Journal: Clinical and Translational Medicine

Article Title: Oncogenic driver and therapeutic target: Prolactin signalling axis in retroperitoneal sarcoma

doi: 10.1002/ctm2.70669

Figure Lengend Snippet: Functional characterisation of PRL‐mediated proliferation and chemoresistance in sarcoma models. (A and B) Secretory PRL quantification by ELISA confirming knockdown efficiency in the culture medium, n = 3. (C–F) Growth suppression following PRL depletion: CCK‐8 time‐course assay ( n = 5) and colony formation capacity ( n = 3) in PRL‐knockdown models. (G–J) Recombinant PRL (50 ng/mL)‐induced proliferative enhancement: (G and H) CCK‐8 ( n = 5) and (I and J) colony formation ( n = 3) in HT1080 and SW872 lines. (K–P) PRLR‐dependent proliferation modulation: (K–N) CCK‐8 dose‐response ( n = 5) and (O‐P) colony formation ( n = 3) analysis post‐PRLR perturbation. (Q and R) After treating SW872 and HT1080 cells with PRLR antibody rolinsatamab talirine (20 µg/mL), the effect on cell proliferation was detected by the CCK8 method, with n = 5. (S and T) Xenograft tumourigenesis assay demonstrating impaired SW872 growth with PRL knockdown ( n = 9). (U) A single SW872 clone exhibiting the lowest PRL expression among the pooled PRL‐knockout cells was isolated by limiting dilution cloning, expanded in culture and validated for PRL protein levels via ELISA. (V) Cell proliferation was assessed using the CCK‐8 assay following stable PRL knockout ( n = 5 biological replicates). (W) Bromocriptine‐mediated antiproliferative effects were evaluated in parallel in wild‐type and PRL‐knockout SW872 cell lines using the CCK‐8 assay ( n = 5). (X) In vivo efficacy was determined in a subcutaneous xenograft mouse model, wherein tumour growth derived from wild‐type or PRL‐knockout SW872 cells was monitored following bromocriptine treatment, n = 7. (Y and Z) Chemosensitisation effects: PRL pretreatment (50 ng/mL) enhances cytotoxicity of RG7112/abemaciclib/doxorubicin/gemcitabine, n = 5, RG7112 (10 µM), abemaciclib (5 µM), doxorubicin (2 µM), gemcitabine (10 µM). (a) Western blot analysis was performed to detect MDM2 expression in human adipocytes, liposarcoma cell lines (SW872, 93T449, 94T778), fibrosarcoma cell line HT1080 and clinically isolated liposarcoma cell lines established in our laboratory. (b) Western blot analysis was performed to detect MDM2 in 12 clinical retroperitoneal liposarcoma tissues and its corresponding paracancerous tissues, 6 clinical retroperitoneal fibrosarcoma tissues and corresponding paracancerous tissues. (c) Therapeutic synergy evaluation: bromocriptine combined with RG7112 in WEHI164 fibrosarcoma murine model, RG7112: 100 mg/kg per day, bromocriptine: 10 mg/kg, twice daily, ( n = 6). Data expressed as mean ± SD unless specified; * p < .05, ** p < .01, *** p < .001 by two‐tailed Student's t ‐test; ns: not significant.

Article Snippet: Antibodies were purchased from the following sources: PRLR (A4121; ABclonal, China), c‐MYC (A19032; ABclonal), c‐MYC (GB13076‐50; Servicebio, China), PRL (GB14138‐50; Servicebio), p‐STAT3 (AP0705; ABclonal), p‐STAT3 (AP0758; ABclonal), STAT3 (9139s; CST, USA), STAT3 (A21228; ABclonal) and GAPDH (TA08; ZSGB‐BIO, China).

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Knockdown, CCK-8 Assay, Recombinant, Expressing, Knock-Out, Isolation, Cloning, In Vivo, Derivative Assay, Western Blot, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: Oncogenic driver and therapeutic target: Prolactin signalling axis in retroperitoneal sarcoma

doi: 10.1002/ctm2.70669

Figure Lengend Snippet: Analysis of the PRL‐regulated pathway. (A) OPLS‐DA score plot distinguishing PRL‐activated (12 h) versus control transcriptional profiles, C: control, P: prolactin, n = 4. (B) Volcano plot analysis. Red denotes up‐regulated genes, blue denotes down‐regulated genes and grey denotes insignificant changed genes. (C) Heatmap: a red colour indicates a higher expression level, while blue colour indicates a lower expression level. (D) KEGG pathway enrichment. (E) Regulatory network mapping PRL‐associated genes (circles) to tumour‐related pathways (squares). (F–L) Tissue microarray validation: IHC staining was performed on RLPS tissue and adipose tissue microarray using c‐MYC antibodies. Positive cell%, histochemistry SCORE and IRS are calculated. Adipose tissue: n = 30; retroperitoneum well‐differentiated liposarcoma (RWDLPS): n = 20; retroperitoneum dedifferentiated liposarcoma (RDDLPS): n = 50. T: sarcoma tissue, N: adipose tissue. (M and N) Clinical correlation: pre‐operative serum up‐regulates c‐MYC versus post‐operative serum in SW872. (O) Rescue experiment: PRL‐induced c‐MYC up‐regulation blocked by PRLR knockdown. (P) Western blot analysis of key proteins in the JAK–STAT signalling pathway were detected following down‐regulation of PRLR and addition of recombinant PRL (50 ng/mL) in SW872 cells. (Q) qPCR was performed to assess mRNA expression levels of PRL, STAT5A, STAT5B and MYC in SW872 cells following treatment with the cAMP agonist bucladesine or the STAT5 inhibitor pimozide, alone or in combination. Bucladesine treatment reversed pimozide‐induced suppression of MYC expression, Buc: bucladesine, Pi: pimozide, n = 3. (R) Combined treatment: PRL reverses c‐MYC inhibitor (APTO‐253, 10058‐F4) mediated c‐MYC induction. (S) Therapeutic synergy: cell survival rates were measured after individual or combined treatment with the MDM2 inhibitor RG7112, PRL recombinant protein and c‐MYC inhibitors (APTO‐253, 10058‐F4), n = 5. Data expressed as mean ± SD unless specified; ** p < .01, *** p < .001 by two‐tailed Student's t ‐test; ns: not significant.

Article Snippet: Antibodies were purchased from the following sources: PRLR (A4121; ABclonal, China), c‐MYC (A19032; ABclonal), c‐MYC (GB13076‐50; Servicebio, China), PRL (GB14138‐50; Servicebio), p‐STAT3 (AP0705; ABclonal), p‐STAT3 (AP0758; ABclonal), STAT3 (9139s; CST, USA), STAT3 (A21228; ABclonal) and GAPDH (TA08; ZSGB‐BIO, China).

Techniques: Control, Expressing, Microarray, Biomarker Discovery, Immunohistochemistry, Knockdown, Western Blot, Recombinant, Two Tailed Test

Journal: Clinical and Translational Medicine

Article Title: Oncogenic driver and therapeutic target: Prolactin signalling axis in retroperitoneal sarcoma

doi: 10.1002/ctm2.70669

Figure Lengend Snippet: Proposed mechanistic model of PRL biosynthesis and oncogenic signalling in retroperitoneal sarcoma. Schematic representation delineates the transcriptional induction of PRL mRNA by SOX4 in retroperitoneal sarcoma cells. The PRL synthesised and secreted by these cells, enabling autocrine/paracrine binding to membrane‐associated PRLR. This ligand–receptor interaction triggers JAK–STAT phosphorylation cascade and up‐regulate c‐MYC proto‐oncogene, ultimately promoting the malignant proliferation of retroperitoneal sarcoma cells. Additionally, the PRL produced by these cells can enter the systemic circulation, potentially leading to widespread physiological effects.

Article Snippet: Antibodies were purchased from the following sources: PRLR (A4121; ABclonal, China), c‐MYC (A19032; ABclonal), c‐MYC (GB13076‐50; Servicebio, China), PRL (GB14138‐50; Servicebio), p‐STAT3 (AP0705; ABclonal), p‐STAT3 (AP0758; ABclonal), STAT3 (9139s; CST, USA), STAT3 (A21228; ABclonal) and GAPDH (TA08; ZSGB‐BIO, China).

Techniques: Binding Assay, Membrane, Phospho-proteomics, Produced