**2.4 Homologous recombination (HR) pathway**

## *2.4.1 BRCA1/BRCA2*

HR is the major DDR mechanism responsible for repairing double-strand DNA breaks (DBSs) [69]. HR repairs DSBs in an error-free manner by using homologous sequence of sister chromatid as an undamaged template [15]. BRCA1, BRCA2 and RAD51 are the key factors involved in this DDR pathway which have shown to be dysregulated in various types of cancers [15, 77–79]. Inherited mutations of the BRCA genes predispose individuals to develop tumors in various organs including breast and ovary [80]. Moreover, the chance of developing cancer significantly increases by acquiring BRCA mutations, and these mutations are commonly seen in patients with breast and ovarian cancers [80]. However, it has been reported that BRCA mutation has potential for inducing synthetic lethality in the cancer cells [81]. PARP inhibition in BRCA-mutated cancer cells (HR-deficient) induces synthetic lethality and cell death and provides a promising opportunity to eliminate cancerous cells (**Figure 2**). Several PARP inhibitors have been approved as monotherapies in HR-deficient ovarian and metastatic breast cancers [81].

As we discussed earlier in this chapter (Section 3.1.2), studies on osteosarcoma demonstrated that BRCA is frequently mutated in osteosarcoma and PARP inhibition either as a monotherapy or in combination with chemotherapy could induce significant anti-tumor effects in BRCA-mutated osteosarcoma cells [48, 82]. Although the significance of BRCA mutation status as a prognostic factor in sarcoma has been reported in a numerous studies [83–85], more clinical trials are warranted to determine the efficacy of PARP inhibitors in BRCA-mutated sarcomas. The importance of BRCA status in sensitivity to the chemotherapeutic drug trabectedin in STS is emphasized in a review paper gathered by Monk et al., and presented that BRCA mutations are significantly associated with favorable clinical response to trabectedin [83]. The frequency of BRCA mutation in soft-tissue sarcoma though, has not found to be significantly high in a study conducted by Seligson et al. [86]. They performed DNA sequencing analysis on 1236 STS patients as well as an additional 1312 leiomyosarcoma patients [86]. The unselected STS analysis revealed that only 1% of patients had BRCA2 mutation [86]. However, subset analysis showed that BRCA2 mutation could be found in 10% of leiomyosarcoma patients [86]. The frequency of BRCA1 mutation was not significant in either analysis [86]. Furthermore, they showed that PARP inhibition demonstrates effective clinical outcomes in BRCA2 deficient leiomyosarcoma patients [86]. Consistently, another study demonstrated a significant correlation between the overexpression of BRCA1,

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sarcoma patients.

*2.4.2 RAD51*

**Figure 2.**

*DNA Repair Defects in Sarcomas*

*DOI: http://dx.doi.org/10.5772/intechopen.94881*

BRCA2, PARP, and γH2AX and higher tumor stage, higher chances of metastasis, lower survival rates, and overall poor prognosis in STS patients [87]. These studies highlighted the significance of BRCA status in sarcoma and underscored the fact that HR mutations should be considered as predictive factors for increasing the

*Schematic role of PARP and PARP inhibition in synthetic lethality. (A) After binding to damaged DNA, PARP undergoes conformational change and poly(ADP-ribosyl)ation which results in recruitment of other DNA Damage Response (DDR) proteins, like DNA ligase 3 (Lig3), DNA polymerase β (polβ) and X-ray repair cross-complementing protein 1 (XRCC1), leading to DNA repair and cell survival. (B) PARP inhibitors block PARP activity, leading to double-strand break (DSB). The cells that have normal homologous recombination (HR) pathway are able to repair the DSBs in a error-free manner, leading to cell survival. However, BRCA-mutated cancer cells (HR-deficient) are unable to efficiently repair DSBs which ultimately results in cell death.*

RAD51 is another key protein in the HR pathway that is also associated with prognosis and treatment response in various cancers. A growing number of studies demonstrate that RAD51 protein is overexpressed in many cancers including breast, prostate, bladder, pancreas, and lung, and this overexpression can up-regulate HR activity and result in resistance to DNA-damaging drugs [88–91]. Increased expression of RAD51 has also been reported in sarcoma patients [92, 93]. Du et al. conducted a study to explore the relationship between RAD51 expression and resistance to radio- or chemotherapy in osteosarcoma [93]. They suppressed the expression of RAD51 using shRNA and found increased sensitivity to chemotherapy and radiation in osteosarcoma cell lines through induction of cell cycle arrest and apoptosis [93]. Hannay et al., investigated the association between RAD51 expression and resistance to chemotherapy in STS patients [92]. They evaluated the RAD51 expression in 62 human primary recurrent and metastatic STS samples [92]. Only 3 tumor samples showed no RAD51 expression, while most of them had overexpressed RAD51 expression levels [92]. They showed that siRNA-mediated RAD51 targeting resulted in STS sensitivity to doxorubicin [92]. Overall, these studies highlighted the significance of RAD51 in chemoresistance and suggested that RAD51 could be considered as a prognostic factor or even a therapeutic target for treatment of

overall survival of patients by choosing the best treatment strategy.

#### **Figure 2.**

*DNA - Damages and Repair Mechanisms*

and possible therapeutic targets in sarcoma.

*2.4.1 BRCA1/BRCA2*

**2.4 Homologous recombination (HR) pathway**

CSE1L-knockdown osteosarcoma cells [75]. The results showed that overexpression of MSH6 significantly increased cell proliferation rate and reversed the anti-tumor effects observed in CSE1L-knockdown cells, indicating that CSE1L activity is dependent on MSH6 expression [75]. Moreover, down-regulation of MSH6 resulted in suppression of cell growth in both *in vitro* and *in vivo* experiments [75]. The prognostic potential of MSH6 and CSE1L was also explored by evaluation of the MSH6 and CSE1L expression levels in tumor samples [75]. They found a significant correlation between the expression of these two proteins and overall poor prognosis in osteosarcoma patients [75]. Similarly, another study on osteosarcoma patients showed that overexpression of MSH2 and MSH6 is significantly associated with shorter survival time, lower sensitivity to chemotherapy, and higher chances of metastasis [76]. These studies underscored the significance of MMR proteins as both prognostic biomarkers

HR is the major DDR mechanism responsible for repairing double-strand DNA breaks (DBSs) [69]. HR repairs DSBs in an error-free manner by using homologous sequence of sister chromatid as an undamaged template [15]. BRCA1, BRCA2 and RAD51 are the key factors involved in this DDR pathway which have shown to be dysregulated in various types of cancers [15, 77–79]. Inherited mutations of the BRCA genes predispose individuals to develop tumors in various organs including breast and ovary [80]. Moreover, the chance of developing cancer significantly increases by acquiring BRCA mutations, and these mutations are commonly seen in patients with breast and ovarian cancers [80]. However, it has been reported that BRCA mutation has potential for inducing synthetic lethality in the cancer cells [81]. PARP inhibition in BRCA-mutated cancer cells (HR-deficient) induces synthetic lethality and cell death and provides a promising opportunity to eliminate cancerous cells (**Figure 2**). Several PARP inhibitors have been approved as mono-

therapies in HR-deficient ovarian and metastatic breast cancers [81].

As we discussed earlier in this chapter (Section 3.1.2), studies on osteosarcoma demonstrated that BRCA is frequently mutated in osteosarcoma and PARP inhibition either as a monotherapy or in combination with chemotherapy could induce significant anti-tumor effects in BRCA-mutated osteosarcoma cells [48, 82]. Although the significance of BRCA mutation status as a prognostic factor in sarcoma has been reported in a numerous studies [83–85], more clinical trials are warranted to determine the efficacy of PARP inhibitors in BRCA-mutated sarcomas. The importance of BRCA status in sensitivity to the chemotherapeutic drug trabectedin in STS is emphasized in a review paper gathered by Monk et al., and presented that BRCA mutations are significantly associated with favorable clinical response to trabectedin [83]. The frequency of BRCA mutation in soft-tissue sarcoma though, has not found to be significantly high in a study conducted by Seligson et al. [86]. They performed DNA sequencing analysis on 1236 STS patients as well as an additional 1312 leiomyosarcoma patients [86]. The unselected STS analysis revealed that only 1% of patients had BRCA2 mutation [86]. However, subset analysis showed that BRCA2 mutation could be found in 10% of leiomyosarcoma patients [86]. The frequency of BRCA1 mutation was not significant in either analysis [86]. Furthermore, they showed that PARP inhibition demonstrates effective clinical outcomes in BRCA2 deficient leiomyosarcoma patients [86]. Consistently, another study demonstrated a significant correlation between the overexpression of BRCA1,

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*Schematic role of PARP and PARP inhibition in synthetic lethality. (A) After binding to damaged DNA, PARP undergoes conformational change and poly(ADP-ribosyl)ation which results in recruitment of other DNA Damage Response (DDR) proteins, like DNA ligase 3 (Lig3), DNA polymerase β (polβ) and X-ray repair cross-complementing protein 1 (XRCC1), leading to DNA repair and cell survival. (B) PARP inhibitors block PARP activity, leading to double-strand break (DSB). The cells that have normal homologous recombination (HR) pathway are able to repair the DSBs in a error-free manner, leading to cell survival. However, BRCA-mutated cancer cells (HR-deficient) are unable to efficiently repair DSBs which ultimately results in cell death.*

BRCA2, PARP, and γH2AX and higher tumor stage, higher chances of metastasis, lower survival rates, and overall poor prognosis in STS patients [87]. These studies highlighted the significance of BRCA status in sarcoma and underscored the fact that HR mutations should be considered as predictive factors for increasing the overall survival of patients by choosing the best treatment strategy.
