Prognostic and therapeutic value of the Hippo pathway, RABL6A, and p53-MDM2 axes in sarcomas

Abstract

Additional prognostic and therapeutic biomarkers effective across different histological types of sarcoma are needed. Herein we evaluate expression of TAZ and YAP, the p53-MDM2 axis, and RABL6A, a novel oncoprotein with potential ties to both pathways, in sarcomas of different histological types. Immunohistochemical staining of a tissue microarray including 163 sarcomas and correlation with clinical data showed that elevated YAP and TAZ independently predict worse overall and progression-free survival, respectively. In the absence of p53 expression, combined TAZ and YAP expression adversely affect overall, progression free, and metastasis free survival more than TAZ or YAP activation alone. RABL6A independently predicted shorter time to metastasis and was positively correlated with p53, MDM2 and YAP expression, supporting a possible functional relationship between the biomarkers. Network analysis further showed that TAZ is positively correlated with MDM2 expression. The data implicate all five proteins as clinically relevant downstream players in the Hippo pathway. Finally, a novel inhibitor of MDM2 (MA242), effectively suppressed the survival of sarcoma cell lines from different histological types regardless of p53 status. These findings suggest both independent and cooperative roles for all five biomarkers across different histological types of sarcoma in predicting patient outcomes and potentially guiding future therapeutic approaches.

Keywords: RABL6A; TAZ; YAP; p53-MDM2; sarcoma.

Figure 1. YAP, TAZ, p53, MDM2 and RABL6A are widely expressed in various sarcomas.

(A) Median H-scores for YAP expression across histological types in descending order of expression. Histological section of an undifferentiated spindle cell sarcoma with YAP nuclear expression by immunohistochemistry (IHC). (B) Median H-scores for TAZ expression arranged in similar order to (A); histological section of an undifferentiated pleomorphic sarcoma with TAZ nuclear expression by IHC. (C) Median H-scores for p53 expression in sarcomas. Histological section of an undifferentiated spindle cell sarcoma with nuclear p53 expression by IHC. (D) Median H-scores for MDM2 expression in sarcomas. Histological section of dedifferentiated liposarcoma with MDM2 nuclear expression by IHC. (E) Median H-scores for RABL6A expression in sarcomas. Histological section of pleomorphic liposarcoma with RABL6A cytoplasmic localization. ES = epithelioid sarcoma; SS = synovial sarcoma; MERT = malignant extrarenal rhabdoid tumor; M/RC LPS = myxoid/round cell liposarcoma; U-LMS = uterine leiomyosarcoma; AS = angiosarcoma; HGOS = high grade osteosarcoma; ST-LMS = soft tissue leiomyosarcoma; DDLPS = dedifferentiated liposarcoma; PLPS = pleomorphic liposarcoma; US = undifferentiated sarcoma (undifferentiated pleomorphic sarcoma/undifferentiated spindle cell sarcoma); MPNST = malignant peripheral nerve sheath tumor; WDLPS = well-differentiated liposarcoma; AVR/AR/SR=alveolar rhabdomyosarcoma, adult-type rhabdomyosarcoma, sclerosing/spindle cell rhabdomyosarcoma; MFS = myxofibrosarcoma; CS = chondrosarcoma; EWS = Ewing sarcoma; CCS = clear cell sarcoma of soft parts.

Figure 2. YAP, TAZ, p53, and RABL6A expression are associated with higher histological grade.

Unadjusted logistic regression showed (A) YAP, (B) TAZ, (C) p53, and (D) RABL6A expression are associated with higher histological sarcoma grade. Grade 1 = low grade, Grade 2 = intermediate grade, Grade 2.5 = intermediate to high grade, and Grade 3 = high grade. Box plots demonstrate the sample median, interquartile range, and outliers if present.

Figure 3. YAP and TAZ are associated with overall and progression free survival across histological types of sarcoma.

(A) Separate univariate analyses for each biomarker showed YAP and TAZ expression are associated with poorer overall survival. (B) A multivariate model including all five biomarkers showed YAP is independently associated with overall survival. (C) Kaplan-Meier curves and log-rank rest showed combined high TAZ and YAP expression is associated with poorer overall survival in sarcomas lacking p53 expression. (D) Separate univariate analysis for each biomarker demonstrated TAZ and YAP are associated with poorer progression free survival. (E) A multivariate model including all five biomarkers showed TAZ is independently associated with progression free survival. (F) Kaplan-Meier curves and log-rank test showed combined high TAZ and YAP expression is associated with poorer progression free survival in sarcomas lacking p53 expression.

Figure 4. RABL6A and the Hippo pathway axis predict metastasis free survival.

(A) Separate analyses for each biomarker show that RABL6A and TAZ are associated with a shorter time to metastasis. MDM2 trends towards statistical significance (p = 0.0530). (B) A model including all five biomarkers showed that RABL6A is independently associated with poorer metastasis free survival. (C) Kaplan-Meier curves and log-rank test showed combined high TAZ and YAP expression is associated with a worse metastasis free survival in sarcomas lacking p53 expression. (D) Kaplan-Meier curves and log-rank test show RABL6A is associated with poorer overall survival when evaluating the upper and low tertiles.

Figure 5. Network analysis supported a model integrating the Hippo pathway, p53/MDM2 axis and RABL6A signaling in sarcomas.

(A) Correlation-based network analysis showed statistically significant, positive correlations between RABL6A and p53, MDM2, and YAP. In addition, a statistically significant positive correlation was identified between TAZ and MDM2. (B) Model diagram depicting the relationship between the five biomarkers integrating the network analysis and previously identified functions of the proteins, with RABL6A serving as a potential link between YAP and TAZ and integrator of the p53-MDM2 axis.

Figure 6. MDM2 can be targeted sarcoma cell lines independent of p53 status.

(A) Expression of RABL6A, MDM2, YAP, TAZ, and p53 in sarcoma cell lines. (B) Cells were treated for 3 days with MA242 and analyzed with AlamarBlue (MTT-style proliferation assay); MA242 similarly inhibits proliferation in p53 wild-type and p53 null sarcoma cell lines. (C) Sarcoma cell lines most sensitive (drug response curves—black) and least sensitive (drug response curves—blue) to MA242 treatment. (D) Positive correlation of IC50 with MDM2 expression trends towards statistical significance.