95% of These data show that b-variant Ciz1 has significant potential for patients with stage 1 lung cancer (in set 2) falling above the mean detecting limited-stage lung cancer among at-risk groups. of the smokers and only 34% of smokers being placed in the The overall performance with set 1 and set 2 is shown in Table suspicious category. Thus, positioning the test either before or 2. Encouragingly, using a threshold set at the mean of 40 indi- after CT could offer significant advantages over the use of viduals with benign lung nodules or inflammatory diseases of the CT alone. lung (set 2), b-variant Ciz1 correctly classified 95% of patients Consistent with the presence of b-variant Ciz1 in patient plasma, with stage 1 lung cancer, with an overall specificity of 71% (set 2). immunohistochemical analysis of primary tumors from patients Moreover, when the threshold is set at the mean of all of the with neuroendocrine lung cancer revealed b-variant–positive noncancers in the discovery set (set 1), 95% of the patients with cells in 34 of 35 patients (Fig. 3A). Staining was heterogeneous in stage 1 lung cancer in the validation set (set 2) are correctly clas- both distribution and level, with positive cells evident throughout sified, with an overall specificity of 75%. Under both circum- the tumor in some individuals and limited to isolated cells in stances the false-positive rate was ∼50%, suggesting that, if it others. These results confirm that b-variant Ciz1 is a tumor antigen

Table 2. Sensitivity and specificity estimates for sample sets 1 and 2 False positive Total in wrong Specificity Thresholds used False negatives Sensitivity (%) False positives (%) group (%)

Set 1 Mean of all noncancers 14.07 3/119 (all cancer) 98 23/51 45 26/170 85 in set 1 Mean of all noncancers 14.07 0/52 (stage 1 NSCLC) 100 23/51 45 23/103 78 in set 1 Set 2 Mean of all noncancers 6.27 2/40 95 48/120 40 50/160 69 in set 2 Mean of all smokers 6.35 2/40 95 27/80 34 29/120 76 in set 2 Mean of benign and 7.45 2/40 95 21/40 53 23/80 71 inflammatory in set 2 Mean of all noncancers 14.07 2/40 (stage 1) 95 40/120 33 42/160 74 in set 1 (all non cancer) Mean of all noncancers 14.07 2/40 (stage 1) 95 23/80 (smokers) 29 25/120 79 in set 1 Mean of all noncancers 14.07 2/40 (stage 1) 95 18/40 (benign and 45 20/80 75 in set 1 inflammatory)

Higgins et al. PNAS Early Edition | 3of8 Downloaded by guest on September 24, 2021 Fig. 2. Variant Ciz1 protein in 160 samples in plasma set 2. (A) Box plot showing results for 80 smokers with more than 10 y of smoking history and for 40 patients with stage 1 NSCLC with similar smoking history (Left) and for 20 individuals diagnosed with stage 1 adenocarcinoma, inflammatory lung disease (granuloma), benign lung nodules (carcinoid, hamartoma), or stage 1 squamous cell carcinoma (Right), showing lower, median, and upper quartiles and outliers (circles). (B) ROC curve with 95% confidence intervals for the indicated comparisons. AUCs are 0.913 when samples from 80 smokers are compared with samples from 40 patients with stage1 lung cancer, 0.905 when samples from 40 patients with benign nodules or inflammatory disease are compared with samples from 40 patients with stage1 lung cancer, and 0.909 when all samples from smokers and patients with benign nodules or inflammatory disease are compared with all samples from patients with stage1 lung cancer, but are only 0.503 when samples from smokers are compared with samples from patients with benign nodules or inflammatory disease. Western blots showing b-variant protein in the 0.5 μL of plasma used for each of the 160 samples are shown in Fig. S3; individual b-variant levels and clinical parameters are given in Dataset S2.Student’s two-tailed t test with unequal variance returned a P value of <0.0001 for the samples from patients with cancer compared with samples either from smokers or from patients with benign nodules or inflammatory disease.

with the potential for exploitation as a lung cancer biomarker. confirming that expression is common in lung cancer cells. Notably, Complementary transcript analysis reported at least 40-fold el- for both the cell lines and the primary tumors, Ciz1 also is elevated evation in bulk tumor RNA from neuroendocrine tumors com- (Fig. 3 B and C), but to a lesser extent. pared with matched adjacent tissue for all three patients tested Visualization by immunofluorescence revealed that the b- (Fig. 3B). In addition, b-variant transcript was elevated in 13 SCLC variant protein resides as subnuclear foci that are somewhat cell lines compared with control lines (Fig. 3C and Fig. S1E), larger than WT Ciz1 in the nucleus in SCLC cell line SBC5 but

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Fig. 3. Expression of Ciz1 b-variant in tumors and cell lines. (A) Sections from SCLC tissue array ARY-HH0117, probed with b-variant antibody 2B (dark brown), showing (i) normal lung negative for b-variant; (ii and iii) b-variant in representative SCLC; (iv) SCLC with positive tumor cells and stromal cells; (v) SCLC with positive stromal cells ; (vi) SCLC negative for b-variant. B-variant was recorded in 29/35 tumors and in stromal cells in 5/35 tumors. Only one pair of tumor sections did not contain b-variant–positive cells. (B) Relative quantification (RQ) of b-variant transcript (primers P1/P2, probe T2) and Ciz1 AD transcript in SCLC tumors of the indicated disease stages plus adjacent tissue from three patients. Results are normalized to actin and calibrated to the first adjacent sample. (C) Ciz1 b-variant AD (primers 6/7, probe 16) and replication domain (13/14, probe 7) in lung-derived cell lines, showing average of three technical replicates after normalization to actin and calibration to WI38 cells, with SEM. Sequences are given in Table S3. All three SCLC lines, plus 10 others (Fig. S1), have elevated b-variant. (D) Detergent-resistant b-variant protein (red) in nuclei of SCLC cells, but not normal lung cells, was detected with antibody 2B. DNA is blue. (Scale bar, 5 microns.) (E) GFP-hCiz1 WT (full length with exon 14a) and GFP-hCiz1 b-variant (with exon 14b). Both proteins form nuclear foci, but foci are larger for the b-variant and are accompanied by localization around the periphery (white arrowhead).

not in the normal lung cell line Wi38 (Fig. 3D)(9).When (5) and human SBC5 cells (Fig. S5). Therefore, if tumor cells that transfected into NIH 3T3 cells, GFP-tagged Ciz1 constructs with express the b-variant rely on it to support S phase, its inhibition exon 14a or 14b produced protein that was exclusively nuclear could selectively restrain their proliferation. From a panel of and punctate and which resisted extraction (Fig. 3E). Thus, we junction-spanning siRNAs that suppress the expression of the saw no gross differences in subcellular localization or attachment b-variant transcript and protein but not Ciz1 isoforms that con- to nonchromatin nuclear structures. However subtle differences tain exon 14a (Fig. S6 and Table S1), we selected the most potent in subnuclear distribution were evident, with the b-variant sequence (Fig. S5A) and tested its effect on the proliferation of forming larger aggregates and localizing to the perimeter of the SBC5 SCLC cells (which express endogenous b-variant; Fig. 3 C nucleus as well as to foci. Therefore, the loss of eight amino acids and D) and also H727 cells (which do not express endogenous from the anchor domain (AD) of Ciz1 does subtly affect the spatial b-variant; Fig. 3C and Fig. S5B) when delivered from an inducible organization of Ciz1, leading us to suggest that it may influence shRNA vector. Like RNAi targeted against a constant region of the functional compartment into which the DNA replication Ciz1, b-variant shRNA effectively restrained cell proliferation in activity of Ciz1 is targeted and possibly, therefore, the execution culture (Fig. 4A). As expected, b-variant transcript, but not other of replication-coupled events. Notably, normal cells do not tol- forms of Ciz1, was suppressed selectively (Fig. 4B), and this erate sustained overexpression of ectopic Ciz1 or b-variant Ciz1 suppression had a significant impact on b-variant protein (Fig. [despite an early positive effect on initiation of DNA replication 4C). Importantly, in a xenograft model of human SCLC in which (5), shown previously for WT Ciz1], because this overexpression s.c. tumors were derived from SBC5 cells (12), b-variant shRNA ultimately results in the loss of nuclear integrity in NIH 3T3 cells dramatically restrained tumor growth. Similar results were obtained and loss of adherence within 33 h of transfection via a mecha- when expression was induced at the time of tumor cell in- nism that is not apoptosis (Fig. S4). oculation or after tumors had formed (Fig. 4D). Together, these The increasingly well-characterized role of Ciz1 in DNA rep- in vitro and in vivo experiments show that the unique junction lication makes it a candidate therapeutic target with the poten- generated by alternative splicing of Ciz1 exon 14 can be exploited tial to modulate cell proliferation. Inhibition of Ciz1 expression to suppress variant expression specifically and that this suppres- using siRNA restrains cell proliferation in murine NIH 3T3 cells sion is sufficient to inhibit tumor cells that express it, identifying

Higgins et al. PNAS Early Edition | 5of8 Downloaded by guest on September 24, 2021 Fig. 4. Suppression of the expression of the Ciz1 b-variant restrains proliferation of lung cancer cells. (A) Effect of luciferase control shRNA (luc) and b-variant junction-selective RNAi sequence 4 on SBC5 cells, expressed as fold increase in cell number over 4 d after induction of shRNA with doxycycline at days 1 and 3 (gray arrowheads). RNAi sequences are described in Fig. S6.(B) RT-PCR analysis of Ciz1 AD (primers P1/P2), b-variant (P4/P3), and actin (P11/P12) in SBC5 b-variant shRNA 4 cells, without (− dox) and with induction of expression at 26 h (+) or as two doses (++) administered at 26 h and 1 h before isolation. (C) Detergent-resistant endogenous b-variant protein (antibody 2B, green) in the nucleus of SBC5 b-variant shRNA 4 cells 2 d after induction of shRNA. DNAis blue. (Scale bar, 10 microns.) (D) Xenograft tumor formation in mice. Control mice were inoculated with SBC5 b-variant shRNA 4 cells and maintained in the absence of doxycycline (open circles, group 1). Additional cohorts (filled circles) were given doxycycline continuously from 3 d before inoculation (group 3, Right) or after 21 d (group 2, Left). For the comparison of groups 1 and 2, mice with tumors less than 100 mg at 21 d were discounted, creating cohorts with low variation. Graphs show mean tumor weight from 10–15 animals, with SEM. (Data are given in Dataset S3.)

b-variant Ciz1 as a functionally relevant driver of tumor cell However, despite their promise, the need remains for markers proliferation. with tumor-specificprofiles that can be used to complement examination of the structure of nuclei. B-variant Ciz1 is one such Discussion molecule, robustly quantifiable in the blood of patients who have The evidence presented here shows that a cancer-associated var- lung cancer, that is both a driver of DNA replication and tumor iant isoform of the nuclear matrix protein Ciz1 holds consider- cell proliferation and a stable derivative of circulating tumor able promise as the basis of a blood test for early-stage lung cells. Notably, this biomarker was identified through intensive cancer. As biomarkers, nuclear matrix proteins offer several gene-focused analysis of expression and function rather than by advantages. They are at the heart of a dysregulated nucleus and mutation- or gene-expression–profiling approaches. are likely to play causative roles in epigenetic control of gene Analysis by high-throughput Western blot offers a direct, visual expression. Perhaps more importantly, nuclear matrix proteins output clarified by sample denaturation that eliminates epitope are inherently stable, offering practical advantages that make masking in native samples. Furthermore, fractionation on the exploitation feasible. As a group they are biochemically defined basis of molecular weight effectively isolates signal from that of as the fraction that remains after extraction to remove lipids, potentially reactive contaminants, giving a clear picture of the soluble proteins, cytoskeleton, chromatin, and associated proteins, potential of the biomarker. Western blot analysis, however, may which when observed microscopically forms a proteinaceous net- not report the full dynamic range of biomarker concentrations work (13) made up of constitutive and cell-type–specificcom- and is not easily applied outside a research laboratory. There- ponents (14). Increasingly nuclear matrix proteins such as NMP22, fore, routine application of a b-variant–based test in a clinical BCLA4, PC1, and NM179 are gaining credibility as tumor markers context will require the development of a more streamlined for bladder (15), cervix (16), and prostate cancer (17), respectively. method, such as ELISA, with a simplified quantitative output.

6of8 | www.pnas.org/cgi/doi/10.1073/pnas.1210107109 Higgins et al. Downloaded by guest on September 24, 2021 Thus, we acknowledge that, although the data presented show iant–positive cancer cells with variable intensity and number, exceeding PNAS PLUS that the biomarker holds considerable discriminatory power, the 10% of cells), PS (containing positive stromal cells), PPS (containing strongly assay ultimately will need to switch platforms and be validated positive stromal cells), or N (containing no positive cells). Images shown in further in that context. Fig. 3A are derived from (i) core H8, (ii) G7. (iii) D10, (iv) G6, and (v)F8. Alteration in the structure of nuclei, detected by light micros- Plasma Analysis. For sample set 1, 1 μL of plasma was resolved by 8% SDS/ copy of tumor biopsies, has remained the most reliable method PAGE through 12 cm × 12 cm maxi-gels (Atto) and was transferred to ni- of cancer diagnosis for more than 100 y (18), but the underlying trocellulose by semidry blotting. For samples in set 2, 0.5 μL of plasma was abnormalities have been difficult to define at the molecular level. denatured using Epage buffer, resolved by electrophoresis through precast Detailed and focused analysis of the relationship between nu- E-PAGE 8% 48-well gels, and was transferred to nitrocellulose using iBlot dry clear architecture and essential nuclear events is beginning to blotting system (all Invitrogen). A more detailed description is given in SI identify new players involved in establishing order (19). More- Materials and Methods. Membranes were probed with anti-Ciz1 b-variant– over, disease-associated molecular changes in these players are specificaffinity purified polyclonal antibody 2B, diluted 1/500 in PBS, 10% now becoming apparent, offering exciting opportunities for the (wt/vol) milk powder, and 0.1% Tween 20. Antibody 2B was detected with fi development of new clinically relevant tools. peroxidase-IgG light chain-speci c, highly cross-adsorbed monoclonal anti- rabbit antibody (211-032-171; Jackson Immunological Research). Western blots Materials and Methods were quantified using Image J (National Institutes of Health), and samples were assigned a value after normalization to loading control and calibration to Additional methods are described in SI Materials and Methods. a constant normal sample (given a value of 0), and to an NSCLC sample (given a value of 100). Cells. NIH 3T3 cells were grown as described (20). SBC5 cells, obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB) were Statistics. Groups were compared using Student’s two-tailed t-tests in which ’ grown in Eagle s minimal essential medium (Sigma), 1% penicillin, strepto- a P value <0.05 is considered significant. Results are displayed in box-and- mycin, glutamine (Gibco), and 10% (vol/vol) FBS (Biosera). RNAi with siRNA whisker plots showing sample minimum and maximum, lower, median, and and inducible shRNA is described in SI Materials and Methods. All other cells upper quartile, and outliers, calculated using www.physics.csbsju.edu/stats/ were from the European Collection of Cell Cultures or JCRB and were cul- (accessed October 21, 2011) and as ROC curves using www.rad.jhmi.edu/ tured as recommended. jeng/javarad/roc/JROCFITi.html (accessed February 7, 2011). Means and SDs were calculated using Excel 2008 for Mac (Microsoft). Quantitative RT-PCR. RNA from cell lines (Table S2) was isolated with TRIzol, and cDNA was synthesized using SuperScript III (Invitrogen). RNA from Study Approval. For sample set 1, blood samples were collected with written, tumors and adjacent tissue (Cytomyx) was reverse transcribed using M-MLV informed consent, according to National Health Service-approved protocols – MEDICAL SCIENCES (Promega) and random primers (Promega) plus oligo-dT primer 12 18 at the Paterson Institute for Cancer Research, University of Manchester, and (Invitrogen). Primers (Table S3) P6/P7 or P1/P2 (Sigma Aldrich) were coupled were processed as described previously (22). Additional plasma samples listed with junction probe T2 (MWG) for b-variant Ciz1 and were verified using in Dataset S1 were obtained from biobanks and were collected according to plasmid templates (Fig. S1). Relative expression was calculated using the −ΔΔ Institutional Review Board-approved protocols. All cases and control samples comparative cycle threshold (Ct) method (2 Ct), and results were expressed in set 2 were collected according to National Cancer Institute Early Detection relative to normal cells or tissue. Research Network standard operating procedures. Samples from patients diagnosed with lung cancer or other nonmalignant lung disorders were Antibodies. – fi B-variant speci c rabbit polyclonal antibodies (2B) were gener- collected at the Thoracic Oncology Laboratories of the New York University ated by Covalab using the splice-junction sequence (C-DEEEIEVRSRDIS) (Fig. Langone Medical Center (New York) at the time of thoracic surgery, and all fi fl S2A). Sera rst were subtracted for antibodies reactive against anking diagnoses were confirmed histologically (as adenocarcinoma, inflammatory sequences in a negative control peptide that includes the amino acids absent lung disease, benign lung nodules, or squamous cell carcinoma) after exci- from b-variant (DEDEEEIEVEEELCKQVRSRDISR) and then were adsorbed to sion of the abnormal lesion. For the smoker cohort, samples were collected fi fi peptide containing the b-variant junction to generate af nity-puri ed anti- from age-matched smokers at the Division of Pulmonary, and Critical Care, fi body 2B. Puri ed antibodies were characterized in a range of assays including and Sleep Medicine, New York University School of Medicine. Study-specific fl immuno uorescence and Western blot on cells expressing WT or b-variant approval was granted by the Department of Biology, University of York, forms of human GFP-Ciz1 (Fig. S2 B and C) and lung cancer cells expressing Research Ethics Committee. endogenous b-variant Ciz1 with and without specific suppression by b-variant shRNA (Figs. 3D and 4C). ACKNOWLEDGMENTS. We thank Rose Wilson, Jian Mei Hou, Faisal Abdel Rahman, and Mark Thornquist; Ellen Eylers, Ting-An Yie, and Nihir Patel (New Immunodetection and Analysis. For immunofluorescence, the b-variant was York University School of Medicine) for sample collection and Alissa Green- detected after paraformaldehyde fixation using affinity-purified polyclonal berg, MD, for clinical follow-up; Lynsey Priest for sample collection at The antibody 2B (Fig. S2) and then anti-rabbit Alexa Fluor 488 (Invitrogen, Folio Christie National Health Service Foundation Trust; Said El Alaou (Covalab) for fi Bioscience). Subcellular fractionation was carried out as described (21). For generation of variant-speci c antibodies; and Yulia Maxuitenko (Southern immunohistochemistry, 2B was applied to SCLC tissue microarray ARY- Research Institute) for in vivo RNAi analysis. Sample collection at The Christie National Health Service Foundation Trust was supported by CHEMORES Sixth HH0117 (Folio Biosciences) and visualized using Ultraview Universal DAB Framework Programme Contract LSHC-CT-2007-037665 (to F.H.B), and at New (Ventana) with hematoxylin counterstain. Duplicate cores representing 35 York University School of Medicine by National Cancer Institute Early De- fi SCLC (grade 4) and tissue from ve normal or cancer-adjacent lungs were tection Research Network Grant U01CA086137 (to W.N.R. and H.I.P.) This work analyzed by a certified pathologist at Phylogeny, Inc. Cores were classified as was funded by Cizzle Biotech, the University of York proof-of-concept fund, PP (containing mostly b-variant–positive cancer cells), P (containing b-var- and Yorkshire Cancer Research.

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