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. 2022 Apr 29;14(9):2217.
doi: 10.3390/cancers14092217.

Nucleolin Overexpression Predicts Patient Prognosis While Providing a Framework for Targeted Therapeutic Intervention in Lung Cancer

Ângela Valério-Fernandes  1   2 , Nuno A Fonseca  1   3 , Nélio Gonçalves  1 , Ana F Cruz  1   4 , Marta I Pereira  1   5 , Ana C Gregório  1   3 , Vera Moura  1   3 , Ana F Ladeirinha  6 , Ana Alarcão  6 , Joana Gonçalves  7 , Antero Abrunhosa  7 , Joana B Melo  8   9 , Lina Carvalho  6 , Sérgio Simões  1   4 , João N Moreira  1   4
Affiliations

Nucleolin Overexpression Predicts Patient Prognosis While Providing a Framework for Targeted Therapeutic Intervention in Lung Cancer

Ângela Valério-Fernandes et al. Cancers (Basel). .

Abstract

Notwithstanding the advances in the treatment of lung cancer with immune checkpoint inhibitors, the high percentage of non-responders supports the development of novel anticancer treatments. Herein, the expression of the onco-target nucleolin in patient-derived pulmonary carcinomas was characterized, along with the assessment of its potential as a therapeutic target. The clinical prognostic value of nucleolin for human pulmonary carcinomas was evaluated through data mining from the Cancer Genome Atlas project and immunohistochemical detection in human samples. Cell surface expression of nucleolin was evaluated by flow cytometry and subcellular fraction Western blotting in lung cancer cell lines. Nucleolin mRNA overexpression correlated with poor overall survival of lung adenocarcinoma cancer patients and further predicted the disease progression of both lung adenocarcinoma and squamous carcinoma. Furthermore, a third of the cases presented extra-nuclear expression, contrasting with the nucleolar pattern in non-malignant tissues. A two- to twelve-fold improvement in cytotoxicity, subsequent to internalization into the lung cancer cell lines of doxorubicin-loaded liposomes functionalized by the nucleolin-binding F3 peptide, was correlated with the nucleolin cell surface levels and the corresponding extent of cell binding. Overall, the results suggested nucleolin overexpression as a poor prognosis predictor and thus a target for therapeutic intervention in lung cancer.

Keywords: lung cancer; nucleolin expression; targeted intracellular drug delivery; tumor microenvironment.

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Conflict of interest statement

V.M. was an employee and shareholder of TREAT U, SA. N.A.F. and A.C.G. were former employees at TREAT U, SA. S.S. and J.N.M. are shareholders of TREAT U, SA. The remaining authors declare no competing interests.

Figures

Figure 1
Figure 1
Epidemiological characterization of lung adenocarcinomas and squamous carcinomas as a function of nucleolin expression. TCGA’s lung adenocarcinomas and squamous carcinomas (PanCancer Atlas datasets, n = 566 and 487, respectively) were analyzed. Data were cleared from missing clinical information. (A–D) Time-to-event analysis (A,C) and respective median overall survival, time to progression, five-year overall, and progression-free survival (B,D) of patients from the lung adenocarcinoma (A,B) and squamous carcinoma (C,D) datasets, according to nucleolin mRNA levels (nucleolinlow and nucleolinhigh) and staging in primary tumors, stratified at identified quantiles (Q). Radar plots represented in (B,D) summarized the data presented in Tables S1 and S2: the closer to zero the points are, the worse the prognosis. The three axes (0–120% in Time-to-event; or 0–80% in Probability) represent the stratification of patients based on whole cohort (dark red axis), Stage I (light grey axis) and Stage ≥ II (dark blue axis). Insert in (A) demonstrates the determination of quantile cut-point by maximally selected rank statistics (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, n.s. p > 0.05, calculated by log-rank Mantel–Cox test). In (D), maximum time to progression is presented for nucleolinlow tumors from the whole cohort or with staging ≥II since median time could not be estimated).
Figure 2
Figure 2
Nucleolin immunoreactivity among four histologically classified human pulmonary carcinomas (n = 58) and adjacent non-malignant lung tissues. (A) Representative images of the tumor cells (×400) and of the adjacent non-malignant tissues (×400) stained for H&E and immunostained for nucleolin. Arrows indicate nucleolar nucleolin; black arrowheads indicate nucleolin spreading throughout the nucleoplasm; white arrowheads indicate extra-nuclear nucleolin. See Supplementary Tables S3 and S4 for complete list of frequencies and global scores of nucleolin immunoreactivities (IR). (B) Prevalence of globally scored nucleolin (IR) (see Materials and Methods) as low/negative (white), weak (light gray), moderate (dark gray), and high (dark) in different sub-types of tumor (cancer and stromal) cells (adenocarcinoma, AD; squamous cell carcinoma, SQ; adenosquamous carcinoma, ADSQ; pleomorphic carcinoma, PM) and in the corresponding surrounding non-malignant tissues, as well as (C) in identifiable stromal cell types, namely tumor endothelial cells (TEC), tumor-infiltrating lymphocytes (TIL), and cancer-associated fusiform cells (CAFs).
Figure 3
Figure 3
Ex vivo cellular association of radiolabeled nucleolin-binding F3-peptide-targeted liposomes to sections of patient-derived pulmonary carcinomas. Tumor sections were incubated for 1 h at room temperature with technetium-99m-labelled liposomes, either targeted with nucleolin-binding F3 peptide (99mTc-F3-L) or non-targeted (99mTc- L). (A) Representative autoradiograph images of bound liposomes to human pulmonary carcinomas and (B) the extent of binding is presented. Data represent the mean ± SEM, bars (n = 7), and were analyzed by two-tailed unpaired t-test (*** p < 0.001).
Figure 4
Figure 4
Density of nucleolin expression in lung cancer cell lines. Immunoblotting of nucleolin in total extracts (A) and in cytoplasm/membrane fractions (B) of non-small cell lung cancer H1975, A549, and H441, nucleolin-overexpressing MDA-MB-435S, and non-tumorigenic MCF12A cell lines. According to the condition, each lane represents an independent experiment. Data represent the mean ± SEM; total extracts were analyzed by 1-way ANOVA and Dunnett’s post hoc test (n = 3–4); cytoplasm/membrane extracts were analyzed by parametric unpaired t-test (n = 3–4). Whole Western blots can be found in Figure S3. (C) Representative dot plots and histograms associated with the strategy for analysis of cell surface nucleolin by flow cytometry. Dead cells were excluded using 7-actinoaminomycin D (7-AAD). (D) Quantification of cell surface nucleolin in the tumorigenic cell lines by flow cytometry. Data represent the mean ± SEM; p-value calculated with parametric unpaired t-test (n = 2–4). ns * p > 0.05, * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 5
Figure 5
In vitro cellular association and internalization of nucleolin-binding F3-peptide-targeted and non-targeted liposomes by lung cancer cells. H1975, A549, and H441 lung cancer cells were left undisturbed (as control) or incubated at 4 °C (not permissive to internalization) or 37 °C, for 1 h, with rhodamine (Rhod) PE-labelled (A) or calcein-labelled (green, B, only at 37 °C) liposomes either non-functionalized (L) or functionalized with nucleolin-binding F3 peptide (F3-L) or non-specific control peptide (NS-L). (A) Results were expressed as the geometric mean value of the cell-associated fluorescence intensity (gMFI), normalized to untreated cells (n = 3–10), as assessed by flow cytometry. gMFIs of liposomal cellular association (F3-L-RhoD, green; NS-L-RhoD, grey; and L-RhoD, white) were represented as mean ± SEM and analyzed as matched data by Friedman test and Dunn’s post hoc test (for H1975) and by 1-way ANOVA and Tukey’s post hoc test (for A549 and H441; * p < 0.05; ** p < 0.01; *** p < 0.001); competitive inhibition study upon pre-incubation with F3-L (black) was compared to F3-L-RhoD, without pre-incubation (green), using paired t-test (* p < 0.05; ** p < 0.01). (B) Colocalization of calcein-labelled (green, aqueous core) liposomes (F3-L-Calcein, NS-L-Calcein, and L-Calcein) with Lysotracker Red (red, lysosomal marker) in H1975 and A549 cells using confocal microscopy. Scale bars: 10 µm (B, H1975) and 20 µm (B, A549).
Figure 6
Figure 6
Cytotoxic potential of different formulations of liposomal doxorubicin against lung cancer cells. H1975, A549, and H441 lung cancer cells were incubated with increasing concentrations of doxorubicin (Dox), free or encapsulated in non-targeted (L[Dox]) or targeted pH-sensitive pegylated liposomes, functionalized either with a non-specific peptide (NS-L[Dox]) or nucleolin-binding F3 peptide (F3-L[Dox]), for the indicated periods. The medium was then replaced with fresh medium and the experiment was prolonged for a total of 96, 120, or 144 h, for A549, H1975, and H441 cells, respectively. Data represent the mean inhibitory concentration for 50% or 90% effect (IC50 and IC90, respectively). Circle size and color reflect the mean Dox concentration value (µM, n = 3): the smaller the size and the greener the color, the higher the cytotoxic potency. See Supplementary Table S5 for detailed data.
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