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Page 6 of 16 Karolak et al. J Cancer Metastasis Treat 2021;7:15 https://dx.doi.org/10.20517/2394-4722.2021.05

[29]
sarcoma subtypes, such as LMS, ES, LS, RMS and MFH . In a study conducted on 17 paediatric RMS and
extraosseous Ewing sarcoma (EES) tumour samples, elevated EZH2 protein levels were associated with
increased aggressiveness of the disease and the presence of metastasis to lymph nodes and/or distant parts
[31]
of the body at the time of diagnosis .

Survival
Higher EZH2 expression in 14 patients with SS was significantly associated with worse survival. However,
the same was not found to be significant in 31 liposarcoma and 36 MFH patients . In a study conducted on
[29]
105 sarcoma tumour samples from patients with no prior adjuvant therapy (28 RMS, 15 Ewing’s Sarcoma,
30 osteosarcoma, 18 SS, 14 epithelioid sarcoma and chondrosarcoma), higher EZH2 expression and higher
[32]
H3K27me3 levels were associated with significantly shortened overall survival . However, the inclusion of
tumour types other than STS means that this may therefore not represent the true OS for these types of
malignancy. Expression of EZH2 was also a marker of lower likelihood of survival in paediatric samples of
[31]
11 RMS and 6 EES tumours, compared to the EZH2-negative cases . Furthermore, 14 out of 29 SS patients
with lower EZH2 levels, had a significantly prolonged overall survival than those of higher EZH2 expression
[30]
profiles .

Histological grade and clinical stage
In a study using 104 patient-derived tumour samples, it was observed that higher EZH2 expression was
associated with higher histological grade of the tumour . This is inconsistent with another study, in which
[29]
[30]
EZH2 high expression did not show significant correlation with respect to histological grade . This may be
due to the smaller sample size (29 SS cases). In another paper, consisting of 50 patients-derived SS tumour
samples, those with II and III clinical stages had significantly higher EZH2 scores by immunohistochemistry
(IHC) than those with stage I with no EZH2 expression found in one case with stage IV . In 17 patients
[3]
with RMS and EES, high intra-tumour EZH2 scores were present in stage III or IV of the malignancy .
[31]
These inequalities may emerge due to the limited sample sizes, so further investigations are needed.

EZH2 and Ki-67
In SS EZH2 expression was correlated with Ki-67 scores, especially in poorly differentiated subtypes .
[28]
Abundant expression of Ki-67 with strong positive correlation to EZH2 scores was also observed in other
types of STS, such as LMS, alveolar and embryonal RMS , LS, ES and MFH . These clearly indicated the
[33]
[29]
positive association between EZH2 expression and proliferative potential in soft tissue malignancies. In
another study including various subtypes of RMS, such as ARMS, ERMS, unclassified, spindle cell and
pleomorphic RMS, the Ki-67 and EZH2 were also parallel in all primary and recurrent RMS tumour
[34]
samples .
TARGETING EZH2 IN STS
EZH2 modulation through RNA interference
Rhabdomyosarcoma
Knockdown of EZH2 through vector-based shRNAs in the embryonal RMS cell line RD resulted in
induction of differentiation due to the increase of muscle specific factors, restored recruitment of
multiprotein complexes at muscle-specific promoters and activated expression of MyoD, as well as its
binding abilities, crucial for myogenesis . Another study showed that depletion of EZH2 with siRNAs in
[1]
the RD cell line led to inhibition of cell proliferation and significant decrease of H3K27me3. These were
followed by the expression of proteins specific to terminal myogenic differentiation [35,36] , indicating that the
ablation of EZH2 restores myogenic differentiation ability of embryonal RMS cells. Alveolar RMS cell lines
(RH30 and RH4, containing PAX3-FOXO1 fusion gene), which underwent EZH2 interference through
siRNA or shRNA manifested inhibition of cellular proliferation, migration, anchorage-independent growth