www.nature.com/scientificreports OPEN Combined analysis of miR‑200 family and its significance for breast cancer Andrea Fontana1, Raffaela Barbano2, Elisa Dama3, Barbara Pasculli2, Michelina Rendina2, Maria Grazia Morritti4, Valentina Melocchi3, Marina Castelvetere5, Vanna Maria Valori4, Sara Ravaioli6, Sara Bravaccini6, Luigi Ciuffreda7, Paolo Graziano5, Evaristo Maiello4, Massimiliano Copetti1, Vito Michele Fazio2, Manel Esteller8,9,10,11, Fabrizio Bianchi3 & Paola Parrella2* While the molecular functions of miR-200 family have been deeply investigated, a role for these miRNAs as breast cancer biomarkers remains largely unexplored. In the attempt to clarify this, we profiled the miR-200 family members expression in a large cohort of breast cancer cases with a long follow-up (H-CSS cohort) and in TCGA-BRCA cohort. Overall, miR-200 family was found upregulated in breast tumors with respect to normal breast tissues while downregulated in more aggressive breast cancer molecular subtypes (i.e. Luminal B, HER2 and triple negative), consistently with their function as repressors of the epithelial-to-mesenchymal transition (EMT). In particular miR-141-3p was found differentially expressed in breast cancer molecular subtypes in both H-CSS and TCGA-BRCA cohorts, and the combined analysis of all miR-200 family members demonstrated a slight predictive accuracy on H-CSS cancer specific survival at 12 years (survival c-statistic: 0.646; 95%CI 0.538–0.754). With five highly conserved miRNAs (i.e. miR-141, miR-200a, miR-200b, miR-200c and miR-429), the miR-200 family is one of the most frequent groups of miRNAs whose expression is altered in cancer. Two gene clusters located at different chromosomes code for miR-200a/miR-200b/miR-429 (at chr1p36) and for miR-200c/miR-141 (at chr12p13). miR-200b, miR-200c and miR-429 share an almost identical seed sequence “AAU​ACU​G”, while the seed of miR-200a and miR-141 differentiates from the other members for only one nucleotide “AAC​ACU​ G”1. The expression regulation of the miR-200 family was associated with the i) suppression of EMT and tumor metastases through the miR-200/ZEB1-2 a­ xis2, ii) inhibition of cancer stem cell self-renewal and d ­ ifferentiation3, and iii) reversal of ­chemoresistance . Despite comprehensive literature that largely described the molecular 4 function of miR-200 family, the precise role of these miRNAs in cancer has not yet completely understood, with some reports suggesting more prevalent oncosuppressive roles while other studies some possible oncogenic functions. Similar inconsistencies are also evident in few studies evaluating miR-200 family expression in tissues and their potential role as prognostic ­biomarkers5. For example, low miR-200b/c expression in breast cancer was correlated with ­death3–5, whereas high miR-200a expression was correlated with the development of distant ­metastases6. By comparing miRNA expression in normal breast tissue, in situ carcinoma, non-metastatic and metastatic breast cancers, Sánchez-Cid et al. also found that miR-200a/b were increased in metastatic tumors as compared to non-metastatic c­ ancer7. 1 Fondazione IRCCS Casa Sollievo della Sofferenza, UO di Biostatistica, San Giovanni Rotondo, FG, Italy. 2Laboratorio Di Oncologia, Fondazione IRCCS Casa Sollievo della Sofferenza, Viale Padre Pio, 71013 San Giovanni Rotondo, FG, Italy. 3Cancer Biomarkers Lab, ISBREMIT, Institute for Stem‑Cell Biology, Regenerative Medicine and Innovative Therapies, Fondazione IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, FG, Italy. 4Fondazione IRCCS Casa Sollievo della Sofferenza, UO di Oncologia, San Giovanni Rotondo, FG, Italy. 5Fondazione IRCCS Casa Sollievo della Sofferenza, UO di Anatomia Patologica, San Giovanni Rotondo, FG, Italy. 6Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy. 7Fondazione IRCCS Casa Sollievo della Sofferenza, UO di Chirurgia Senologica, San Giovanni Rotondo, FG, Italy. 8Josep Carreras Leukaemia Research Institute (IJC), Badalona, Barcelona, Catalonia, Spain. 9Centro de Investigación Biomédica en Red Cáncer (CIBERONC), Madrid, Spain. 10Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona, Barcelona, Catalonia, Spain. 11Institució Catalana de Recerca I Estudis Avançats (ICREA), Barcelona, Catalonia, Spain. *email: pparrella@operapadrepio.it Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ Variable Category Mean ± SD 59.02 ± 13.60 Age (years) Median (IQR) 59.68 (47.39–70.54) Range 29.7–89.3 Mean ± SD 2.97 ± 1.65 Tumor dimension (cm) Median (IQR) 2.5 (2.0–3.5) Range 0.5–11 Mean ± SD 35.69 ± 23.16 Ki67 Median (IQR) 30 (18–50) Range 1–95 Primitive 282 (98.26) Tumor history—N(%) Recurrence 5 (1.74) No 86 (29.97) Menopause—N(%) Yes 201 (70.03) NST 261 (90.94) Histotype—N(%) NST + ILC 5 (1.74) ILC 21 (7.32) Bilateral 2 (0.7) Site—N(%) Right 136 (47.39) Left 149 (51.92) Stage I 43 (14.98) Stage IIa 99 (34.49) Stage IIb 45 (15.68) Stage (WHO 7)—N(%) Stage IIIa 22 (7.67) Stage IIIb 30 (10.45) Stage IIIc 32 (11.15) Stage IV 16 (5.57) Missing values 30 1 27 (10.51) Histological grade—N(%) 2 125 (48.64) 3 105 (40.86) Negative 70 (24.39) Estrogen receptor—N(%) Positive 217 (75.61) Negative 87 (30.31) Progesterone receptor—N(%) Positive 200 (69.69) Missing values 7 HER2neu—N(%) AMP 65 (23.21) NEG 215 (76.79) Missing values 15 HER2-amplified 34 (12.5) Surrogate molecular classification—N(%) Luminal A-like 104 (38.24) Luminal B-like 96 (35.29) Triple Negative 38 (13.97) Table 1.  Clinicopathological characteristics of the H-CSS patient cohort (N = 287). These contrasting results led us to perform an extensive expression analysis of the entire miR-200 family in two large cohorts of breast cancer patients, the first collected in our hospital (H-CSS cohort, N = 283) and the second from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA cohort, N = 451), in order to clarify the extent of miR-200 family deregulation in breast cancer, and its specific association with clinico- pathological parameters. Results Patients and treatment. Table 1 summarizes the clinicopathological information obtained from the review of medical records and descriptive statistics for the 287 enrolled cases (H-CSS cohort). Metastases at diagnosis were present in 16 cases; among non-metastatic patients (N = 271), 55 experienced disease progres- sion (Incidence Rate, IR of 3.5 events per 100 person-years), and 30 of them died for the disease (IR of 1.7 events per 100 person-years). The median time to disease progression was 69.8 months (IQR: 33.8–107.5), whereas the overall follow-up time was 75.1 months (IQR: 40.6–109.7). Hormone receptor positive breast Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 2 Vol:.(1234567890) www.nature.com/scientificreports/ tumors were defined as cases expressing estrogen (ER) or progesterone (PgR) receptors in ≥ 1% of neoplastic cells as indicated by international ­guidelines8, and HER2 status assessment was carried out according to standard ­recommendations9. Cases were staged according to the World Health Organization staging system version ­7th10. The surrogate molecular classification was performed as described by Pasculli et al.11. Overall, 104 cases (38%) were classified as Luminal A, 96 cases (35%) as Luminal B; 34 cases (12%) were HER2-amplified, and 38 cases (14%) were Triple Negative (Table 1). Fifteen cases were not classified because the HER2 and or ki67 status was not reported in the medical records. All patients received breast-conserving surgery or total mastectomy, plus sentinel node biopsy or complete axillary dissection. Post-surgery treatments were performed according to the following guidelines: San Gallen, NCCN and ASCO. Recurrence was defined as evidence of loco-regional and/ or distant disease over 4 months from diagnosis and after curative-intent surgical treatment. Selection of TCGA‑BReast invasive CAncer (TCGA‑BRCA) cohorts. We selected a cohort of 1053 women with breast cancer not treated with neoadjuvant therapy from the TCGA data portal (https​://porta​l.gdc. cance​r.gov/). All tumors had available expression profile for all the five miRNAs of the miR-200 family. The log2 read counts were used for miRNA expression analysis. The TCGA-BRCA cohort was harmonized with the H-CSS cohort by using a two-step approach (Supplemental Fig. 2 and Supplemental Table 1): (i) the TCGA- BRCA cohort was limited to those histotypes (NST and ILC) and stages (I-IIa/b, IIIa/c and IV) represented in H-CSS (N = 822); (ii) we performed a random disproportionate sampling to align the distribution of histotypes and stages between the two cohorts; weights were overall based on H-CSS distribution, with the exception that we reduced the weights for late stages not to deplete the final cohort’s size extensively. We ultimately selected 451 patients for all subsequent analyses. Characteristics of these cohorts are reported in Supplemental Table 1. Analysis of variance, run through a general linear model, was performed to evaluate the association of miRNAs expression and clinicopathological features, considering both main effects and interaction terms. Cox regression model was implemented to estimate hazard ratios for overall survival, defined as the time from the date of tumor resection until death from any cause. We also downloaded miRNA expression data for 104 avail- able normal breast tissues from TCGA-BRCA data portal. Forty-eight of these normal samples were matched to 48 tumor counterparts among the cohort of 451 women considered. miR‑200 family expression in tumor samples as compared with normal tissues. Following eval- uation of RNA quality, 283 out of the 287 samples from the H-CSS cohort showing an RNA Integrity Number (RIN) > 7.0 were suitable for the analysis (Supplemental Fig. 1). Thus, the expression profile of the entire miR-200 family could be performed in 283 breast cancers, and in 13 normal breast tissues (NBTs) obtained from reduc- tive mammoplasty. As shown in Supplemental Table 2A and Fig. 1, all miRNAs were significantly overexpressed in tumors (p < 0.001) when compared to NBTs. Furthermore, almost all miR-200 family members (except for miR-200b-3p) were overexpressed in tumors as compared to normal tissue adjacent to tumor (Margin) (Sup- plemental Table 2B). Accordingly, in the TCGA-BRCA cohort, we confirmed the overexpression of miR-200 family in breast tumors vs. normal breast tissues (p < 0.0001) (Supplemental Table 3A and Fig. 2) and in matched tumor-normal pairs (N = 48; Supplemental Table 3B). Association among miR‑200 family expression and tumor clinicopathological features. Next, we analyzed the correlation of miR-200 family expression with the clinicopathological characteristics in both the H-CSS (Table 2) and TCGA-BRCA cohorts (Supplemental Table 4A). In the H-CSS cohort (Table 2), the analysis across breast cancer (BC) molecular subtypes (Luminal A, Luminal B, HER2-amplified, and basal/Triple-negative (TNEG); see “Methods”) showed a lower expression of miR-141-3p (p = 0.0306) and miR-200a-3p (p = 0.0381) in those tumors associated to more aggressive subtypes (e.g. LUMB, HER2-amplified and TNEG) Table 2). In particular, miR-141-3p was less expressed in HER2-amplified and TNEG tumors, while miR-200a-3p was less expressed in HER2-amplified, Luminal B and TNEG subtypes (Fig. 3). Consistently, in the TCGA-BRCA cohort, we found that miR-141-3p was downregulated in the Normal-like subgroup (p = 0.0210) while miR-200a-3p was downregulated in HER2-amplified and Luminal B tumors (p = 0.0200). In addition, miR-200b-3p was found down regulated in the HER2-amplified subgroup (p = 0.0060) (Supplemental Table 4A; Fig. 4). In the H-CSS cohort, miR-141-3p, miR-200a-3p and miR-429 expression was increased in advanced stage disease (stage IV) (p = 0.037, p = 0.0011 and p = 0.0078 respectively) (Table 2). In the TCGA cohort, miR-200a-3p (p = 0.0128), miR-200b-3p (p = 0.0009) and miR-200c-3p (p = 0.0013) were increased in invasive lobular carci- noma (Supplemental Table 4A). To evaluate whether these differences may affect the association with molecular subtypes, we performed a multivariable analysis adjusting for stage, histotype and molecular subtype. Overall, our results indicate that miR-200a-3p and miR-141-3p remain significantly associated with the molecular subtypes in breast cancer after the adjustments in the H-CSS cohort (Table 3), whereas an association with miR-141-3p and miR-200c-3p was found in the TCGA cohort (Supplemental Table 4B). Evaluation of miR‑200 family prognostic value in breast cancer cases. The association with time- to-event outcomes (i.e. CSS, DFS, and MFS) was evaluated in the H-CSS cohort without metastases at diagno- sis and with complete information about survival outcomes (Supplemental Fig. 1). As shown in Supplemental Tables 5 and 6, tumor dimension, stage, hormone receptor status, HER2-amplification, and surrogate molecular classification were associated with DFS, MFS, and CSS, while high Ki67 was associated with DFS and MFS only. No statistically significant associations were found with any miRNA of miR-200 family in the overall population. Next, we investigated the prognostic role of the miR-200 family in the TCGA-BRCA cohort by considering the subgroup of patients without metastases at diagnosis (cohort C1, N = 435; Supplemental Fig. 2). We scored 56 deaths and a median follow-up for surviving women of 2.4 years (Q1 = 1.2; Q3 = 4.6 years). Although this cohort Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 3 Vol.:(0123456789) www.nature.com/scientificreports/ A B 12 T vs NBT: p<0.001 12 T vs NBT: p<0.001 log(hsa−miR−200a) T vs M: p<0.001 T vs M: p<0.001 log(hsa−miR−141) 10 10 8 8 6 6 4 4 2 2 0 0 −2 −2 −4 −4 T M NBT T M NBT C D 12 T vs NBT: p<0.001 12 T vs NBT: p<0.001 log(hsa−miR−200b) log(hsa−miR−200c) T vs M: p<0.001 10 10 8 8 6 6 4 4 2 2 0 0 −2 −2 −4 −4 T M NBT T M NBT E 12 T vs NBT: p<0.001 T vs M: p<0.001 log(hsa−miR−429) 10 8 6 4 2 0 −2 −4 T M NBT Figure 1.  miR-200 family members expression in H-CSS cohort in tumor tissues (T), normal breast tissues distant from tumor (M) and normal breast tissue from reductive mammoplasty (NBT). Plots were performed using the R Foundation for Statistical Computing (version 3.6, packages: ggplot2, gridExtra). showed a shorter follow-up and limited number of events, we were able to confirm the prognostic role of stage, estrogen and progesterone receptor status, HER2 status, and molecular subtypes (Supplemental Table 7A). In line with the results obtained in the H-CSS, we did not observe a statistically significant association of any of the miR-200 family members with overall survival (OS) in multivariate analysis (Supplemental Table 7B). These figures were confirmed in the larger cohort of N = 806 subjects without metastases (cohort B1, Supplemental Fig. 2), scoring 101 events, and with a follow-up length (median = 2.4 years; Q1 = 1.2; Q3 = 4.7) comparable to the smaller C1 cohort (Supplemental Table 7A, B). Last, we evaluated whether the combined expression of miR-200 family members was able to predict survival outcomes. As shown in Table 4, when all miRNAs were jointly considered for the building of the weighted scores, only a slight predictive accuracy on H-CSS outcome at 12 years was found (survival c-statistic: 0.646; 95%CI 0.538–0.754). Regression coefficients (weights) used to calculate the scores were reported in Table 5. Discussion In the attempt to elucidate the extent of miR-200 family deregulation in breast cancer and, hence, its potentiality as clinically significant biomarker, we profiled a large series of breast cancer cases with a long follow-up (H-CSS cohort) and the TCGA-BRCA cohorts. First, we found in both H-CSS and TCGA-BRCA cohorts that the global miR-200 family expression is increased in tumors as compared with normal breast tissue or margin. Since miR- 200 family is mainly expressed in epithelial cells, these results are most likely due to the enrichment in fibrous connective adipose tissue typical of the normal breast. The enrichment in normal breast epithelial component might also explain some inconsistencies among literature data. Consistently with our data, Amorim et al.12 found Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 4 Vol:.(1234567890) www.nature.com/scientificreports/ A B 20 20 log(hsa-miR-200a-3p) T vs N: p<0.0001 T vs N: p<0.0001 log(hsa-miR-141-3p) 15 15 10 10 5 5 0 0 T N T N C D 20 T vs N: p<0.0001 20 T vs N: p<0.0001 log(hsa-miR-200b-3p) log(hsa-miR-200c-3p) 15 15 10 10 5 5 0 0 T N T N E 20 T vs N: p<0.0001 log(hsa-miR-429) 15 10 5 0 T N Figure 2.  miR-200 family members expression in TCGA-BRCA cohort in tumor tissues (T), and normal breast tissues distant from tumor (N). Plots were performed using the R Foundation for Statistical Computing (version 3.6, packages: ggplot2, gridExtra). an increased expression of miR-200b-3p and miR-141-3p in tumors as compared with margin, whereas other studies reported reduced expression of miR-200b-3p3 and miR-200c-3p5 in tumors as compared with margins. In both H-CSS and TCGA-BRCA cohorts, we found a differential expression of miR-200 family members within the molecular subgroups identified by the surrogate molecular classification (H-CSS cohort) and intrinsic molecular subtypes (TCGA-BRCA cohort). In particular, lower expression of miR-141-3p/miR-200a-3p was associated with HER2-amplified, Luminal B, and Triple Negative (H-CSS cohort) or Normal Like (TCGA-BRCA cohort) breast cancer subtypes. This is consistent with reports describing that miR-200 family loss of expression unleashes ZEB1 ­expression13, which in turn induces epithelial-to-mesenchymal transition (EMT), which is an important step forward in the initial phase of the metastatic spreading from the primary tumor. Functionally speaking, the association between miR-200 family and metastatic processes have been widely investigated in different tumor types, including breast cancer and, once again, conflicting results have been reported. Indeed, the ectopic expression of miR-200a and miR-200b was shown to inhibit EMT features in undifferentiated, non-tumorigenic breast cancer cells, and impair proliferation, migration, and invasion in triple negative breast c­ ancer14. Accordingly, miR-200c/141 cluster deletion affects breast cancer stem cell heterogeneity by promoting the generation of EMT-like stem cells, which resulted in increased tumor ­metastasis3. miR-200 family members were also found to support Epidermal Growth Factor (EGF)-driven invasion, with the miR- 200bc/429 cluster showing stronger effects than the miR-200a/141 c­ luster1,15. Moreover, miR-200a suppressed cell proliferation in breast cancer by targeting mitochondrial transcription factor A ­ 16, and impaired EMT-like 17 transformation, thus migration, by regulating SIRT1 in breast epithelial c­ ells . Nevertheless, while these stud- ies likely suggest a tumor suppressor role for miR-200 family members, others indicate that higher expression Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 5 Vol.:(0123456789) www.nature.com/scientificreports/ miRNA Clinical variable Category N Statistic# p value* Age –- 283 r = 0.016 0.7948 Tumor dimension –- 283 r = 0.035 0.5616 Ki67 –- 263 r =  − 0.128 0.0379 No 85 38.61 (26.49–76.48) Menopause 0.0961 Yes 198 53.51 (25.9–93.03) NST 257 50.63 (27.35–81.07) Tumor Histotype NST + ILC 5 34.39 (28.98–48.48) 0.7499 ILC 21 56.83 (20.8–109.97) Bilateral 2 98.04 (56.14–139.94) Site Right 134 49.71 (26.49–89.27) 0.4226 Left 147 52.02 (25.9–78.94) Negative 69 36.69 (20.98–65.17 ) Estrogen receptor 0.0146 Positive 214 53.84 (28.01–93.37 ) Negative 85 37.95 (24.14–65.88 ) Progesterone reeceptor 0.0352 Positive 198 54.05 (28.05–94.51 ) miR-141-3p Amplified 64 44.64 (26.2–72.35 ) Her2neu 0.1379 Negative 212 52.98 (27.05–91.71 ) Stage I 41 57.35 (39.33–88.36) Stage IIa 98 42.55 (23.1–78.4) Stage IIb 45 50.77 (28.05–65.08) Stage (WHO 7) Stage IIIa 22 43.66 (17.62–54.23) 0.0370 Stage IIIb 30 65.2 (28.74–128.88) Stage IIIc 31 45.74 (24.65–80.52) Stage IV 16 80.58 (29.82–189.07) 1 26 47.56 (28.74–103.33) Histological grade 2 124 53.66 (28.52–97.22) 0.1401 3 103 38.61 (23.1–72.73) HER2 amplified 33 31.94 (18.63–51.91) Luminal A 104 56.99 (28.4–105.55) Surrogate molecular classification 0.0306 Luminal B 93 53.51 (27.72–87.22) Triple Negative 38 38.32 (26.74–73.94) Age –- 283 0.028 0.6335 Tumor dimension –- 283 0.005 0.9334 Ki67 –- 263 − 0.105 0.0893 No 85 43.13 (24.73–75.42) Menopause 0.2043 Yes 198 50.8 (24.68–90.39) NST 257 48.76 (25.46–84.75) Tumor Histotype NST + ILC 5 35.79 (25.72–36.42) 0.5089 ILC 21 44.65 (19.03–166.08) Bilateral 2 147.61 (94.3–200.92) Site Right 134 42.36 (25.72–76.84) 0.3743 Left 147 53.33 (23.69–86.71) Negative 69 33.37 (21.48–78.68 ) Estrogen receptor 0.2981 Positive 214 51.77 (26.26–86.71 ) Negative 85 44.17 (23.02–76.59 ) Progesterone receptor 0.4315 Positive 198 51.02 (26.36–90.39 ) hsa-miR-200a Amplified 64 40.18 (23.21–69.3 ) Her2neu 0.1009 Negative 212 52.97 (24.7–95.68 ) Stage I 41 44.93 (23.02–85.32) Stage IIa 98 36.49 (20.96–67.7) Stage IIb 45 55.65 (28.99–85.68) Stage (WHO 7) Stage IIIa 22 38.81 (21.48–78.68) 0.0011 Stage IIIb 30 56.9 (33.36–161) Stage IIIc 31 44.17 (27.28–80.1) Stage IV 16 105.65 (36.9–204.02) 1 26 58.14 (25.72–96.78) Histological grade 2 124 52.57 (26.27–87.14) 0.2894 3 103 43.13 (19.23–67.7) HER2 amplified 33 33.37 (18.44–55.43) Luminal A 104 58.18 (26.86–97.92) Surrogate molecular classification 0.0381 Luminal B 93 41.71 (26.19–73.5) Triple Negative 38 45.24 (25.72–94.59) Continued Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 6 Vol:.(1234567890) www.nature.com/scientificreports/ miRNA Clinical variable Category N Statistic# p value* Age –- 283 − 0.031 0.6072 Tumor dimension –- 283 0.039 0.5088 Ki67 –- 263 − 0.095 0.1226 No 85 196.75 (116.38–279.00) Menopause 0.7207 Yes 198 200.15 (112.31–301.69) NST 257 197.42 (111.71–289.32) Tumor histotype NST + ILC 5 222.86 (175.98–233.38) 0.4229 ILC 21 219.44 (119.66–569.52) Bilateral 2 242.05 (204.54–279.55) Site Right 134 190.94 (106.23–273.31) 0.7886 Left 147 201.83 (126.28–330.57) Negative 69 178.88 (119.12–266 ) Estrogen receptor 0.5246 Positive 214 203.34 (111.71–319.75 ) Negative 85 178.88 (113.62–264.23 ) Progesterone receptor 0.5849 Positive 198 204.09 (112.31–323.37 ) hsa-miR-200b Amplified 64 199.09 (115.02–273.7 ) Her2neu 0.3643 Negative 212 200.37 (113.41–307.4 ) Stage I 41 182.02 (119.66–279) Stage IIa 98 188.17 (108.48–268.28) Stage IIb 45 248.55 (135.31–330.57) Stage (WHO 7) Stage IIIa 22 177.43 (75.74–365.43) 0.0684 Stage IIIb 30 224.37 (142.77–476.98) Stage IIIc 31 203.04 (113.62–267.86) Stage IV 16 235.35 (134.93–511.01) 1 26 180.52 (91.35–301.69) Histological grade 2 124 202.44 (110.15–338.3) 0.5903 3 103 194.95 (113.2–264.23) HER2 amplified 33 176.36 (126.28–242.73) Luminal A 104 224.15 (120.3–352.84) Surrogate molecular classification 0.1588 Luminal B 93 197.15 (101.09–285.24) Triple Negative 38 199.11 (123.11–272.04) Age –- 283 − 0.033 0.5780 Tumor dimension –- 283 0.044 0.4606 Ki67 –- 263 − 0.068 0.2738 No 85 781.21 (572.53–1125) Menopause 0.5383 Yes 198 814.66 (534.54–1237.5) NST 257 793.15 (556.83–1191.88) Tumor histotype NST + ILC 5 786.62 (768.82–1068.29) 0.6936 ILC 21 992.52 (542.84–1486.69) Bilateral 2 1831.45 (1475.34–2187.57) Site Right 134 790.84 (522.03–1216.31) 0.3406 Left 147 794.84 (562.58–1191.88) Negative 69 781.21 (452.92–1194.55 ) Estrogen receptor 0.6537 Positive 214 804.54 (561.25–1216.31 ) Negative 85 768.82 (452.92–1194.55 ) Progesterone receptor 0.6473 Positive 198 808.92 (572.53–1210.46 ) hsa-miR-200c Amplified 64 881.74 (604.47–1286.37 ) Her2neu 0.9829 Negative 212 787.79 (550.85–1187.66 ) Stage I 41 744.36 (576.41–1119.53) Stage IIa 98 791.57 (528.96–1232.86) Stage IIb 45 867.48 (626.76–1227) Stage (WHO 7) Stage IIIa 22 796.29 (420.18–1128.67) 0.2718 Stage IIIb 30 878.03 (595.54–1411.15) Stage IIIc 31 773.83 (562.58–1092.21) Stage IV 16 787.49 (527.48–1355.04) 1 26 667.19 (488.12–1097.21) Histological grade 2 124 805.55 (572.36–1167.01) 0.4365 3 103 812.15 (574.36–1229.08) HER2 amplified 33 791.69 (497.88–1194.55) Luminal A 104 811.76 (582.91–1229.93) Surrogate molecular classification 0.5229 Luminal B 93 836.18 (595.54–1175.58) Triple Negative 38 780.99 (465.26–1205.38) Continued Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 7 Vol.:(0123456789) www.nature.com/scientificreports/ miRNA Clinical variable Category N Statistic# p value* Age –- 282 − 0.05 0.4004 Tumor dimension –- 282 0.046 0.4451 Ki67 –- 262 − 0.049 0.4313 No 85 47.03 (28.57–75.29) Menopause 0.6537 Yes 198 49.1 (24.94–99.57) NST 257 47.03 (25.41–91.93) Tumor histotype NST + ILC 5 48.48 (45.43–87.35) 0.4162 ILC 21 55.37 (19.03–108.22) Bilateral 2 89.15 (15.14–163.17) Site Right 134 44.61 (24.94–87.71) 0.9737 Left 147 50.75 (25.44–99.57) Negative 69 46.69 (22.94–87.71 ) Estrogen receptor 0.4817 Positive 214 49.6 (26.91–93.37 ) Negative 85 46.26 (23.28–87.71 ) Progesterone receptor 0.4184 Positive 198 49.97 (27.2–99.57 ) hsa-miR-429 Amplified 64 44.54 (26.16–84.92 ) Her2neu 0.2805 Negative 212 50.42 (25.42–102.58 ) Stage I 41 48.48 (22.94–83.93) Stage IIa 98 41.94 (22.92–66.79) Stage IIb 45 58.16 (32.02–90.82) Stage (WHO 7) Stage IIIa 22 46.48 (28.81–136.24) 0.0078 Stage IIIb 30 77.09 (37.76–152.36) Stage IIIc 31 42.25 (28.57–76.99) Stage IV 16 97.96 (37.67–169.11) 1 26 48.99 (23.28–104.25) Histological grade 2 124 49.97 (26.34–101.45) 0.2133 3 103 44.39 (25.41–74.27) HER2 amplified 33 43.79 (23.52–56.63) Luminal A 104 50.68 (26.34–106.23) Surrogate molecular classification 0.4289 Luminal B 93 45.73 (24.94–85.39) Triple negative 38 53.89 (25.41–117.92) Table 2.  Association between miRNAs and clinicopathological variables. # In case of continuous clinical variables (i.e. age, tumour dimension and Ki67), r denotes Pearson correlation coefficient with log-transformed miRNA expression whereas in presence of categorical clinical variables, median along with interquartile range (IQR, i.e. first-third quartiles) of the miRNA expression was reported. *p values from Pearson correlation or two-sample t test (or ANOVA model as appropriate) using log-transformed miRNA expressions was reported for continuous and categorical variables, respectively. p-value <0.05 are reported in bold. of miR-200 family members might induce rather than prevent metastases formation. For instance, the forced expression of miR-200a/miR-200b in MCF10 mammary cells induced an enhanced epithelial program, aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo, suggesting that miR-200 family mem- bers may promote traits of highly proliferative breast luminal progenitor ­cells7. Likewise, miR-200c/141 cluster overexpression induced by SerpinB2 was shown to foster breast cancer cell m ­ etastasis18. Furthermore, miR-200a overexpression was found to enhance malignant transformation of immortalized human mammary epithelial ­cells19, to protect tumor cells from apoptosis, and promote ­metastases4 and c­ hemoresistance20. Altogether, these discrepancies lead to hypothesize that the biological functions of miR-200 family members may depend on the cellular context, tumor molecular subtype, and stage of tumor ­progression21. In our study, the association between miR-200 family expression and patients’ outcome was evaluated in terms of DFS, MFS, and CSS in the H-CSS cohort including 283 non-metastatic breast cancer cases with a median follow-up of 75 months. In the TCGA-BRCA cohort, only overall survival data were available instead. Our analyses did confirm the prognostic role of lymph node status, estrogen and progesterone receptors status, HER2 status, and molecular subtypes in both H-CSS and TCGA-BRCA cohorts. However, we did not observe any statistically significant association of the miR-200 family members with patients’ outcome in multivariable analyses. Indeed, in the H-CSS cohort, the combined expression of miR-200 family members only showed a slight predictive accuracy on CSS outcome at 12 years (Table 4). To date, only a minority of ­studies22 have performed the expression analysis of miR-200 family members in breast cancer tissues, and evaluated its association with patients’ outcomes (Fig. 5, and Supplemental Table S8). Among those, only one study reported an hazard ratio of 0.231 (95%CI 0.094–0.564) in univariable analysis for miR-200c in a patient cohort including only luminal ­tumors12. Other three ­studies23–25 evaluated the association between miR-200 family members expression in plasma samples and patient’s outcome (Supplemental Table S8). Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 8 Vol:.(1234567890) www.nature.com/scientificreports/ A B 12 HER2 vs LUMA: p=0.004 12 HER2 vs LUMA: p=0.011 log(hsa−miR−200a) log(hsa−miR−141) HER2 vs. LUMB p=0.045 HER2 vs. TNEG p=0.024 10 10 8 8 6 6 4 4 2 2 0 0 −2 −2 HER2 LUMA LUMB TNEG HER2 LUMA LUMB TNEG C D 12 12 log(hsa−miR−200b) log(hsa−miR−200c) 10 10 8 8 6 6 4 4 2 2 0 0 −2 −2 HER2 LUMA LUMB TNEG HER2 LUMA LUMB TNEG E 12 log(hsa−miR−429) 10 8 6 4 2 0 −2 HER2 LUMA LUMB TNEG Figure 3.  miR-200 family members expression within the surrogate molecular classification subgroups identified in the H-CSS cohort: HER2 amplified tumors (HER2), Luminal A (LUMA), Luminal B (LUMB), Triple Negative (TNEG). Plots were performed using the R Foundation for Statistical Computing (version 3.6, packages: ggplot2, gridExtra). In particular, Medhavan et al.24 found an association between increased expression of miR-200a, miR-200b and miR-200c and higher risk of overall mortality in univariable analyses (Fig. 5). Conclusion To the best of our knowledge, this is the first study evaluating the expression of all miR-200 family members in breast cancer tissues in order to identify potential combination biomarkers of clinical relevance. Our results suggest a differential expression of miR-200 family in breast cancer as compared to normal breast, and within the breast cancer molecular subgroups identified by either surrogate classification (H-CSS cohort) or intrinsic molecular classification (TCGA-BRCA cohort). Nevertheless, the correlation analyses with breast cancer patients’ prognosis exclusively found a weak predictive accuracy of the combined expression of miR-200 family on CSS outcome at 12 years in the H-CSS cohort. Although these results seem not to encourage the use of miR-200 family members as combination biomarkers in breast cancer, we cannot rule out that such a role might be held within a single breast cancer subgroup. Indeed, in the H-CSS cohort the number of cases and event outcome is not sufficient for subgroup analyses, whereas only partial information about overall survival and no data on Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 9 Vol.:(0123456789) www.nature.com/scientificreports/ A B 20 p=0.021 20 p=0.020 log(hsa-miR-200a-3p) log(hsa-miR-141-3p) 15 15 10 10 5 5 0 0 HER2 LUMA LUMB BASAL NORMAL HER2 LUMA LUMB BASAL NORMAL LIKE LIKE C D 20 p=0.006 20 p=0.052 log(hsa-miR-200b-3p) log(hsa-miR-200c-3p) 15 15 10 10 5 5 0 0 HER2 LUMA LUMB BASAL NORMAL HER2 LUMA LUMB BASAL NORMAL LIKE LIKE E 20 p=0.1437 log(hsa-miR-429) 15 10 5 0 HER2 LUMA LUMB BASAL NORMAL LIKE Figure 4.  miR-200 family members differential expression within the intrinsic molecular classification subgroups in the TCGA-BRCA cohort: HER2 enriched tumors (HER2), Luminal A (LUMA), Luminal B (LUMB), Basal like, and NORMAL LIKE tumors. Plots were performed using the R Foundation for Statistical Computing (version 3.6, packages: ggplot2, gridExtra). H-CSS COHORT Univariate analysis—p value* Multivariable analysis—p value* Histotype 0.7499 0.8906 miR-141-3p Stage 0.0370 0.2186 Surrogate Molecular Classification 0.0306 0.0233 Histotype 0.5089 0.6958 miR-200a-3p Stage 0.0011 0.0318 Surrogate molecular classification 0.0381 0.0235 Histotype 0.4162 0.6375 miR-429 Stage 0.0078 0.0864 Surrogate molecular classification 0.4289 0.1134 Table 3.  Univariate and multivariable analysis of the association between miRNAs and clinicopathological characteristics in the TCGA cohort. *p-values <0.05 are reported in bold. Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 10 Vol:.(1234567890) www.nature.com/scientificreports/ Outcome Time horizon (years) N.events/total Survival c-statistic (95%CI*) 7 (median) 27/263 0.650 (0.535–0.766) Cancer specific survival (CSS) 12 (max) 29/263 0.646 (0.538–0.754) 7 (median) 45/263 0.590 (0.497–0.682) Progression free survival (PFS) 12 (max) 53/263 0.528 (0.402–0.654) 7 (median) 43/259 0.613 (0.527–0.699) Distant metastases free survival (MFS) 12 (max) 52/258 0.572 (0.479–0.664) Table 4.  Prognostic accuracy of each outcome-specific weighted miRNA score at median and maximum time horizons. *95% confidence interval after 1000 perturbation-resamplings of the data. Outcome miRNA (log expressions) coefficients (weights) p value hsa-miR-141 − 0.77834 0.0073 hsa-miR-200a 0.77455 0.0495 Cancer specific survival (csS) hsa-miR-200b − 1.22087 0.0433 hsa-miR-200c 0.71390 0.1173 hsa-miR-429 0.23035 0.4661 hsa-miR-141 − 0.50129 0.0170 hsa-miR-200a 0.57881 0.0540 Progression free survival (pfs) hsa-miR-200b − 0.78914 0.0730 hsa-miR-200c 0.48904 0.1450 hsa-miR-429 0.09213 0.6720 hsa-miR-141 − 0.52081 0.0110 hsa-miR-200a 0.44872 0.1270 Distant metastases free survival (mfs) hsa-miR-200b − 0.90106 0.0410 hsa-miR-200c 0.66320 0.0470 hsa-miR-429 0.12025 0.5670 Table 5.  Estimated regression coefficients used to compute multiple weighted miRNA scores. progression are available within the TCGA-BRCA cohort. Thus, this possibility needs to be further investigated in studies specifically designed to evaluate miR-200 family expression in each of the breast cancer subtypes. Materials and methods Study design, setting and eligibility criteria. This study is part of the project TRANSCAN Joint Trans- national Call (JTC) 2013-BREMIR initiated in 2015 at the Fondazione IRCCS Casa Sollievo della Sofferenza (H-CSS), aimed to identify novel biomarkers predicting disease progression and metastases development in breast cancer patients. In this study, we evaluated the miR-200 family expression in a retrospective consecutively collected cohort of 287 breast cancer cases (H-CSS cohort) with a median age of 60 years (Supplemental Table 1). We conducted the study according to the REporting of tumor MARKer Studies (REMARK) ­guidelines8,26, and a prospectively written research (TRANSCAN-BREMIR) plan. Breast cancer tissues were collected between January 2006 and December 2014 at the Breast-Unit, Fondazione IRCCS Casa Sollievo della Sofferenza. Following pathological evaluation, tissue samples were snap-frozen in liquid nitrogen and stored at − 80 °C. For legal reasons, only women older than 18 years of age with tumors greater than 1.0 cm in diameter were included in the study. For each sample, a 5 μm hematoxylin/eosin stained section was visually inspected by light microscopy to select tumor areas with at least 70% viable cancer cells rather than normal specimens, obtained from reductive mammoplasty, to check for the absence of tumor cells among normal epithelial. The study meth- odologies using these samples were carried out following the international of Helsinki Declaration 7th revision (2013, EU Directive 2004/23/EC) and the Italian (D. Lgs. 30/06/2003, n. 196) regulations for research on human subjects. All experimental procedures of this study were approved by the Ethical Committee of the Fondazione IRCCS Casa Sollievo della Sofferenza (Prot N 140/CE). A written informed consent was obtained from all patients following the experimental protocol approved by the Ethical Committee. RNA isolation and RT‑qPCR analysis. RNA was isolated from H-CSS tissue samples by Trizol reagent (Invitrogen) according to the manufacturer’s instructions. Total RNA concentration was determined by the absorbance measurement at 260 nm and 280 nm using the NanoDropTM 1000 spectrophotometer (Thermo Fisher Scientific). The RNA quality and integrity were analyzed through 2100 Expert Analyzer (Agilent Technol- ogy), and only RNAs with RIN (RNA Integrity Number) ≥ 7.0 were considered acceptable. Then, 10 ng of total RNA was reverse transcribed to single stranded cDNA by using TaqMan MicroRNA Reverse Transcription Kit Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 11 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 5.  Forest plot of hazard ratios (HR) for studies on plasma samples from breast cancer patients. Plots were performed using the R Foundation for Statistical Computing (version 3.6, packages: ggplot2, gridExtra). (Thermo Fisher Scientific) and 5 × specific stem-loop RT primers for both individual miR-200 family members and the endogenous control, according to the manufacturer’s instructions. RT positive and negative controls were included in each batch of reactions. To assess miR-200 family expression levels in the H-CSS cohort, we applied a relative quantification method with a standard ­curve27. The expression levels of each miR-200 fam- ily member were assessed by using TaqMan MicroRNA Assays that were as follows: hsa-miR-200a-3p, assay ID: 000502; hsa-miR-200b-3p, assay ID: 002251; hsa-miR-200c-3p, assay ID: 002300; hsa-miR-141-3p, assay ID: 000463; hsa-miR-429, assay ID: 001024, and normalized to RNU48 endogenous control, assay ID: 001006 (Thermo Fisher Scientific). Each qPCR run was performed by using 0.5 μl of TaqMan microRNA (20X), 5 μl of TaqMan Universal PCR Master Mix II, No AmpErase UNG, and 1 μl of cDNA. The PCR conditions were as follows: at 95 °C for 10 min, followed by 40 cycles (95 °C for 15 s, 60 °C for 1 min). All samples were run in triplicates. Each plate included positive and negative controls of reverse transcription and multiple water blanks. qPCR reactions were per- formed on ABI PRISM 7900HT Sequence Detection System and the SDS 2.4 software (Thermo Fisher Scientific) was used for post-run analyses. For each miR-200 family member and RNU48 control, standard curves were Scientific Reports | (2021) 11:2980 | https://doi.org/10.1038/s41598-021-82286-1 12 Vol:.(1234567890) www.nature.com/scientificreports/ constructed by plotting the threshold cycle (Ct) values against log10 of the copy number, and fitting by linear least square regression. For each sample, miR-200 family member expression was determined as the ratio of any single miR-200 family member’s copy number to the RNU48 copy number. Then, it was multiplied by 1000 for more straightforward tabulation (i.e. miRNA target/RNU48) × 1000)27. Statistical analysis. Patients’ clinicopathological characteristics were reported as median along with inter- quartile range (IQR, i.e. first-third quartiles) or frequencies and percentages for continuous and categorical variables, respectively. Normal distribution assumption of miRNA expression was evaluated by Q-Q plots and Shapiro-Wilks test, and a log-normal distribution for all miR-200 family members was detected. The two-sample t test (or ANOVA model as appropriate) was used to assess comparisons of log-transformed miRNA expression among patient groups. Pearson correlation coefficient was estimated to assess the correlation between natural log of miRNA expression and continuous variables. Time-to-event analyses were performed by univariable and multivariable proportional hazards Cox regression models and risks were reported as Hazard Ratios (HR) along with their 95% Confidence Interval (95%CI). The individual overall follow-up time was defined as the time between the enrollment date (i.e. at the time of snap-frozen fresh tissue collection) and the occurrence of the death due to cancer (Cancer Specific Survival, CSS), whereas the individual time to tumor progression or distant metastasis was defined as the time between the enrollment date and the occurrence of the first disease progression (Disease Free Survival, DFS), or the first distant metastasis (Metastasis Free Survival, MFS). For patients who did not experience any event as above, their individual follow-up time was defined as the time between the enrollment date and the end of the observational period (i.e. last available examination). Furthermore, annual mortality and disease progression rates were defined as the number of events divided by the number of person-years × 100. When each miRNA expression was considered as the main covariate into a univariable Cox model, HRs were reported with respect to patients groups defined by miRNA’s median value (i.e. above vs. below the median). Moreover, multivariable Cox models were also performed with the inclusion of lymph node and surrogate molecular classification as further covariates. A weighted miRNA score was computed for each survival outcome at issue by the assessment of a multivariable Cox model, which included all miRNAs (natural log of expression) of the miR-200 family as main covariates. Weighted scores were calculated as the linear combination of the regression coefficients by the value of each miRNA (natural log of expression). The prognostic accuracy of each miRNA score was assessed at 7 (i.e. the median time horizon) and at 12 years (i.e. the maximum time horizon) by survival C-statistic, along with its 95% CIs derived following 1000 perturbation- resampling28. A two-sided p value < 0.05 was considered for statistical significance. All statistical analyses were performed using SAS Release 9.4 (SAS Institute, Cary, NC, USA). Plots were performed using the R Foundation for Statistical Computing (version 3.6, packages: ggplot2, gridExtra). Ethics approval and consent to participate. The study methodologies using human samples were car- ried out following the international of Helsinki Declaration 7th revision (2013, EU Directive 2004/23/EC) and the Italian (D. Lgs. 30/06/2003, n. 196) regulations for research on human subjects. All experimental procedures of this study were approved by the Ethical Committee of the IRCCS Casa Sollievo della Sofferenza (Prot N 140/ CE). 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V.M., F.B.: performed bioinformatics analyses; V.M.V., M.M., E..M., L.C.: collected clinical follow up data and reviewed the manuscript, M.C., P.G.: pathological evaluation of specimens and review of the manuscript, A.F., P.P., F.B., S.B.: analysis and interpretation of data. P.P., A.F., R.B.., S.B.: manuscript writing, V.M.F., M.E.: critical review of the manuscript. P.P.: Study supervision. All authors reviewed the manuscript. Funding This work was supported by Italian Ministry of Health (MoH) TRANSCAN Joint Transnational Call (JTC) 2013 co-funded by the European Regional Development Fund, “A way of making Europe” RRC-2014-2354565, Ital- ian Ministry of Health (MoH) “Ricerca Corrente 2019” and “5 × 1000″ voluntary contributions” and partially supported by Associazione Italiana Ricerca sul Cancro (AIRC) Project Code: 16747. Competing interests The authors declare no competing interests. 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