The repair gene BACH1 - a potential oncogene

Abstract

BACH1 encodes for a protein that belongs to RecQ DEAH helicase family and interacts with the BRCT repeats of BRCA1. The N-terminus of BACH1 functions in DNA metabolism as DNA-dependent ATPase and helicase. The C-terminus consists of BRCT domain, which interacts with BRCA1 and this interaction is one of the major regulator of BACH1 function. BACH1 plays important roles both in phosphorylated as well as dephosphorylated state and functions in coordination with multiple signaling molecules. The active helicase property of BACH1 is maintained by its dephosphorylated state. Imbalance between these two states enhances the development and progression of the diseased condition. Currently BACH1 is known as a tumor suppressor gene based on the presence of its clinically relevant mutations in different cancers. Through this review we have justified it to be named as an oncogene. In this review, we have explained the mechanism of how BACH1 in collaboration with BRCA1 or independently regulates various pathways like cell cycle progression, DNA replication during both normal and stressed situation, recombination and repair of damaged DNA, chromatin remodeling and epigenetic modifications. Mutation and overexpression of BACH1 are significantly found in different cancer types. This review enlists the molecular players which interact with BACH1 to regulate DNA metabolic functions, thereby revealing its potential for cancer therapeutics. We have identified the most mutated functional domain of BACH1, the hot spot for tumorigenesis, justifying it as a target molecule in different cancer types for therapeutics. BACH1 has high potentials of transforming a normal cell into a tumor cell if compromised under certain circumstances. Thus, through this review, we justify BACH1 as an oncogene along with the existing role of being a tumor suppressant.

Introduction to BACH1

BACH1/BRIP1 /FANCJ/hCHLR1 (BRCA1 associated C-terminal helicase 1), which is the homolog of yeast Chl1p helicase, is a phosphoprotein located on chromosome 17q22.^1-3 It consists of 1249 amino acid residues with the protein size of 130 KDa, the gene length of 180kb and contains 20 exons (Figure 1).^1,4,5 BACH1 helicase is present in both active and inactive forms depending on the phosphorylation status at the K52 position of the protein. The dephosphorylated BACH1 leads to the activation of helicase, which is involved in the timely progression of S-phase, repair of DNA cross-links and secondary structures formed during replication and replication induced stress.^6,7 Thus phosphorylateddephosphorylated state of BACH1 plays a major role in cell cycle regulation through activation of various pathways in BRCA1 dependent and independent manner.⁴ During replication stress, it acts with DNA topoisomerase-2-binding protein TOPBP1 to load replication protein A (RPA) onto the chromatin. Presence of RPA is required for activation and control of replication checkpoints and to undergo repair by homologous recombination.^8,9 In the case of management of DNA damage responses like interstrand crosslinks (ICLs), the helicase activity of BACH1 and its interaction with the mismatch repair protein MLH1 provides ICL resistance. ¹⁰

BACH1 also acts as a tumor suppressor in different cancer types.^7,11 It maintains chromosomal integrity and prevents genomic instability by resolving the G-quadruplexes and processing replication intermediates.^12-14 It has the ability to recognize Gquadruplexes mostly those formed upon replication and mediates their stepwise unfolding and refolding to modulate epigenetic programming and chromatin remodeling also.^12-15 BACH1 maintains as well as preserves the chromatin structure and its epigenetic information hence facilitating the smooth progression of the replication fork when it encounters altered/ damaged/ complex DNA structures (Figure 2).^11,12,14 As it is involved in regulating many vital pathways, any aberration to it can cause multifactorial diseases like cancer. BACH1/BRIP1 plays a role in hereditary breast and ovarian cancer suppression as well as instrumental in progressive bone marrow failure disorder, Fanconi anemia (FA).¹⁷ Germline mutations in the BACH1/FANCJ gene leads to chromosomal instability which results in bone marrow failure defects, developmental abnormalities and sets up favorable conditions to develop cancer.¹⁸ Clinical data analysis of BRIP1 mutations by Seal et al. indicates that majority of the BRIP1 missense mutants/variants are not linked with a risk of familial breast cancer, whereas the truncated variant of BACH1 are more susceptible alleles of breast cancer running in the family.¹⁹ The biological explanations for the differences in cancer risk for mutant variant and truncated variant are unclear. Moreover, the group identified that, biallelic BRIP1 mutations confers less risk of breast cancer compared to the monoallelic truncated version.¹⁹ Mutations in BACH1 also lead to liver carcinogenesis, among patients with viral cirrhosis, due to impaired DNA mismatch repair pathway.²⁰ In summary to the above lines, the mutated version of BACH1 gene leads to the development of oncogenicity (Figure 1).

BACH1 functionally and physically interact with a bunch of proteins, like BRCA1, MUTLα, MLH1, PMS2, MMS19, TOPBP1, TLS polymerase, BLM, RPA1, MRE11 and FANCD2 and plays a significant role in regulating the metabolic pathways in combination with them.^10,21,22 The BACH1 interactors play major and minor roles in maintenance of genomic integrity, cell cycle regulation, DNA damage detection and repair processes.¹¹ The interactors MUTLα, MLH1, PMS2 and RPA1 play important role in mismatch repair.^21,23,24 The BACH1 homolog, BRIP1 interacts with the mismatch repair heterodimer complex, MUTLα, which is composed of mismatch repair proteins MLH1 and PMS2. It also interacts with MLH1 directly independent of BRCA1. The interaction with the single-stranded DNA-binding protein RPA (Replication Protein A) through its helicase domain enhances the DNA unwinding activities at the difficult sites of replication.²⁵ The other interactors MMS19 and TOPBP1 maintains genomic integrity with FANCD2 in Fanconi anemia DNA damage repair pathway. ²⁴ The interaction of DNA helicase BACH1 with BLM, another helicase, coordinated with DNA damage signaling protein molecules, structure-specific nucleases, polymerases, RPA, and RAD51 imparts a delicate balance between homologous recombination (HR) and non-homologous end-joining (NHEJ) to repair double strand breaks (DSBs) and maintain genomic stability.²⁶

The most important event is the physical interaction of BACH1 with BRCA1 which justifies the possible role of BACH1 in cancer development.^7,11 This interaction is dependent on the phosphorylation-dephosphorylation function of BACH1, as the interaction increases in presence of phosphatase inhibitors, whereas in the presence of λ phosphatase the interaction is lost,²⁷ which proves that the phosphorylated form of BACH1 interacts with BRCA1. BRCA1 is localized to the site of DNA double strand break by forming a complex with different interacting molecules like RAP80, CTIP and FANCJ. The BRCA1-RAP80 complex comes through Abraxas ubiquitinase and follows the non-homologous end-joining at the DSBs. The parallel pathway of homologous recombination repair is followed by the BRCA1–FANCJCTIP complex. This complex is also regulated by heterochromatin binding protein 1 (HP1) pathway in response to DNA damage for its accumulation at the site of DNA double strand break which mediates DNA repair. FANCJ interacts with HP1 in a BARD1 dependent manner and mediates homologous recombination.²⁸ The association of BRCA1 with BACH1 adds on to the functioning of the G2/M checkpoint.²⁹ The BRCA/BACH1 complex prevents DNA breakage resulting in lowering of genomic instability.³⁰ BACH1 status affect the recruitment of BRCA1 to double strand breaks depending on the type of damage.³¹ Presence of change in amino acid sequence in the BRCA1 binding domain of the protein BACH1, in case of tumorigenesis, proves that recognition of BRCT phosphoprotein by BACH1 is necessary for tumor suppression activity of BRCA1.³²

Figure 1.

Schematic representation of BACH1 gene with conserved domains and reported pathogenic mutations in different cancer types. The BACH1 gene comprises of 20 exons of which exon 3&4 belongs to DNA polymerase domain (18-61aa residue), exon 5 nuclear localization signal (NLS; 158-175aa), exon7-19 HBB domain (245-881aa), exon9 DEAH box (393-396aa) and exon 19 &20 DNA helicase/BRCA1 interacting domain (888-1063aa) respectively. The large boxes represent exons and small colored thick lines represent verified pathogenic mutations in the respective exons in different cancers (data analyzed from cBioPortal.org).

BACH1 in cell cycle regulation and replication

The most fascinating characteristic of BACH1 is observed during its regulation of the cell cycle through its helicase activity.³³ Though the expression of BACH1 protein remains the same throughout the cell cycle, its association with the chromatin increases in S-phase only.⁶ The helicase activity of BACH1 is regulated by its phosphorylated-dephosphorylated state. The sequence of steps explaining the functioning of BACH1 in the cell cycle is represented in Figure 3. At G1-phase, BACH1 is phosphorylated leading to the interaction with BRCA complex with low ATPase/helicase activity. As a result, the movement of the replication complex slows down enhancing the proof reading of the polymerase. Adversely, during the slowdown of the fork, the nascent leading and lagging strands tend to anneal to each other due to fork regression or reversal to form secondary structures.³⁴ The complex of BACH1/BRCA along with the combination of BLM1, a helicase with opposite polarity, resolves these difficult structural motifs encountered by the replication forks during DNA replication. Once the proofreading and resolving activity of the secondary structures are over, the de-phosphorylation of BACH1 takes place. On dephosphorylation, the BACH1/BRCA complex breaks down, leaving behind BACH1 at the fork generating the space for the replication machinery to start replication. Simultaneously dephosphorylated BACH1 regains the helicase activity to unwind the DNA for timely progression through S-phase. The helicase and translocase activities of BACH1 are also modulated by protein-protein interactions. ¹ Replication protein A (RPA), comes with BACH1 to facilitate the removal of the DNA bound protein obstacles like the replication complex and increases the ability of BACH1 to unwind the secondary structures.^35,36 So BACH1 plays a major role in regulating the kinetics of replication in S-phase (Figure 3).⁶ BACH1 also have a role during replication stress when there is a damage to DNA or forks are blocked by blocking molecules. It resolves the blocked forks so that they can progress into S-phase and complete the duplication of the genome within a defined period of time.³⁷ BACH1 alone is unable to unwind partial duplex DNA structures formed due to double-strand breaks, when the strands are bound by DNA double-strand break interacting proteins or blocking molecules. ^8,38 Similarly, as happens during replication, the presence of RPA, stimulates BACH1/FANCJ for the displacement of the DNA interacting proteins resulting in unwinding of complex structures. ³⁹ RPA also stimulates BLM1 to efficiently dislodge protein bound to duplex DNA to alleviate replication stress imposed by stalling of the replication forks.^8,36,40 So, during the formation of the secondary structures, these helicases, BACH1 and BLM1, displace proteins bound near double-stranded ends and resolve secondary structure or damaged DNA to enable error-free and kinetically efficient end-joining leading to the restoration of DNA replication. ²⁵

Figure 2.

BACH1- a multifaceted protein. The schematic representation depicts the multi-functional role of BACH1. The inner circle (white) shows BACH1 and its interacting partners. The first ring represents various functions of BACH1 (half green ring) and altered function of aberrant BACH1 (half red ring). The second ring (multicolor) represents the group of functions and its correlation with different pathways where red represents DNA damage, blue represents mutation burden, yellow represents DNA damage repair and grey represents BACH1 expression. The third ring (white) represents the various pathways and its alterations, arrow represents ↑ increase, ↓ decrease and ↕ stable functional effects. The outermost colored ring represents the collective outcome of normal and aberrant BACH1 function in cancer formation. Left outermost arc represent various cancers with significant BACH1 aberrations.

In conclusion, genomic integrity is maintained by the helicase and the phosphorylation property of BACH1. During diseased state with perturbed BRCA-BACH interaction due to dephosphorylation of BACH1, genomic instability develops by increased helicase activity and faster progression through S-phase. Accelerated S-phase leads to error-prone resolving of the secondary structures at the forks leading to genomic instability. BACH1 interacts with BRCA1, through the BRCT domain and contributes to the DNA repair function of BRCA1.⁴¹ Loss in interaction also happens due to mutation in the interacting domains of the genes and so brings in less repair and helicase molecules at the damaged sites. As a result, the secondary structures form during the replication fail to resolve, resulting in more breaks and more damage to the DNA strands. High burden of damage leads to the activation of alternate repair pathways other than homologous recombination repair which results in accumulation of mutations followed by the development of cancer.^42,43

BACH1 in DNA repair

The repair function of BACH1 along with its helicase activity maintains the genomic integrity. Unlike dephosphorylation of BACH1 for the helicase activity, the repair property is regulated through its acetylation. This acetylation of BACH1 dependent on the BRCA1-BACH1 interaction.⁴⁴ The BRCA1-BACH1 interaction ensures to suppress the mutation prone end-joining and promote double-strand DNA repair via the activation of homologous recombination.^1,45,46 Phosphorylated BACH1, interacts with BRCA1 and activates the G2/M checkpoint, results in stalled replication forks, which may signal for delayed entry into S phase which is shown in Figure 3.^6,27,29 On the other hand, delay in the S-phase leads to increased secondary structures and breaks resulting to activation of the DNA damage checkpoint.^6,47,48 This fine balance of BRCA1 interaction with phospho-BACH1 promotes a damage-free S-phase progression by activating the G2/M and damage checkpoints and ensuring error free HR repair mechanism.^44,45 Both BRCA1 and BACH1 are recruited to the site of damage depending on the type of damage.^9,11,31 Recruitment of BRCA1 to laser-induced DSBs or Psoralen (Pso) Interstrand Crosslink (ICLs) is dependent on BACH1 whereas the recruitment is independent when the damage is caused by exposure to IR. Also, the recruitment of BACH1 at the damage sites is dependent on the interactor proteins to the site of damage. For laser-induced DSBs but not Psoralen (Pso)-Interstrand Crosslink (ICLs), DNA double strand break repair proteins, MRE11 and its associated nuclease activity function with or in parallel to BRCA1 for efficient BACH1 recruitment at the sites of damage.³¹ In absence of MRE11 exonuclease, loading of another BRCA1 interactor, CTIP, to DSBs is also delayed. CTIP is another protein associated with BRCA1 and modulates BRCA1s functions in DNA repair and/or cell cycle checkpoint control.⁴⁹ BRCA1 deficient cells also leads to less localization of CTIP at damage sites. This indicates that at laser induced damage sites, FANCJ/ BACH1 join hands with CTIP to remove secondary structures and helps CTIP to efficiently repair DNA ends with the help of repair protein MRE11 after interacting with BRCA1.^50,51 At the site of Pso-ICLs, BACH1 is also localized with the help of another mismatch repair (MMR) protein MLH1.^10,33 In case of UV light induced DNA crosslink, both MLH1 and upstream MMR protein MSH2 along with BACH1 is required, hence preventing aberrant DNA damage response.⁵² So, BACH1 helps in the repair of damaged DNA through homologous recombination. In any of the situations like, absence of BACH1, mutation in the BACH1 or loss of BACH1-BRCA1 interaction, the repair of damage through HR is perturbed. As a result, the alternate errorprone repair pathways like non-homologous end-joining (NHEJ) gets activated. The error-prone repair leads to mutations, which promotes the development, progression, recurrence and metastasis of cancer.

Figure 3.

Mechanism of action of BACH1 through its dual states. At G1-phase of the cell cycle, phosphorylated BACH1 interacts with BRCA - complex to form the BRCA-BACH complex. This complex have low ATPase/helicase activity which results in slowdown in the S-phase progression enhancing the proof reading of the polymerase. The complex of BACH1/BRCA supports in resolving difficult structural motifs (right side of the figure). With de-phosphorylation or defect in phosphorylation, the BACH1/BRCA complex breaks down, leaving behind only BACH1 at the fork generating the space for the replication machinery to start replication. Dephosphorylated BACH1 regains the helicase activity to unwind the DNA for timely progression through S-phase (left side). Active helicase represented in dark green color, Inactive helicase-light green color and BRCT domain- blue color respectively. Arrow represents ↑ increase; ↓ decrease and ↕ stable functional effects.

BACH1 in chromatin remodeling

Chromatin remodeling is another important event, which takes place in and around the replication complex or the double-strand DNA breaks aiding in genomic stability, unperturbed replication and DNA repair.^53,54 The proteins involved in maintaining genomic integrity bind to replication forks and damaged sites through highly organized signaling pathways.^54,55 BACH1 belongs to the group of human XPD-like helicases, which include XPD, RTEL1 and CHLR1, which have a role in chromatin remodeling as well as repair and regulates replication at difficult sites.⁵⁶ Structural and biochemical studies have proved that the XPD like helicases have an affinity towards single stranded DNA and forked DNA and plays a vital role in their arrangements.^57-59 BACH1 has direct interaction with BRCA1 through its BRCT domain, and also to the DNA through histone H3. This justifies that these proteins may have a role in the remodeling of chromatin along with repair, that takes place in and around the region of DNA damage.⁶⁰ Among the XPD like helicases, BACH1 possesses a G-quadruplex specific recognition site.¹³ The G- quadruplexes are proven as epigenetic modulators and chromatin remodelers.¹⁵ The affinity between G4s and BACH1/FANCJ helicase strongly justify the involvement of FANCJ in chromatin remodeling through G4s.^12,13,15 FANCJ mostly recognizes the replication linked transient G4s which plays role in CpG island methylation maintenance as well as de novo CpG methylation control.¹⁵ FANCJ binds to the G4s through G4-binding peptide sequence, RHAU18 which unwinds the branched DNA structures by repeated rounds of stepwise G4-unfolding and refolding. ¹³ It specifically binds to the 5’ flaps and D-loops facilitating the fork movement through replication barriers and helps in processing of the replication intermediates. This results in suppression of the heterochromatin spreading and proper maintenance of chromatin structure.^13,14 BACH1 also coordinates the functioning of polymerase REV1 and helicases WRN/BLM of opposite polarity near G4 DNA motifs to maintain epigenetic stability.¹² The BACH1 sequence is significantly homologous to the DEAH helicase CHLR1.¹ CHLR1, which belongs to FANCJ helicase family also plays a role in heterochromatin organization.^3,16 This helicase affects epigenetic modification of the genetic content and chromatin organization in the mammalian nucleus. In absence of CHLR1, defects in localization and organization of the chromatin have been observed. Aberrant localization of pericentric heterochromatin accompanied by perturbed centromere clustering happens in absence of CHLR1, the homolog of BACH1.¹⁶ Pericentric heterochromatin is loaded with chromatin binding proteins like heterochromatin-binding protein 1 (HP1) isoforms. Epigenetic modifications like histone methylation at sites H3K9 and H4K20 also takes place at the pericentric heterochromatin.^61,62 CHLR1 plays a role in the chromatin association of HP1 at the pericentric regions but it does not affect the histone modifications resulting into no alteration in the level of heterochromatin marker H3K9me3. Interestingly at the telomere regions, the association of both HP1 and histone modification is affected with depletion of FANCJ helicase family. Therefore BACH1/FANCJ helicase family certainly do play a role in targeting HP1 to the correct genomic regions.¹⁶ Proper localization and binding of HP1 protein are required for the global organization of heterochromatin and centromere clustering. Its presence at the constitutive heterochromatin forms an obstacle for DNA replication providing a regulation to it.^63,64 Epigenetic modification like methylation is severely impaired at the pericentric/heterochromatin regions in absence of CHLR1, whereas the centromere/kinetochore regions are unaffected with the absence of these FANCJ like helicase. So, BACH1/ChlR1/FANCJ plays a heterochromatin specific role in epigenetic events.^12,16 Heterochromatin organization and accessibility to replication is regulated by BACH1 through HP1.²⁸ FANCJ helicases might function in facilitating DNA replication at difficult sites, such as cohesion binding sites as well as condensed heterochromatic sites, stalled replication forks with secondary structures or at the DNA double strand breaks, by participating in both cohesion establishment and heterochromatin arrangement.^12-14 The BACH1 interactor BRCA1 is capable of functioning as a histone deacetylase.⁶⁵ So combination of epigenetic modifications like deacetylation and methylation at the heterochromatin or difficult to replicate regions lead to chromatin remodeling for accessibility of the DNA for repair and replication.^66,67 All these functions of BACH1 lead to the conclusion that BACH1 has a role in heterochromatin organization during replication, regulation and repair of complex sites.

BACH1 - an important player in cancer biology

The very basic cause of cancer is the mutations in the genetic material. While mutations in tumorigenesis is very well characterized, but very little is known about the development of mutations that initiate tumorigenesis. In very simple terms cancer can develop due to genetic mutations transmitted through generations or it can be defined as a disease of ageing fueled by the accumulation of somatic mutations.^68,69 Mutations can develop by the variations or differential expression of the DNA damage repair molecules. Aberrations in the genetic material can also develop by the compromised proofreading activity of the replication machinery or inability to resolve the secondary structures at the difficult to repli cate site. In this era of cancer management, next generation sequence testing (NGS) and multi-gene ‘panel’ germline mutation testing in different cancers have identified the increase in mutations. ^70,71 There is a rise in the number of mutations that leads to variants of uncertain significance (VUS) and needs further characterization. Characterizing these VUS will help to identify additional genes associated with an increased risk of cancer. These mutations which are till date insignificant are mostly present in the genes which play a role in DNA damage repair or cell cycle regulation pathways. Mutations in genes affecting these type of signaling pathways can significantly affect the molecular pathogenesis of diseases like cancer. The helicase BACH1, which has a significant role in repair through homologous recombination, is instrumental in the molecular pathogenesis of cancer in different tissue types (Figure 2). Analysis from 2 different datasets, cBioPortal and COSMIC, shows a significant number of mutations in BACH1 which are VUS. These VUS mutations significantly falls in the HBB domain, which has a role in DNA damage repair and BRCA1-BRCT interacting domain, which plays a role in helicase by considering cBioPortal and COSMIC datasets respectively (Figure 4). This picture of distribution of mutation becomes clear by analyzing the characterized mutation along with the VUS. Furthermore, Analysis of mutations (SNPs) which are characterized and predicted including VUS conclude that the HBB domain of BACH1 is the most susceptible site of mutation in different cancer types followed by the BRCT interacting domain, which is the BRCA1 binding site (Figure 5). This conclusion justifies that BACH1s repair activity significantly plays an independent role in the tumor biology of different cancer types apart from the BRCA1 interaction. Mutational analysis reveals that the BRIP1 locus is strongly associated with hepatocellular carcinoma (HCC) risk in patients with hepatitis B virus (HBV) and/or hepatitis C virus (HCV)-induced liver disease.²⁰ The variants of BACH1 which are linked with viral cirrhosis have mutations in the domains which interacts with the DSB repair protein or DNA mismatch repair protein like MRE11 or MUTL α (Figure 2) [20]. Alternately, mutations in the mismatch repair proteins like MLH1 and MSH2, which regulate the localization of BACH1 at the DSBs has been recognized as bladder cancer driver genes.⁷² SNPs in the BRIP1 gene influences cervical cancer susceptibility by regulating the RHOA GTPase activity, which is a player in cell proliferation, adhe sion, apoptosis, cell polarity, invasion and metastasis (Figure 2).⁷³ Several somatic mutations are found in FANCJ which are associated with skin cancer also. Mutational analysis of the melanoma candidate genes and BRIP1 gene justifies the role of DNA damage response as an important factor in melanoma etiology (Figure 2).⁷⁴ Very recent case studies confirm the role of BRIP1 in colon cancer also. Germline mutations in BRIP1 lead to its truncated variants, which have an association with the colon cancer predisposition (Figure 2).⁷⁵ A brief analysis of the somatic mutations present in different cancer obtained from different databases shows an increase in BACH1 mutation and its overexpression, confirming its involvement in different cancer type (Figure 6). Many evidences confirm the significant role of BACH1 in the development and progression of lung cancer and most of the gynecological cancers, which are explained, in the following paragraphs.

Figure 4.

Mutation analysis of BACH1 variants of uncertain significance, predicting it’s mutational hot spots. A) SNP’s of BACH1 gene were analyzed from cBioPortal. These mutations are variants of uncertain significance. The mutation position was mapped with the domains of BACH1 respectively. X-axis represents BACH1 domains and Y-axis represents number of mutations. Different color code is used to represent cancer types (Number of mutations= 611). B) SNP’s of BACH1 gene were analyzed from COSMIC. These mutations are variants of uncertain significance. The mutation position was mapped with the domains of BACH1 respectively. X-axis represents BACH1 domains and Y-axis represents number of mutations. Different color code is used to represent cancer types (Number of mutations= 560).

Figure 5.

The bar graph represents the domain specific mutation in different cancers. A) SNP’s of BACH1 gene were analyzed from cBioPortal. These mutations are of clinical and non-clinical conditions like pathogenic, non-pathogenic and variants of uncertain significance. The mutation position was mapped with the domains of BACH1 respectively. X-axis represents BACH1 domains and Y-axis represents number of mutations. Different color code is used to represent cancer types (Number of mutations= 611). B) SNP’s of BACH1 gene were analyzed from COSMIC. These mutations are of clinical and non-clinical conditions like pathogenic, non-pathogenic and variants of uncertain significance. The mutation position was mapped with the domains of BACH1 respectively. X-axis represents BACH1 domains and Y-axis represents number of mutations. Different color code is used to represent cancer types (Number of mutations= 560).

BACH1 in breast cancer

BRCA1 gene is well established for its role in breast cancer (BC),⁷⁶ but recent scientific developments show that BRCA1 interactor genes like BRIP1 (BACH1/FANCJ), ATM, BRCC45, CTIP, MERIT40, NBS1, RAD50 and TOPBP1 plays an important role as modifiers of breast cancer risk.⁷⁷ The meta-analysis study reveals that the BACH1 polymorphism at the 919-serine position may reduce the danger of breast cancer in the Caucasian populations, mainly in postmenopausal females with a family history of breast cancer and without BRCA1/2 mutations.⁷⁸ Literature shows that BRCA1 and TP53 are the major genetic players in case of Triple- Negative Breast Cancer (TNBC). Though there is no significant correlation between BRCA1 and TP53 expression in TNBCs but their expression have a high prognostic significance.⁷⁹ BRCA1+/TP53+ patients had better overall survival than BRCA1– /TP53–TNBC patients.⁸⁰ Further research would reveal the pathways and the associated players to provide the molecular explanation behind their interactions and role in disease pathology. Low expression of BRCA1 leads to loss of BRCA1 protein interaction resulting in more free BACH1 repair protein. Efficient repair is affected as BRCA1 interactor BACH1 cannot be loaded at the site of DNA damage. Also less damage sensing leads to less efficient homologous repair by BACH1 and the damage is repaired through some other pathways like NHEJ, with compromised proofreading activity developing mutations.^81,82 Accumulation of mutations leads to favorable condition for developing transformed tumor cells which are resistant to chemotherapy-induced apoptosis. This results in the recurrence of breast cancer, which would explain why negative BRCA1 expression is associated with poorer prognosis.⁸³ In case of TP53, the expression of the gene does not directly correlates with the proper function of the gene. Missense mutation of TP53 yields a highly stable mutant TP53 protein that can give high TP53 expression, whereas TP53 proteins resulting from truncating TP53 mutations are unstable and cannot be detected.⁸⁴ The prevalence of TP53 mutation types varies among different breast cancer subgroups. There is a high prevalence of missense mutations in luminal tumors whereas the prevalence of truncated mutations are there in basal tumors.⁸⁵ Also truncated mutations are strongly associated with poor survival and these mutations are not easy to be detected. In other words, though the mutations in checkpoints are undetected they can lead to an increase in DNA damage, which will increase the activity of repair genes like BACH1. Increased BACH1 transcript levels were found in tumors with an estrogen receptor-negative, progesterone receptor-negative or HER-2-positive status. BRIP1/BACH1 overexpression is also detected in primary invasive breast carcinomas.⁸⁶ The 2014 COSMIC data on breast cancer and case studies reports overexpression of BACH1 is 18.75% of patient samples and the mutation rate is around 3.8%, confirming the role of BACH1 in breast cancer biology (Figure 6). The appearance of BACH1 and its target genes was correlated to an increased risk of breast cancer reappearance in patients.⁸⁷ Mutations in exon 20 of BRCA1 are identified in BC patient which alter the stability of the BRCT domain at the binding site of BACH1.⁸⁸ The male breast cancer patients are generally identified to be normal for BRCA1/2 gene but silent mutations are found on BACH1 tumor suppressor gene and DNA helicase.⁸⁹ Susceptibility towards breast cancer also develops in carriers of the C47G polymorphism and Pro-Ser genotype of BACH1 in premenopausal women.⁹⁰ Deletion mutations in BRIP1 are also identified in earlyonset of breast cancer.⁹¹ In conclusion, these data from different research group justifies that BRIP1/BACH1 is a genuine target gene for breast cancer disease pathology.

Figure 6.

Mutation and overexpression of BACH1 in different cancer types. The schematic bar graph represents the total percentage of samples with mutated BACH1 and the total percentage of the sample with overexpressed BACH1 in different cancer types. X-axis represents the type of cancer and Y-axis represents the percentage(%) of BACH1 alteration. The data was extracted from COSMIC data base.

BACH1 in ovarian cancer

Cancer in ovary is the leading cause of cancer associated mortality among woman.⁹² Reproductive factors such as high parity, use of oral contraceptive, breastfeeding, removal of the uterus and tubal ligation are few ways to protect against ovarian cancer,⁹³ whereas infertility and endometriosis are the major risk factors.⁹⁴ The mechanism for the development of this cancer at the molecular level is not well studied and understood, but inflammationrelated oxidative stress has been proposed as a unifying theory by which these risk factors could cause genomic damage leading to the development of tumorigenesis in the ovary.⁹⁵ In other words, the efficacy of the DNA damage repair pathway may play a major role in ovarian carcinogenesis.⁹⁶ The above statement is supported by the COSMIC data, which shows that in a sample size of 266 patients the percentage of BACH1 overexpression, DNA damage repair gene, is 65.75 and in 1268 patients the percentage of BACH1 mutation is 3.84 (Figure 6). Several evidences link DNA repair with ovarian cancer- most of the ovarian cancer susceptible genes, like BRCA1 and BRCA2 have been identified to regulate DNA repair. TP53 is another susceptible gene, which plays a role in maintaining genomic integrity via several mechanisms including induction of cell cycle arrest in response to DNA damage, DNA repair and regulation of apoptosis.⁹⁷ Statistical analysis from a population-based North Carolina Ovarian Cancer Study (NCOCS) support for strong associations between ovarian cancer and polymorphisms in the repair genes. They identified two SNPs in CHEK2, two SNPs in TP53, and one SNP each in BACH1 and LIG4 repair genes. Few weak targets like NBS1, MSH6, RAD52, XRCC5 and GADD45B are also identified by some other group.⁹⁸ As BACH1 is a major player in HR repair pathway, it will be of great diagnostics and prognostic value to find the exact role and the underlying molecular mechanism of BACH1 in ovarian cancer, which remains unclear. The bioinformatics data indicates that the BACH1 SNP found in ovarian cancer patients is predicted to affect splicing and also mi-RNA binding site.⁹⁹ These findings reflects that BRCA1-BACH1 interaction plays an important role in the etiology of ovarian cancer.

BACH1 in lung cancer

Lung cancer is the most commonly diagnosed cancer and it is one of the reasons for cancer death worldwide. Approximately 1.6 million case results in deaths per year.¹⁰⁰ The molecular mechanisms which play the role in malignancy are unknown. The important genes which have a role in lung cancer are the cell cycle and the repair genes like TP53, RB, BRD7, PCNA and NFKB1.¹⁰¹ BRIP1 is found to be overexpressed in lung cancer (COSMIC data, 2014, Figure 6). Homozygous deletions are observed in lung adenocarcinoma in the BRIP1 gene (3%). Also, BRCAness, i.e. HR defects in absence of any germline mutation in BRCA, is usually seen in non-small cell lung cancer (NSCLC).¹⁰² High transcript level expression of BRCA1 is a helpful tool for choosing NSCLC patients for individualized chemotherapy, as it is the only independent prognostic variable for NSCLC patients.¹⁰³ The findings of Zhang group highlights that the integrity of the FA-BRCA pathway is a determinant of sensitivity/resistance to DNA crosslinking agents in lung cancer cells and may represent a mechanism underlying the resistance to chemotherapy of DNA crosslinking agents.¹⁰⁴ Ubiquitous type of mutation having BRIP1 variants are identified from tumor and blood sample obtained from NSCLC patients.¹⁰⁵ In lung cancer, germline mutations are observed in the CHK1 gene which is involved in Fanconi anemia and BRCA1/2 signaling pathways.¹⁰⁶ Methylation in FANCF promoter is a significant predictor for poor survival in adenocarcinoma of the lung, so inactivation of FANC-BRCA pathways may result in the poorer survival rate of patients with lung cancer.¹⁰⁷ These findings justify the important role played by FANC/BACH1 in cancer metabolism of lungs.

Concluding remarks and future perspective

Our current understanding indicates that BACH1 nuclear protein differentially participate in complex networks that regulate cell growth, cell cycle, DNA replication, DNA repair, mitotic chromatin dynamics, and also epigenetic modifications at the specific heterochromatin sites. BACH1 functions in the replication of the difficult sites and during stress, damage and secondary structures, because of its characteristics as a helicase, repair gene and as chromatin remodeler. Cancer mutation data (COSMIC) shows the widespread mutation of this gene in different cancer types. The overexpression of this gene in different cancer types clearly explains the increase in damage in the process of tumorigenesis and the proper repair activity is highly abrogated leading to accumulation of mutations. High mutation burden provides a favorable environment for the development, progression and recurrence of tumor. Further analysis reveals that the HBB domain of BACH1 is the most affected domain and the hot spot for characterized as well as uncharacterized mutations, explaining its role in cancer biology. Since the HBB domain has no link with BRCA1 interaction, so the effect conferred by this domain in different cancer types is independent of the BRCA1 function. The BRCA1 binding domain or the DNA helicase comes as the second most affected region of BACH1. The analysis of variants with uncertain significance also shows HBB domain as the most susceptible sites in BACH1 which justifies the emerging role of DNA repair through BACH1 in cancer biology. A deep insight into the functional aspect of the HBB domain along with BRCA1 interaction will open new avenues in the treatment of most of the deep-rooted cancers. Mutations or defect in this gene affects major molecular pathways that regulates and maintains the genomic integrity of the cells. With an aberration in the genetic integrity tumorigenesis develops. So, BACH1/BRIP1/FANCJ/ChlR1 gene has high potentials of transforming a normal cell into a tumor cell if compromised under certain circumstances, thus justified to be named as an oncogene. Even-though BACH1 has a substantial role in cancer biology and has a major role to play in different types of cancer, very few studies have been completed towards understanding the mechanism of how the proteins interact among themselves. In few of the cancers, BACH1 is analyzed as the major interactor protein of BRCA1, so, a detailed analysis of the interaction study is required to identify its role in tumorigenesis and metastasis. Current literature and our ongoing studies indicate that BRCA1-BACH1 interaction is lost due to diseased condition or a mutation at the interactor domain results in downregulation of DNA proofreading activity leading to more mutations, and hence increasing the risk of tumorigenesis. So, to understand BACH1, it is essential to explore this protein, its functional and interacting domains and critically evaluate its involvement to physiology and identify the potential roles in human pathologies, such as cancer.

Funding Statement

Funding: this work is supported by Minority/fellowship/CR-97/2018-19 from Directorate of Minority, Government of Karnataka to K.M.N.