The role of BiP and the IRE1α-XBP1 axis in rhabdomyosarcoma pathology

Background: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children, and is associated with a poor prognosis in patients presenting with recurrent or metastatic disease. The unfolded protein response (UPR) plays pivotal roles in tumor development and resistance to therapy, including RMS.

Methods: In this study, we used immunohistochemistry and a tissue microarray (TMA) on human RMS and normal skeletal muscle to evaluate the expression of key UPR proteins (GRP78/BiP, IRE1α and cytosolic/nuclear XBP1 (spliced XBP1-sXBP1)) in the four main RMS subtypes: alveolar (ARMS), embryonal (ERMS), pleomorphic (PRMS) and sclerosing/spindle cell (SRMS) RMS. We also investigated the correlation of these proteins with the risk of RMS and several clinicopathological indices, such as lymph node involvement, distant metastasis, tumor stage and tumor scores.

Results: Our results revealed that the expression of BiP, sXBP1, and IRE1α, but not cytosolic XBP1, are significantly associated with RMS (BiP and sXBP1 p-value = 0.0001, IRE1 p-value = 0.001) in all of the studied types of RMS tumors (n = 192) compared to normal skeletal muscle tissues (n = 16). In addition, significant correlations of BiP with the lymph node score (p = 0.05), and of IRE1α (p value = 0.004), cytosolic XBP1 (p = 0.001) and sXBP1 (p value = 0.001) with the stage score were observed. At the subtype level, BiP and sXBP1 expression were significantly associated with all subtypes of RMS, whereas IRE1α was associated with ARMS, PRMS and ERMS, and cytosolic XBP1 expression was associated with ARMS and SRMS. Importantly, the expression levels of IRE1α and sXBP1 were more pronounced in ARMS than in any of the other subtypes. The results also showed correlations of BiP with the lymph node score in ARMS (p value = 0.05), and of sXBP1 with the tumor score in PRMS (p value = 0.002).

Conclusions: In summary, this study demonstrates that the overall UPR is upregulated and, more specifically, that the IRE1/sXBP1 axis is active in RMS. The subtype and stage-specific dependency on the UPR machinery in RMS may open new avenues for the development of novel targeted therapeutic strategies and the identification of specific tumor markers in this rare but deadly childhood and young-adult disease.

Methods
2.1. Tissue Microarray (TMA)

The tissue microarray (TMA) analysis was conducted on rhabdomyosarcoma (RMS) and normal (control) striated muscle samples. A rhabdomyosarcoma tissue array kit containing 104 cases and 208 cores was utilized, purchased from US Biomax, Inc. (Catalog no. SO2082a; RKV, MD, USA). This array provides detailed information on tumor characteristics, including TNM (tumor, node, metastasis) staging, clinical stages, and pathology grades, ensuring comprehensive clinical and pathological data for each sample.

The TMA includes cores from four subtypes of RMS:

15 cases of spindle cell/sclerosing RMS (SRMS)

27 cases of embryonal RMS (ERMS)

30 cases of pleomorphic RMS (PRMS)

24 cases of alveolar RMS (ARMS)

Additionally, it includes 8 normal skeletal muscle tissue cores as controls, with all samples processed in duplicate. Each of the 208 tissue cores is embedded on a single slide, with two cores derived from each case or control. Detailed histopathological characteristics for all samples are presented in Supplementary Table S1.

2.2. Immunohistochemistry (IHC)

TMA slides (5-μm thick) were subjected to immunohistochemistry (IHC) analysis to evaluate protein expression profiles. Slides were first deparaffinized by incubation at 60°C for 30 minutes and subsequently rehydrated using xylene and a graded alcohol series. Heat-mediated antigen retrieval was performed using a 0.01 M sodium citrate buffer (pH 6.0), following previously described protocols.

To minimize non-specific background staining, slides were washed with phosphate-buffered saline (PBS) and blocked with a blocking solution (composition: 1.5 mL maleic acid buffer, 0.5 mL FBS, 0.5 mL stock blocking solution, 50 μL 10% Tween-20, and 2.5 mL PBS) at room temperature for 30 minutes. Endogenous peroxidase activity was quenched by incubation in freshly prepared 3% hydrogen peroxide (H₂O₂). Slides were then incubated sequentially with Avidin blocking solution (15 minutes; Vector SP-2001) and Biotin blocking solution (15 minutes; Vector WP-2001).

The following primary antibodies were used for IHC analysis:

Mouse monoclonal antibody (mAb) against IRE1 (1:100; Abcam, cat. no. ab96481)

Rabbit monoclonal antibody against BiP (1:200; Cell Signaling, cat. no. 3177)

Rabbit polyclonal antibody against XBP1 (1:200; Abcam, cat. no. ab37152)

The ab37152 antibody against XBP1 is validated for the detection of both cytosolic (unspliced) and nuclear (spliced) isoforms of XBP1, allowing for a comprehensive analysis of its localization and functional activity.

Slides were incubated with these primary antibodies overnight at 4°C. Following thorough PBS washes, slides were incubated with biotinylated secondary antibodies specific to each primary antibody for 1 hour at room temperature. After additional PBS washes, slides were treated with horseradish peroxidase-labeled streptavidin (1:200 dilution) for 30 minutes at room temperature.

Signal detection was performed using 3,3′-diaminobenzidine (DAB) substrate for 2 minutes at room temperature. Slides were then counterstained with Mayer’s Hematoxylin (10 drops in 1.25 mL PBS; Vector H-3404) for 1–4 minutes. Negative controls, processed without primary antibodies, were included to ensure specificity of staining.

2.3. Tissue Microarray Scoring

IHC results were independently evaluated by three pathologists in a blinded manner. Scoring was based on the intensity of staining, categorized as follows:

None (N)

Weak (W)

Moderate (M)

Strong (S)

For XBP1, both cytosolic and nuclear staining were assessed, ensuring a detailed analysis of its functional isoforms and their differential localization.

2.4. Statistical Analysis

Statistical analysis was performed using SPSS software (version 16.0; SPSS, Inc., Chicago, IL, USA). Data are expressed as numbers (n) and percentages (%). Group comparisons were conducted using a chi-square test or Fisher’s exact test, as appropriate. All p-values are presented as two-tailed, with a significance threshold of p < 0.05.