Cancer Biol Med 2021. doi: 10.20892/j.issn.2095-3941.2019.0324

ORIGINAL ARTICLE

Reduced anoctamin 7 (ANO7) expression is a strong and independent predictor of poor prognosis in prostate cancer

Andreas Marx1,*, Lena Koopmann2,*, Doris Höflmayer2, Franziska Büscheck2, Claudia Hube-Magg2, Stefan Steurer2, Till Eichenauer3, Till S. Clauditz2, Waldemar Wilczak2, Ronald Simon2, Guido Sauter2, Jakob R. Izbicki4, Hartwig Huland5, Hans Heinzer5, Markus Graefen5, Alexander Haese5, Thorsten Schlomm6, Christian Bernreuther2, Patrick Lebok2, Sarah Bonk3 1Institute of Pathology, Klinikum Fürth, Fürth 90766, Germany; 2Institute of Pathology, University Medical Center Hamburg- Eppendorf, Hamburg 20246, Germany; 3Department of Urology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany; 4General, Visceral and Thoracic Surgery Department and Clinic, University Medical Center Hamburg- Eppendorf, Hamburg 20246, Germany; 5Martini-Clinic, Prostate Cancer Center, University Medical Center Hamburg- Eppendorf, Hamburg 20246, Germany; 6Department of Urology, Charité – Universitätsmedizin Berlin, Berlin 10117, Germany

ABSTRACT Objective: Anoctamin 7 (ANO7) is a calcium2+-dependent chloride ion channel . Its expression is restricted to prostate epithelial cells. The exact function is unknown. This study aimed to analyze ANO7 expression and its clinical significance in prostate cancer (PCa). Methods: ANO7 expression was assessed by immunohistochemistry in 17,747 clinical PCa specimens. Results: ANO7 was strongly expressed in normal prostate glandular cells but often less abundant in cancer cells. ANO7 staining was interpretable in 13,594 cancer tissues and considered strong in 34.4%, moderate in 48.7%, weak in 9.3%, and negative in 7.6%. Reduced staining was tightly linked to adverse tumor features [high classical and quantitative Gleason grade, lymph node metastasis, advanced tumor stage, high Ki67 labeling index, positive surgical margin, and early biochemical recurrence (P < 0.0001 each)]. The univariate Cox hazard ratio for prostate-specific antigen (PSA) recurrence after prostatectomy in patients with negative vs. strong ANO7 expression was 2.98 (95% confidence interval 2.61–3.38). The prognostic impact was independent of established pre- or postoperatively available parameters (P < 0.0001). Analysis of annotated molecular data showed that low ANO7 expression was linked to TMPRSS2:ERG fusions (P < 0.0001), elevated androgen receptor expression (P < 0.0001), as well as presence of 9 of 11 chromosomal deletions (P < 0.05 each). A particularly strong association of low ANO7 expression with phosphatase and tensin homolog (PTEN) deletion may indicate a functional relationship with the PTEN/AKT pathway. Conclusions: These data identify reduced ANO7 protein expression as a strong and independent predictor of poor prognosis in PCa. ANO7 measurement, either alone or in combination, might provide clinically useful prognostic information in PCa. KEYWORDS ANO7; immunohistochemistry; prognosis; prostate cancer

Introduction disease still represents the third most common cause of can- cer-related death1. Treatment options vary from radical sur- gery to active surveillance and are chosen based on the per- With an estimated 1.3 million new cases worldwide in 2018, ceived cancer aggressiveness. Gleason grade and tumor extent prostate cancer (PCa) is the most common cancer in men. on biopsies, preoperative prostate-specific antigen (PSA), and Despite a rather indolent clinical course of most PCa, this clinical stage are currently the established pretreatment para- meters. Although these data are statistically powerful, they do *These authors contributed equally to this work. Correspondence to: Ronald Simon not always allow optimal treatment decisions in retrospect. E-mail: [email protected] Thus it is hoped that new molecular biomarkers will enable a ORCID ID: https://orcid.org/0000-0003-0158-4258 precise prediction of PCa aggressiveness. Received September 25, 2019; accepted February 7, 2020. Anoctamins are a family of at least 10 calcium2+-depend- Available at www.cancerbiomed.org ©2021 Cancer Biology & Medicine. Creative Commons ent chloride ion channels with physiological roles in epithelial Attribution-NonCommercial 4.0 International License and other cell types2. Anoctamin 7 (ANO7) is of particular 246 Marx et al. ANO7 expression in prostate cancer interest for PCa biology. It was originally termed NGEP (new used in accordance with the local laws (HmbKHG, §12,1) and expressed in prostate), as its expression is virtually approved by the local ethics committee (Ethics commission restricted to prostate epithelial cells3. Early immunohisto- Hamburg, WF-049/09). All work has been carried out in com- chemical studies revealed that ANO7 is a transmembrane cell pliance with the Helsinki Declaration. junction protein strongly expressed at the apical pole and on lateral surfaces of the epithelial cells of prostate glands4, but Immunohistochemistry its exact function is still unknown. ANO7 resides at 2q37, a genomic region that has been associated with PCa risk Freshly cut TMA sections were stained on one day and in before5. Immunohistochemical analyses on 120–160 samples one experiment. Slides were deparaffinized and exposed to of normal and cancerous prostate tissues found that ANO7 heat-induced antigen retrieval for 5 min at 121 °C in pH 7.8 expression is retained during PCa development but decreases Tris-EDTA buffer. Primary antibody specific for ANO7 (rab- with progression to high Gleason grade6,7, suggesting a role bit polyclonal antibody, Sigma-Aldrich, St. Louis, MO, USA, of ANO7 loss for PCa biology. In conflict with these data, a HPA035730; dilution 1:900) was applied at 37 °C for 60 min. recent analysis of ANO7 messenger RNA (mRNA) expression Bound antibody was then visualized using the EnVision Kit from 289 PCa patients included in The Cancer Genome Atlas (Dako, Glostrup, Denmark) according to the manufacturer’s project8 suggested that increased ANO7 expression might directions. All tissues were analyzed by one trained patholo- indicate poor patient outcome9. gist. Anti-ANO7 showed cytoplasmic staining and the staining These contradictory findings prompted us to evaluate the intensity (0, 1+, 2+, 3+) as well as the percentage of stained potential prognostic impact of ANO7 expression in PCa by cells were recorded for each tissue spot. A final score was using our tissue microarray (TMA) comprising tumor sam- built from these two parameters as follows: lack of any stain- ples from more than 17,000 individual patients. ing (intensity 0) was considered “negative”, 1+ staining in ≤ 70% of tumor cells or 2+ staining in ≤ 30% of tumor cells was Materials and methods considered “weak”, 1+ staining in > 70% of tumor cells or 2+ staining in > 30% but ≤ 70% of tumor cells or 3+ staining in ≤ Patients 30% of tumor cells was considered “moderate”, and 2+ stain- ing in > 70% of tumor cells or 3+ staining in > 30% of tumor The 17,747 patients had prostatectomy between 1992 and cells was considered “strong”. 2015 (Department of Urology and the Martini Clinic at the University Medical Center Hamburg-Eppendorf). PSA recur- Statistical analysis rence was defined as the time point when postoperative PSA was at least 0.2 ng/mL and increasing at subsequent meas- Contingency tables and χ² tests were used to examine associ- urements. Supplementary Table S1 summarizes the patient ations between molecular and histopathological tumor para- characteristics on the TMA. Quantitative Gleason grading meters. Kaplan–Meier survival curves were calculated, and the supplemented classical Gleason categories10. The TMA was log-rank test was applied to detect differences between groups. produced with a single 0.6-mm core taken from a tumor-con- Cox proportional hazards regression analysis was performed taining tissue block from each patient11. The attached molecu- to test for independence between pathological, molecular, and lar database included data on Ki67 labeling index (Ki67LI)12, clinical variables. Calculations were done with JMP 12 (SAS, erythroblast transformation-specific-related gene (ERG) Cary, NC, USA). All conducted analyses are referred to the protein expression, and ERG rearrangement analysis by flu- overall P values. orescence in situ hybridization (FISH)13,14, as well as dele- tion status of 3p13 [forkhead box protein F1 (FOXP1)]15, Results 5q21 [chromodomain-helicase-DNA-binding protein 1 (CHD1)]16, 6q15 [mitogen-activated protein kinase kinase A total of 67.6% of 17,747 tumor samples were interpretable kinase 7 (MAP3K7)]17, 8p2118, 10q23 [phosphatase and ten- in the TMA analysis. Reasons for non-informative cases (n = sin homolog (PTEN)]19, 12p1320, 12q2421, 13q1422, 16q2423, 4,153; 32.4%) included lack of tissue samples or absence of 17p1324, and 18q2125. Archived diagnostic leftover tissues were unequivocal cancer tissue in the TMA spot. Cancer Biol Med Vol 18, No 1 February 2021 247

ANO7 expression in normal and cancerous Supplementary Figure S2B, S2C). A particularly strong asso- prostate tissues ciation was found between reduced ANO7 staining and the presence of PTEN (10q23) deletions, which was highly signifi- In normal prostate glands, luminal and basal cells stained cant in all cancers as well as in ERG-negative and ERG-positive moderately to strongly positive for ANO7. In PCa, cytoplasmic cancers (P < 0.0001 each). Reduced or lost ANO7 staining was staining was considered strong in 34.4%, moderate in 48.7%, significantly linked to increased cell proliferation as measured weak in 9.3%, and negative in 7.6% of 13,594 interpretable by Ki67LI (P < 0.0001; Figure 4). However, this association did tumors. Representative images of ANO7 staining are given in not hold true in subsets of cancers with identical Gleason score. Figure 1. Lost or reduced ANO7 staining showed significant associations with adverse tumor features. Low ANO7 expres- Multivariable analysis sion was linked to advanced tumor stage (P < 0.0001), high classical and quantitative Gleason grade (P < 0.0001 each), The potential importance of ANO7 staining was analyzed in presence of lymph node metastasis (P < 0.0001), high preoper- four different scenarios (Table 2, Supplementary Table S4). ative PSA level (P < 0.0001), and positive surgical margin (P < Scenario 1 evaluated all postoperatively available parameters 0.0001; Table 1). Low ANO7 staining was also strongly linked including pathological tumor (pT), pathological lymph node to early biochemical recurrence (P < 0.0001, Figure 2A). (pN), surgical margin status, preoperative PSA value, and Gleason grade obtained on the prostatectomy specimen. In ANO7, TMPRSS2:ERG fusion status, and scenario 2, all postoperatively available parameters except pN androgen receptor expression were included. The rationale for this approach was that the indication and extent of lymph node dissection is not stand- Data from 5656 tumors with ERG FISH and immunohis- ardized in the surgical therapy of PCa and may introduce a tochemistry (IHC) were available and showed concordant bias toward high-grade cancers. Two additional scenarios results in 95.5% of cancers. Strong ANO7 staining was less were to model the preoperative situation as much as possible. prevalent in cancers harboring TMPRSS2:ERG rearrange- Scenario 3 included ANO7 expression, preoperative PSA, clin- ments (24%) and ERG expression (25%) than in cancer lack- ical tumor stage (cT stage), and Gleason grade obtained on ing ERG fusions (43%) or ERG expression (45%, P < 0.0001 the prostatectomy specimen. As postoperative determination each, Supplementary Figure S1). This observation prompted of a tumor’s Gleason grade is superior to the preoperatively us to determine whether associations between ANO7 expres- determined Gleason grade (subjected to sampling errors and sion and tumor phenotype were dependent on the ERG sta- consequently under grading in more than one third of cases), tus. Separate analyses of ERG-negative and -positive cancers this parameter was replaced by the preoperative Gleason showed, however, that similarly strong associations with unfa- grade obtained on the original biopsy in scenario 4. All these vorable tumor type (Supplementary Tables S2 and S3) and analyses identified ANO7 as a strong independent prognos- patient outcome (P < 0.0001 each; Figure 2B, 2C) were seen tic feature in the entire cohort and also in the subgroups of in both subsets. Low ANO7 staining was also linked to high ERG-negative and -positive cancers (P < 0.0001 each). The androgen receptor (AR) expression. This was seen in all can- striking prognostic role of ANO7 loss is also demonstrated cers (P < 0.0001) and in ERG-negative cancers (P = 0.0002) by its prognostic relevance in multiple subgroups of cancers but was not significant in ERG-positive cancers (Figure 3). with identical traditional (3+4, 4+3, ≥ 8; P < 0.0005 each; Supplementary Figure S3A) and quantitative Gleason grade Associations with chromosomal deletions (3 of 7 groups, Supplementary Figure S3B–S3H). A striking and Ki67 labeling index prognostic impact of ANO7 loss was also seen in the subset of 982 PTEN-deleted cancers (P = 0.0033; Figure 2E). For 9 of 11 analyzed chromosomal regions, ANO7 stain- ing was weaker and more often negative in cases of deletion Discussion (Supplementary Figure S2A). In ERG-negative and ERG- positive cancers, a statistically significant difference was still The present data identify reduced ANO7 expression as a strong seen for 5 of 11 and 8 of 11 analyzed deletions (P < 0.05 each, and independent predictor of poor patient prognosis in PCa. 248 Marx et al. ANO7 expression in prostate cancer

A

B C

DE

Figure 1 Examples of anoctamin 7 (ANO7) staining: (A) ANO7 staining of cancerous (1) and normal prostate gland (2) in a single tissue microarray (TMA) spot. (B–E) cancer spots with (B) lack of staining, (C) weak staining, (D) moderate staining, and (E) strong staining. Spot size 0.6 mm at 100× and 400× of origin.

ANO7 was strongly expressed in normal prostate glands tissues6,7,26. Only a few studies had analyzed ANO7 expression in our study. This fits well to earlier studies reporting 100% in PCa before. Das et al.6 reported 91% positive PCa in a cohort positivity in 10–44 samples of normal or benign prostate of 126 patients. In two subsequent studies, Mohsenzadegan Cancer Biol Med Vol 18, No 1 February 2021 249

Table 1 ANO7 expression and prostate cancer phenotype Parameter n Fraction of ANO7 expression (%) P Negative Weak Moderate Strong All cancers 13,594 7.6 9.3 48.7 34.4

Tumor stage < 0.0001

pT2 8889 5.5 7.0 47.4 40.2

pT3a 2984 9.8 11.0 52.1 27.1

pT3b-pT4 1668 14.5 18.5 49.6 17.3

Gleason grade < 0.0001

≤ 3+3 2730 4.9 5.6 46.2 43.3

3+4 7372 5.3 7.7 49.8 37.3

3+4 Tert.5 614 7.5 12.2 47.4 32.9

4+3 1292 14.5 12.8 49.6 23.1

4+3 Tert.5 898 13.0 18.3 50.8 17.9

≥ 4+4 591 24.0 18.8 44.8 12.4

Quantitative Gleason grade

≤ 3+3 2730 4.9 5.6 46.2 43.3 < 0.0001

3+4 ≤ 5% 1927 3.9 5.3 46.4 44.4

3+4 (6%–10%) 1845 4.3 6.0 50.4 39.3

3+4 (11%–20%) 1614 4.8 7.7 51.4 36.1

3+4 (21%–30%) 813 6.8 11.6 51.4 30.3

3+4 (31%–49%) 663 8.9 8.9 53.4 28.8

3+4 Tert.5 614 7.5 12.2 47.4 32.9

4+3 (50%–60%) 556 9.9 10.4 50.0 29.7

4+3 Tert.5 898 13.0 18.3 50.8 17.9

4+3 ≥ 61% 559 17.4 12.9 50.8 19.0

≥ 4+4 511 23.3 17.0 46.8 12.9

Lymph node metastasis < 0.0001

N0 8150 7.9 9.7 48.9 33.4

N+ 864 17.4 19.9 45.7 17.0

Preoperative PSA level (ng/mL) < 0.0001

< 4 1657 8.8 8.6 49.3 33.3

4–10 8140 6.3 8.3 48.2 37.2

10–20 2817 8.9 10.7 49.7 30.6

> 20 898 12.6 15.0 48.6 23.8

Surgical margin < 0.0001

Negative 10,922 6.9 8.5 48.5 36.0

Positive 2624 10.1 12.5 49.4 28.0

ANO7, anoctamin 7; PSA, prostate-specific antigen. 250 Marx et al. ANO7 expression in prostate cancer

A All B ERG negative cancers 1.0 1.0 P 0.0001 < P < 0.0001 0.8 0.8

0.6 0.6

0.4 0.4 Negative (n = 897) Negative (n = 326) Weak (n = 922) Weak (n = 233) 0.2 0.2 Moderate (n = 5,547) Moderate (n = 2,020) PSA recurrence free survival

PSA recurrence free survival Strong (n = 3,891) Strong (n = 1,952) 0.0 0.0 012243648 60 72 84 96 108 120 0122436486072 84 96 108 120 Time (month) Time (month)

C ERG positive cancers D PTEN normal 1.0 1.0 P < 0.0001 P < 0.0001 0.8 0.8

0.6 0.6

0.4 0.4 Negative (n = 272) Negative (n = 256) Weak (n = 260) Weak (n = 210) 0.2 Moderate (n = 2,176) 0.2 Moderate (n = 2,072) Strong (n = 873) Strong (n = 1,619) PSA recurrence free survival PSA recurrence free survival 0.0 0.0 0122436486072 84 96 108 120 0122436486072 84 96 108 120 Time (month) Time (month) E PTEN deleted 1.0 P = 0.0033 0.8

0.6

0.4 Negative (n = 108) Weak (n = 96) 0.2 Moderate (n = 613)

PSA recurrence free survival Strong (n = 165) 0.0 0122436486072 84 96 108 120 Time (month)

Figure 2 Association between anoctamin 7 (ANO7) expression and biochemical recurrence in (A) all cancers, (B) the erythroblast transfor- mation-specific-related gene (ERG)-negative subset, (C) the ERG-positive subset, (D) phosphatase and tensin homolog (PTEN) normal cancers, and (E) PTEN-deleted cancers. et al.7,26 analyzed 123–152 PCa and also reported reduced prognostic impact in cancers with comparable traditional expression in a fraction of cancers as compared to normal or quantitative Gleason grade. In our patient cohort, where epithelial cells. Overall, these data strongly suggest that ANO7 Gleason grading has been very thoroughly performed, most expression can be reduced or lost during PCa development or prognostic molecular parameters lose their prognostic role progression. in cancers with homogeneous morphology27-29. A strong The comparison with clinical data in 13,594 successfully prognostic role of ANO7 loss in PCa is also in line with analyzed patients identifies reduced ANO7 as one of the earlier data from Mohsenzadegan et al.7,26 who reported strongest prognostic molecular features in PCa. This is not an inverse correlation between ANO7 staining intensity only demonstrated by the independent prognostic value of and tumor stage or Gleason score. We cannot explain why ANO7 staining in all applied models but also by its retained a recent meta-analysis of ANO7 mRNA expression data on Cancer Biol Med Vol 18, No 1 February 2021 251

100.0 90.0 ) 80.0 70.0 60.0

cancers (% 50.0 40.0 30.0 20.0 Fraction of 10.0 0.0 ( n = 696) ( n = 643) ( n = 714) ( n = 385) ( n = 960) ( n = 142) ( n = 1,504) ( n = 1,216) ( n = 1,257) ong ( n = 2,879) ong ( n = 1,027) ong ( n = 1,584) AR weak AR weak AR weak AR str AR str AR str AR negative AR negative AR moderate AR moderate AR negative AR moderate All cancers, P < 0.0001 ERG negative, P = 0.0002 ERG positive, P = 0.5086

Strong ModerateWeak Negative ANO7 expression

Figure 3 Anoctamin 7 (ANO7) staining and androgen receptor (AR) expression in all cancers, the erythroblast transformation-specific-­ related gene (ERG)-negative, and ERG-positive subsets.

3.50 3.00 2.50 2.00 1.50

(mean + SEM) 1.00 Ki67 labeling index 0.50 0.00 Negative Weak Moderate Strong n = 394 n = 345 n = 3,140 n = 2,104

ANO7 expression level (P < 0.0001)

Figure 4 Anoctamin 7 (ANO7) staining and Ki67 labeling index.

289 tumors obtained from The Cancer Genome Atlas pro- The reduction of ANO7 expression from well-differenti- ject database8 found a correlation between increased ANO7 ated to poorly differentiated prostate cells may suggest its total expression and early biochemical relapse9. It cannot be expression loss in aggressive PCa. This indicates that ANO7 excluded that mRNA expression does not always translate could be a surrogate of molecular driver of tumor aggression in immunohistochemically detectable ANO7 protein, and in PCa that is yet to be discovered. The physiologic function of that true differences exist between the prognostic value of ANO7 is not sufficiently known. A Medline search on March ANO7 mRNA and protein analysis. Alternatively, technical 28, 2019 identified fewer than 20 publications on ANO7 or issues such as a variable contamination with ANO7-positive NGEP. Being an ion channel protein, ANO7 may influence normal glandular cells or ANO7-negative stromal cells, a levels and compartmentalization of intracellular calcium30. possible selection bias for larger advanced cancers that can Intracellular calcium levels can affect the activity of that be more easily used for next-generation sequencing, or the are relevant for cancer development and progression30. Ion comparatively low number of patients may account for these channel with a well-accepted role in cancer biology, discrepant findings. for example, include potassium channels, Cl− channels, and 252 Marx et al. ANO7 expression in prostate cancer

Table 2 Hazard ratios (95% confidence intervals) for biochemical relapse after prostatectomy for established risk factors andANO7 expression Model Analyzable (n) Scenario 4 Scenario 3 Scenario 2 Scenario 1 Variable 9527 11,032 11,213 7214 Gleason grade 3+4 vs. ≤ 3+3 1.8 (1.6–2.0), P < 0.0001 biopsy 4+3 vs. ≤ 3+3 3.0 (2.7–3.4), P < 0.0001

≥ 4+4 vs. ≤ 3+3 3.9 (3.4–4.4), P < 0.0001 cT stage T2a vs. T1c 1.3 (1.1–1.4), P < 0.0001 1.3 (1.1–1.4), P < 0.0001

T2b vs. T1c 1.9 (1.6–2.1), P < 0.0001 1.7 (1.5–1.9), P < 0.0001

T2c vs. T1c 1.9 (1.5–2.4), P < 0.0001 1.9 (1.5–2.4), P < 0.0001

T3a vs. T1c 1.3 (1.0–1.7), P = 0.0464 1.5 (1.2–2.0), P = 0.0011

Preoperative 4–10 vs. < 4 1.6 (1.3–1.9), P < 0.0001 1.4 (1.2–1.7), P < 0.0001 1.3 (1.1–1.5), P = 0.0008 PSA level 10–20 vs. < 4 2.4 (2.0–2.8), P < 0.0001 2.0 (1.7–2.4), P < 0.0001 1.7 (1.4–2.0), P < 0.0001

> 20 vs. < 4 3.8 (3.1–4.6), P < 0.0001 2.9 (2.4–3.4), P < 0.0001 2.0 (1.7–2.4), P < 0.0001

ANO7 Moderate vs. strong 1.3 (1.2–1.5), P < 0.0001 1.2 (1.1–1.3), P = 0.000 1.1 (1.0–1.2), P = 0.0373 1.1 (1.0–1.2), P = 0.0645 expression Weak vs. strong 1.8 (1.6–2.1), P < 0.0001 1.5 (1.3–1.8), P < 0.0001 1.3 (1.1–1.5), P = 0.0003 1.3 (1.4–1.6), P = 0.0003

Negative vs. strong 2.0 (1.7–2.3), P < 0.0001 1.6 (1.4–1.9), P < 0.0001 1.5 (1.3–1.7), P < 0.0001 1.4 (1.2–1.7), P < 0.0001

Gleason grade 3+4 vs. ≤ 3+3 2.9 (2.5–3.3), P < 0.0001 2.3 (2.0–2.7), P < 0.0001 2.1 (1.7–2.7), P < 0.0001 prostatectomy 4+3 vs. ≤ 3+3 7.3 (6.2–8.6), P < 0.0001 4.9 (4.1–5.8), P < 0.0001 4.3 (3.4–5.4), P < 0.0001

3+4 Tert. 5 vs. ≤ 3+3 5.2 (4.1–6.6), P < 0.0001 3.5 (2.8–4.4), P < 0.0001 3.2 (2.4–4.2), P < 0.0001

4+3 Tert. 5 vs. ≤ 3+3 11.3 (9.4–13.5), P < 0.0001 6.1 (5.1–7.4), P < 0.0001 5.1 (4.0–6.5), P < 0.0001

≥ 4+4 vs. ≤ 3+3 14.0 (11.7–16.9), P < 0.0001 6.8 (5.6–8.3), P < 0.0001 5.5 (4.3–7.1), P < 0.0001 pT stage T3a vs. T2 2.0 (1.8–2.2), P < 0.0001 2.0 (1.8–2.2), P < 0.0001

T3b-4 vs. T2 3.3 (2.9–3.7), P < 0.0001 2.9 (2.5–3.3), P < 0.0001

Resection R1 vs. R0 1.4 (1.3–1.5), P < 0.0001 1.2 (1.1–1.4), P < 0.0001 margin status

Nodal stage N+ vs. N0 1.5 (1.3–1.7), P < 0.0001

ANO7, anoctamin 7; cT, clinical tumor; pT, pathological tumor.

Na+ channels31,32. Some other anoctamins have been earlier ANO7 share a complete specificity for prostate epithelial cells linked to cancer biology. For example, anoctamin 1 is particu- and the very high level of expression in these cells. Although larly expressed in gastrointestinal stromal tumors and head some authors have speculated on a possible cancer preventive and neck squamous cell carcinomas, where it contributes to role of PSA, this has never been proven37. The main physio- cell proliferation, poor prognosis, and metastasis33,34. A splice logical role of PSA is semen liquidification. The prognostic variant of anoctamin 6 has been associated with poor progno- impact of reduced PSA was very strong, comparable to the sis in breast cancer35. role of ANO7 loss, and also found in PTEN-deleted cancers. It is also possible that the striking prognostic impact of For PSA, we had speculated that a measurable loss of one of ANO7 loss is not related to its – so far unknown – function. the most important cellular proteins might represent a subtle We had recently found a strong link between reduced cellu- sign of cellular dedifferentiation. In analogy to the PSA situa- lar expression of PSA and poor PCa prognosis36. PSA and tion, we consider it possible that ANO7 is another important Cancer Biol Med Vol 18, No 1 February 2021 253 component of prostate epithelial cells without a cancer-spe- linked to ANO7 expression and significant associations could cific role and that a reduced expression might indicate cellular often not be found in homogeneous subgroups. We consider it dedifferentiation. thus unlikely that ANO7 exerts a potential tumor suppressive The abundant data available from earlier studies using our function through impacting tumor cell proliferation. Thus, we PCa cohort enabled us to compare ANO7 expression with favor the notion that ANO7 loss indicates loss of polar orien- several important molecular features. TMPRSS2:ERG fusions tation in prostate epithelial cells as a form of dedifferentiation. occur in about 50% of PCa resulting in permanent overex- The strong independent prognostic role found for ANO7 pression of the transcription factor ERG. This overexpression expression in this study suggests that measuring this protein modulates the expression of more than 1600 genes but lacks could result in useful prognostic information for PCa patients. prognostic relevance by itself. Our data suggest that loss of Further studies using different antibodies and independent ANO7 expression is ERG dependent as ANO7 protein levels patient cohorts potentially also including patients treated with were clearly lower in ERG-positive than in ERG-negative can- radiotherapy or radiotherapy and hormonal therapy, as well as cers. Such inverse associations found by IHC are particularly premalignant prostate conditions, would be helpful to confirm certain to represent true associations. This observation thus the validity of ANO7 as a routine diagnosis marker in prostate provides further evidence for the validity of our experimental and potentially even other human cancer types. If immuno- procedures. A mild positive association between immunohis- histochemical analyses are meant to provide clinically relevant tochemically determined parameters could always be due to information to trigger treatment decisions, it will be critical to a fraction of samples that are non-reactive to IHC resulting apply highly reproducible assays. The advent of multiplex IHC in “negative” staining results for all parameters measured. The enabling the simultaneous application of multiple antibodies same might hold true also for the inverse association between and a computerized quantification of individual proteins may ANO7 expression and AR positivity. That this weak association assist in making this possible. was statistically significant only in the subset of ERG-negative In summary, reduced ANO7 expression is tightly linked cancers might be first of all related to the strong link between to an unfavorable disease course in PCa. The statistical inde- AR and ERG, which might obscure less strong interactions. pendence from pre- and postoperatively available parameters However, AR does not belong to the more than 100 transcrip- argues for a possible diagnostic application of ANO7 measure- tion factors that are known to impact ANO7 expression37. ment, either alone or combined with other parameters. Chromosomal deletions represent the most common recur- rent genomic alterations in PCa after TMPRSS2:ERG fusions. Grant support That most deletions were significantly associated with reduced ANO7 expression could suggest a role of ANO7 in mechanisms The study was supported by the Wilhelm Sander-Stiftung regulating genomic instability. Alternatively, these findings (Grant No. 2015.010.1). would also be consistent with the notion of reduced ANO7 expression representing a dedifferentiation surrogate. The Conflict of interest statement particularly low levels of ANO7 expression in PTEN-deleted cancers are consistent with a direct or indirect functional inter- No potential conflicts of interest are disclosed. action between ANO7 and the PTEN/AKT pathway. That the interaction with PTEN is not responsible for the prognostic References impact of reduced ANO7 expression is demonstrated by its retained prognostic role in PTEN-deleted cancers. This is unu- 1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. sual and further argues for a particularly strong prognostic role Global cancer statistics 2018: GLOBOCAN estimates of incidence of ANO7 expression loss in PCa. PTEN deletions are strongly and mortality worldwide for 36 cancers in 185 countries. CA linked to poor prognosis in PCa. Many prognostic features fail Cancer J Clin. 2018; 68: 394-424. 2. Wanitchakool P, Wolf L, Koehl GE, Sirianant L, Schreiber R, to further stratify patient outcome in molecular subgroups that Kulkarni S, et al. Role of anoctamins in cancer and apoptosis. 38 are already defined by PTEN deletion . Several studies had Philos Trans R Soc Lond B Biol Sci. 2014; 369: 20130096. 39-41 suggested an impact of ANO1 on tumor cell proliferation . 3. Bera TK, Das S, Maeda H, Beers R, Wolfgang CD, Kumar V, et al. In our patients, tumor cell proliferation was only marginally NGEP, a gene encoding a membrane protein detected only in 254 Marx et al. ANO7 expression in prostate cancer

prostate cancer and normal prostate. Proc Natl Acad Sci U S A. 18. Kluth M, Amschler NN, Galal R, Moller-Koop C, Barrow P, 2004; 101: 3059-64. Tsourlakis MC, et al. Deletion of 8p is an independent prognostic 4. Das S, Hahn Y, Nagata S, Willingham MC, Bera TK, Lee B, et al. parameter in prostate cancer. Oncotarget. 2017; 8: 379-92. NGEP, a prostate-specific plasma membrane protein that promotes 19. Krohn A, Diedler T, Burkhardt L, Mayer PS, De Silva C, Meyer- the association of lncap cells. Cancer Res. 2007; 67: 1594-601. Kornblum M, et al. Genomic deletion of PTEN is associated with 5. Cropp CD, Simpson CL, Wahlfors T, Ha N, George A, Jones MS, tumor progression and early PSA recurrence in ERG fusion- et al. Genome-wide linkage scan for prostate cancer susceptibility positive and fusion-negative prostate cancer. Am J Pathol. 2012; in finland: evidence for a novel on 2q37.3 and confirmation 181: 401-12. of signal on 17q21-q22. Int J Cancer. 2011; 129: 2400-7. 20. Kluth M, Ahrary R, Hube-Magg C, Ahmed M, Volta H, Schwemin 6. Das S, Hahn Y, Walker DA, Nagata S, Willingham MC, Peehl DM, C, et al. Genomic deletion of 12p is an independent et al. Topology of NGEP, a prostate-specific cell: cell junction prognostic marker in prostate cancer. Oncotarget. 2015; 6: 27966-79. protein widely expressed in many cancers of different grade level. 21. Weischenfeldt J, Simon R, Feuerbach L, Schlangen K, Weichenhan Cancer Res. 2008; 68: 6306-12. D, Minner S, et al. Integrative genomic analyses reveal an androgen- 7. Mohsenzadegan M, Madjd Z, Asgari M, Abolhasani M, Shekarabi driven somatic alteration landscape in early-onset prostate cancer. M, Taeb J, et al. Reduced expression of NGEP is associated with Cancer Cell. 2013; 23: 159-70. high-grade prostate cancers: a tissue microarray analysis. Cancer 22. Kluth M, Scherzai S, Buschek F, Fraune C, Moller K, Hoflmayer Immunol Immunother. 2013; 62: 1609-18. D, et al. 13q deletion is linked to an adverse phenotype and poor 8. Cerami E, Gao J, Dogrusoz U, Gross BE, Sumer SO, Aksoy BA, et al. prognosis in prostate cancer. Gene Chromosome Canc. 2018; 57: The cbio cancer genomics portal: an open platform for exploring 504-12. multidimensional cancer genomics data. Cancer Discov. 2012; 2: 23. Kluth M, Jung S, Habib O, Eshagzaiy M, Heinl A, Amschler N, et al. 401-4. Deletion lengthening at 6q and 16q targets multiple 9. Kaikkonen E, Rantapero T, Zhang Q, Taimen P, Laitinen V, tumor suppressor genes and is associated with an increasingly poor Kallajoki M, et al. ANO7 is associated with aggressive prostate prognosis in prostate cancer. Oncotarget. 2017; 8: 108923-35. cancer. Int J Cancer. 2018; 143: 2479-87. 24. Kluth M, Harasimowicz S, Burkhardt L, Grupp K, Krohn A, 10. Sauter G, Steurer S, Clauditz TS, Krech T, Wittmer C, Lutz F, et al. Prien K, et al. Clinical significance of different types of p53 gene Clinical utility of quantitative gleason grading in prostate biopsies alteration in surgically treated prostate cancer. Int J Cancer. 2014; and prostatectomy specimens. Eur Urol. 2016; 69: 592-8. 135: 1369-80. 11. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml 25. Kluth M, Graunke M, Moller-Koop C, Hube-Magg C, Minner P, Leighton S, et al. Tissue microarrays for high-throughput S, Michl U, et al. Deletion of 18q is a strong and independent molecular profiling of tumor specimens. Nat Med. 1998; 4: 844-7. prognostic feature in prostate cancer. Oncotarget. 2016; 7: 86339-49. 12. Tennstedt P, Koster P, Bruchmann A, Mirlacher M, Haese A, Steuber 26. Mohsenzadegan M, Shekarabi M, Madjd Z, Asgari M, Abolhasani T, et al. The impact of the number of cores on tissue microarray M, Tajik N, et al. Study of NGEP expression pattern in cancerous studies investigating prostate cancer biomarkers. Int J Oncol. 2012; tissues provides novel insights into prognostic marker in prostate 40: 261-8. cancer. Biomark Med. 2015; 9: 391-401. 13. Minner S, Enodien M, Sirma H, Luebke AM, Krohn A, Mayer 27. Gobel C, Ozden C, Schroeder C, Hube-Magg C, Kluth M, Moller- PS, et al. ERG status is unrelated to PSA recurrence in radically Koop C, et al. Upregulation of centromere protein F is linked operated prostate cancer in the absence of antihormonal therapy. to aggressive prostate cancers. Cancer Manag Res. 2018; 10: Clin Cancer Res. 2011; 17: 5878-88. 5491-504. 14. Minner S, Wittmer C, Graefen M, Salomon G, Steuber T, Haese 28. Neubauer E, Latif M, Krause J, Heumann A, Armbrust M, Luehr A, et al. High level PSMA expression is associated with early PSA C, et al. Up regulation of the steroid hormone synthesis regulator recurrence in surgically treated prostate cancer. Prostate. 2011; 71: HSD3B2 is linked to early PSA recurrence in prostate cancer. Exp 281-8. Mol Pathol. 2018; 105: 50-6. 15. Krohn A, Seidel A, Burkhardt L, Bachmann F, Mader M, Grupp 29. Hoflmayer D, Willich C, Hube-Magg C, Simon R, Lang D, K, et al. Recurrent deletion of 3p13 targets multiple tumour Neubauer E, et al. Snw1 is a prognostic biomarker in prostate suppressor genes and defines a distinct subgroup of aggressive erg cancer. Diagn Pathol. 2019; 14: 33. fusion-positive prostate cancers. J Pathol. 2013; 231: 130-41. 30. Kunzelmann K, Ousingsawat J, Benedetto R, Cabrita I, Schreiber R. 16. Burkhardt L, Fuchs S, Krohn A, Masser S, Mader M, Kluth M, et al. Contribution of anoctamins to cell survival and cell death. Cancers CHD1 is a 5q21 tumor suppressor required for erg rearrangement (Basel). 2019; 11: 382. in prostate cancer. Cancer Res. 2013; 73: 2795-805. 31. Bates E. Ion channels in development and cancer. Annu Rev Cell 17. Kluth M, Hesse J, Heinl A, Krohn A, Steurer S, Sirma H, et al. Dev Biol 2015; 31: 231-47. Genomic deletion of MAP3K7 at 6q12-22 is associated with early 32. Cheng Q, Chen A, Du Q, Liao Q, Shuai Z, Chen C, et al. Novel PSA recurrence in prostate cancer and absence of TMPRSS2:ERG insights into ion channels in cancer stem cells (review). Int J Oncol. fusions. Mod Pathol. 2013; 26: 975-83. 2018; 53: 1435-41. Cancer Biol Med Vol 18, No 1 February 2021 255

33. Ardeleanu C, Arsene D, Hinescu M, Andrei F, Gutu D, Luca L, et al. 38. Burdelski C, Menan D, Tsourlakis MC, Kluth M, Hube-Magg C, Pancreatic expression of dog1: a novel gastrointestinal stromal Melling N, et al. The prognostic value of SUMO1/Sentrin specific tumor (GIST) biomarker. Appl Immunohistochem Mol Morphol. peptidase 1 (senp1) in prostate cancer is limited to ERG-fusion 2009; 17: 413-8. positive tumors lacking pten deletion. BMC Cancer. 2015; 15: 538. 34. Carles A, Millon R, Cromer A, Ganguli G, Lemaire F, Young J, et al. 39. Wu H, Wang H, Guan S, Zhang J, Chen Q, Wang X, et al. Cell- Head and neck squamous cell carcinoma transcriptome analysis by specific regulation of proliferation by Ano1/TMEM16A in breast comprehensive validated differential display. Oncogene. 2006; 25: cancer with different ER, PR, and HER2 status. Oncotarget. 2017; 8: 1821-31. 84996-5013. 35. Dutertre M, Lacroix-Triki M, Driouch K, de la Grange P, 40. Guan L, Song Y, Gao J, Gao J, Wang K. Inhibition of calcium- Gratadou L, Beck S, et al. Exon-based clustering of murine activated chloride channel ANO1 suppresses proliferation breast tumor transcriptomes reveals alternative exons whose and induces apoptosis of epithelium originated cancer cells. expression is associated with metastasis. Cancer Res. 2010; 70: Oncotarget. 2016; 7: 78619-30. 896-905. 41. Britschgi A, Bill A, Brinkhaus H, Rothwell C, Clay I, Duss S, et al. 36. Bonk S, Kluth M, Hube-Magg C, Polonski A, Soekeland G, Calcium-activated chloride channel ANO1 promotes breast cancer Makropidi-Fraune G, et al. Prognostic and diagnostic role of progression by activating EGFR and CAMK signaling. Proc Natl PSA immunohistochemistry: a tissue microarray study on 21,000 Acad Sci U S A. 2013; 110: E1026-34. normal and cancerous tissues. Oncotarget. 2019; 10: 5439-53. 37. Fishilevich S, Nudel R, Rappaport N, Hadar R, Plaschkes I, Iny Cite this article as: Marx A, Koopmann L, Höflmayer D, Büscheck F, Hube- Stein T, et al. GeneHancer: genome-wide integration of enhancers Magg C, Steurer S, et al. Reduced anoctamin 7 (ANO7) expression is a strong and target genes in . Database (Oxford). 2017; 2017: and independent predictor of poor prognosis in prostate cancer. Cancer Biol bax028. Med. 2021; 18: 245-255. doi: 10.20892/j.issn.2095-3941.2019.0324 Cancer Biol Med Vol 18, No 1 February 2021 1

Supplementary materials

Table S1 Composition of the prostate prognosis tissue microarray No. of patients (%) Study cohort Biochemical relapse on TMA* among categories Follow-up

n 14,464 3,612 (25%)

Mean/median (months) 56/48 –

Age (years)

≤ 50 433 66 (15.2%)

51–59 4,341 839 (19.3%)

60–69 9,977 2,073 (20.8%)

≥ 70 2,936 634 (21.6%)

Pretreatment PSA (ng/mL)

< 4 2,225 313 (14.1%)

4–10 10,520 1,696 (16.1%)

10–20 3,662 1,043 (28.5%)

> 20 123 545 (44.3%) pT stage (AJCC 2002)

pT2 11,518 1,212 (10.5%)

pT3a 3,842 1,121 (29.2%)

pT3b 2,233 1,213 (54.3%)

pT4 85 63 (74.1%)

Gleason grade

≤ 3+3 3,570 264 (7.4%)

3+4 9,336 1,436 (15.4%)

3+4 Tert.5 1,697 165 (9.7%)

4+3 2,903 683 (23.5%)

4+3 Tert.5 1,187 487 (41%)

≥ 4+4 999 531 (53.2%) pN stage

pN0 10,636 2,243 (21.1%)

pN+ 1,255 700 (55.8%)

Surgical margin

Negative 14,297 2,307 (16.1%)

Positive 3,388 1,304 (38.5%)

AJCC, American Joint Committee on Cancer; cT, clinical tumor; pT, pathological tumor; pN, pathological lymph node; PSA, prostate- specific antigen; TMA, tissue microarray. *Numbers do not always add up to 17,747 in the different categories because of cases with missing data. 2 Marx et al. ANO7 expression in prostate cancer

Table S2 ANO7 expression and prostate cancer phenotype in the ERG-negative subset Parameter n Fraction of ANO7 expression (%) P Negative Weak Moderate Strong All cancers 4915 7.0 5.3 44.5 43.2

Tumor stage < 0.0001

pT2 8889 5.5 7.0 47.4 40.2

pT3a 2984 9.8 11.0 52.1 27.1

pT3b-pT4 1668 14.5 18.5 49.6 17.3

Gleason grade < 0.0001

≤ 3+3 1002 3.7 3.6 40.4 52.3

3+4 2670 5.1 4.5 44.0 46.3

3+4 Tert.5 216 5.6 7.4 41.7 45.4

4+3 509 15.1 6.3 48.7 29.9

4+3 Tert.5 282 11.3 8.2 54.3 26.2

≥ 4+4 233 20.6 13.7 50.2 15.5

Quantitative Gleason grade < 0.0001

≤ 3+3 1002 3.7 3.6 40.4 52.3

3+4 ≤ 5% 730 4.5 3.8 41.1 50.5

3+4 (6%–10%) 697 3.4 3.6 43.3 49.6

3+4 (11%–20%) 595 4.5 4.7 45.5 45.2

3+4 (21%–30%) 291 7.2 7.6 46.7 38.5

3+4 (31%–49%) 261 8.8 5.4 46.7 39.1

3+4 Tert.5 216 5.6 7.4 41.7 45.4

4+3 (50%–60%) 226 11.9 4.9 44.7 38.5

4+3 Tert.5 282 11.3 8.2 54.3 26.2

4+3 (61%–100%) 235 16.6 7.2 53.2 23.0

≥ 4+4 209 20.6 12.0 50.7 16.7

Lymph node metastasis < 0.0001

N0 2876 7.0 5.5 45.8 41.7

N+ 248 19.8 13.3 46.4 20.6

Preoperative PSA level (ng/mL) < 0.0001

< 4 523 8.0 6.1 44.9 40.9

4–10 2936 5.8 4.5 43.4 46.4

10–20 1082 8.0 5.3 47.4 39.3

> 20 349 12.0 10.9 45.6 31.5

Surgical margin < 0.0001

Negative 3951 6.1 4.9 44.5 44.5

Positive 952 10.6 6.7 44.9 37.8

ANO7, anoctamin 7; ERG, erythroblast transformation-specific-related gene; PSA, prostate-specific antigen. Cancer Biol Med Vol 18, No 1 February 2021 3

Table S3 ANO7 expression and prostate cancer phenotype in the ERG-positive subset Parameter n Fraction of ANO7 expression (%) P Negative Weak Moderate Strong All cancers 3895 7.4 7.4 60.3 24.9

Tumor stage < 0.0001

pT2 2367 5.1 5.1 58.9 31.0

pT3a 1027 10.3 9.2 63.9 16.7

pT3b-pT4 485 12.8 14.2 59.8 13.2

Gleason grade < 0.0001

≤ 3+3 815 4.3 2.8 58.4 34.5

3+4 2261 6.0 6.6 62.2 25.2

3+4 Tert.5 116 10.3 10.3 59.5 19.8

4+3 377 14.6 12.7 57.3 15.4

4+3 Tert.5 205 10.7 16.1 60.0 13.2

≥ 4+4 118 24.6 16.9 47.5 11.0

Quantitative Gleason grade < 0.0001

≤ 3+3 815 4.3 2.8 58.4 34.5

3+4 ≤ 5% 569 2.8 4.2 57.8 35.1

3+4 (6%–10%) 600 5.3 7.2 62.8 24.7

3+4 (11%–20%) 492 5.7 7.1 66.1 21.1

3+4 (21%–30%) 283 7.4 8.1 63.3 21.2

3+4 (31%–49%) 197 11.2 6.1 67.5 15.2

3+4 Tert.5 116 10.3 10.3 59.5 19.8

4+3 (50%–60%) 173 6.4 12.7 59.0 22.0

4+3 Tert.5 205 10.7 16.1 60.0 13.2

4+3 (61%–100%) 163 20.2 14.1 54.6 11.0

≥ 4+4 98 21.4 16.3 52.0 10.2

Lymph node metastasis < 0.0001

N0 2248 9.2 7.9 58.9 24.1

N+ 220 12.7 15.9 59.5 11.8

Preoperative PSA level (ng/mL) < 0.0001

< 4 530 8.3 5.5 62.3 24.0

4–10 2409 6.0 6.9 59.5 27.6

10–20 694 9.8 8.1 62.0 20.2

> 20 233 12.9 12.9 58.4 15.9

Surgical margin 0.0001

Negative 3072 7.2 6.8 59.5 26.5

Positive 806 8.2 9.2 63.3 19.4

ANO7, anoctamin 7; ERG, erythroblast transformation-specific-related gene; PSA, prostate-specific antigen. 4 Marx et al. ANO7 expression in prostate cancer

Table S4 Multivariate Cox regression analysis with established prognostic parameters and the ANO7 expression in all prostate cancers, the ERG-negative, and ERG-positive subsets Tumor subset Scenario n analyzable P-value pT stage cT stage Gleason grade Gleason grade pN stage R stage ANO7 Preoperative prostatectomy biopsy expression PSA level All cancers 1 7173 < 0.0001 < 0.0001 – < 0.0001 – < 0.0001 < 0.0001 < 0.0001

2 11,141 < 0.0001 < 0.0001 – < 0.0001 – - < 0.0001 < 0.0001

3 10,965 < 0.0001 – < 0.0001 < 0.0001 – – – < 0.0001

4 9527 < 0.0001 – < 0.0001 – < 0.0001 – – < 0.0001

ERG-negative 1 2877 0.0054 < 0.0001 – < 0.0001 – 0.0004 0.00537 0.0032 cancers

2 4518 < 0.0001 < 0.0001 – < 0.0001 – – 0.0004 0.0028

3 4474 < 0.0001 – < 0.0001 < 0.0001 – – – 0.0002

4 4404 < 0.0001 – < 0.0001 – < 0.0001 – – < 0.0001

ERG-positive 1 2274 0.0016 < 0.0001 – < 0.0001 – 0.0166 < 0.0001 0.0002 cancers

2 3567 < 0.0001 < 0.0001 – < 0.0001 – – < 0.0001 0.0001

3 3511 < 0.0001 – < 0.0001 < 0.0001 – – – < 0.0001

4 3455 < 0.0001 – < 0.0001 – < 0.0001 – – < 0.0001

ANO7, anoctamin 7; ERG, erythroblast transformation-specific-related gene. Scenario 1 includes all postoperatively available parameters: pathological tumor (pT) stage, lymph node (pN), surgical margin (R) status, preoperative prostate-specific antigen (PSA) value, and Gleason grade obtained after the morphological evaluation of the entire resected prostate. Scenario 2 excluded the nodal status from analysis. Scenario 3 included preoperative PSA, clinical tumor (cT) stage, and Gleason grade obtained on the prostatectomy specimen. In scenario 4, the preoperative Gleason grade obtained on the original biopsy was combined with preoperative PSA and cT stage.

100

90

80 ) 70 ANO7 60 Negative Weak

samples (% 50 Moderate 40 Strong 30 Fraction of 20

10

00 ERG negative ERG positive ERG normal ERG BA (n = 2,635) (n = 4,915) (n = 3,895) (n = 3,250) ERG-IHC P < 0.0001 ERG-FISH P < 0.0001

Figure S1 Anoctamin 7 (ANO7) expression and erythroblast transformation-specific-related gene (ERG) status [immunohistochemistry (IHC)/fluorescence in situ hybridization (FISH)]. Cancer Biol Med Vol 18, No 1 February 2021 5

A All cancers 100 90 80 70 60 50 40 30 20 10 0 ( n = 5,472) ( n = 6,099) ( n = 4,174) ( n = 3,756) ( n = 4,544) ( n = 4,518) ( n = 6,020) ( n = 4,964) ( n = 3,734) ( n = 5,747) ( n = 5,428) Deletion ( n = 586) Deletion ( n = 659) Deletion ( n = 983) Deletion ( n = 735) Deletion ( n = 179) Deletion ( n = 858) Deletion ( n = 467) Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Deletion ( n = 2,230) Deletion ( n = 1,054) Deletion ( n = 1,323) Deletion ( n = 1,066) 3p13, 5q21, 6q15, 8p21, 10q23, 12p13, 12q24, 13q14, 16q24, 17p13, 18q21, P = 0.0081 P = 0.0062 P = 0.0897 P < 0.0001 P < 0.0001 P = 0.0131 P = 0.0256 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.1455

ANO7 strong (%) ANO7 moderate (%) ANO7 weak (%) ANO7 negative (%)

B ERG-negative subset

100 90 80 70 60 50 40 30 20 10 0 ( n = 2,831) ( n = 2,693) ( n = 1,873) ( n = 1,940) ( n = 2,558) ( n = 2,312) ( n = 3,096) ( n = 2,413) ( n = 2,082) ( n = 3,304) ( n = 2,660) Deletion ( n = 79) Deletion ( n = 186) Deletion ( n = 509) Deletion ( n = 688) Deletion ( n = 326) Deletion ( n = 361) Deletion ( n = 742) Deletion ( n = 314) Deletion ( n = 320) Deletion ( n = 250) Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Deletion ( n = 1,063) 3p13, 5q21, 6q15, 8p21, 10q23, 12p13, 12q24, 13q14, 16q24, 17p13, 18q21, P = 0.0557 P = 0.0006 P = 0.0828 P = 0.0025 P < 0.0001 P = 0.3023 P = 0.1401 P = 0.0445 P < 0.0001 P = 0.5192 P = 0.5538

ANO7 strong (%) ANO7 moderate (%) ANO7 weak (%) ANO7 negative (%)

C ERG-positive subset 100 90 80 70 60 50 40 30 20 10 0 ( n = 2,150) ( n = 2,770) ( n = 2,024) ( n = 1,554) ( n = 1,793) ( n = 1,935) ( n = 2,672) ( n = 2,274) ( n = 1,466) ( n = 2,292) ( n = 2,432) Deletion ( n = 95) Deletion ( n = 379) Deletion ( n = 118) Deletion ( n = 255) Deletion ( n = 691) Deletion ( n = 329) Deletion ( n = 501) Deletion ( n = 494) Deletion ( n = 722) Deletion ( n = 177) Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Normal Deletion ( n = 1,066) 3p13, 5q21, 6q15, 8p21, 10q23, 12p13, 12q24, 13q14, 16q24, 17p13, 18q21, P = 0.3473 P = 0.0245 P < 0.0001 P < 0.0001 P < 0.0001 P = 0.0023 P = 0.1672 P < 0.0001 P < 0.0001 P < 0.0002 P < 0.1371

ANO7 strong (%) ANO7 moderate (%) ANO7 weak (%) ANO7 negative (%)

Figure S2 Anoctamin 7 (ANO7) staining and genomic deletions. 6 Marx et al. ANO7 expression in prostate cancer

A 1.0 B 1.0 al al ≤ 3 + 3 (P = 0.3117) iv iv 0.8 0.8 3 + 4 (P < 0.0001) ee su rv ee su rv 0.6 0.6 ence fr ence fr 0.4 4 + 3 (P < 0. 0001) 0.4 ecurr ecurr ≥ 4 + 4 (P = 0.0002) 0.2 0.2 P = 0.7635 PSA r PSA r 0.0 0.0 0122436486072 84 96 108 120 0122436486072 84 96 108 120 Time (month) Time (month) C D 1.0 1.0 al al iv 0.8 iv 0.8 ee su rv 0.6 ee su rv 0.6 nce fr 0.4 nce fr re 0.4 re cur 0.2 ecur 0.2 P = 0.0085 P = 0.0157 PSA re PSA r 0.0 0.0 0122436486072 84 96 108 120 0122436486072 84 96 108 120 Time (month) Time (month) E F 1.0 1.0 al al iv 0.8 iv 0.8 ee su rv 0.6 ee su rv 0.6 ence fr 0.4 ence fr 0.4 ecurr ecurr 0.2 0.2

P = 0.0785 P = 0.9823 PSA r PSA r 0.0 0.0 0122436486072 84 96 108 120 0122436486072 84 96 108 120 Time (month) Time (month) G 1.0 H 1.0 al al iv 0.8 iv 0.8 ee su rv 0.6 ee su rv 0.6 ence fr 0.4 ence fr 0.4 ecurr ecurr 0.2 0.2

PSA r P = 0.1978 P < 0.0001 PSA r 0.0 0.0 0122436486072 84 96 108 120 0122436486072 84 96 108 120 Time (month) Time (month)

Figure S3 Prognostic impact of anoctamin 7 (ANO7) expression defined by the Gleason score. (A) Impact of ANO7 expression as compared to the classical Gleason score categories; (B–H) as compared to the quantitative Gleason score categories defined by (B) ≤ 5%, (C) 6%–10%, (D) 11%–20%, (E) 21%–30%, (F) 31%–49 %, (G) 50%–60%, and (H) ≥ 61% Gleason 4 patterns. Dotted line shows the reference for the respective Gleason category.