Keywords

Introduction

Eukaryotes encompass distinct organelle within the cells. Among them, the nucleus accommodates a huge amount of DNA in a small space of approximately 6.0 μm. The human DNA is composed of ~3.5 billion base pairs, giving rise to 46 chromosomes which if laid end to end would measure up to 2 meters in length. Positively charged histone molecules bind to DNA resulting in a higher-order compacted conformation that allow it to reside in the tiny nucleus. Canonical histones including histones H2A, H2B, H3, and H4 form an octamer around which 147 bp of DNA wraps and form the basic unit of a nucleosome. These nucleosomes form “beads on string structure” resulting in chromatin fiber and which further undergoes compaction to form the chromosomes. Although canonical histones help DNA to be packaged inside the nucleus, covalent modifications to these histones add up another level of regulation based on the chromatin architecture. Depending upon the extent of the compaction of chromatin, it is classified into euchromatin and heterochromatin. Euchromatin is a relaxed state of the chromatin, while heterochromatin represents a highly compacted state. Euchromatin being loosely compacted offers access to the transcription factors allowing active transcription. Heterochromatin restricts access to the transcription factors and represses transcription. The state of chromatin is regulated by different parameters or factors, together termed as epigenetic factors. Epigenetic factors include DNA methyltransferase-mediated DNA methylation, histone epigenetics involving modifications at the canonical histones, or non-canonical histone variants deposited by histone chaperones, mediated by histone-modifying enzymes and non-coding RNAs. Chromatin compaction status thus regulates the expression and the repression of genes implicated in physiological processes and cell fate decisions. Chromatin architecture is dynamic and undergoes a multitude of changes which are highly regulated and orchestrated by key epigenetic players.

Improvement in the whole genome sequencing technology has enabled us to understand the mutational landscape of a cancerous cell to understand mutational hotspots. Mutations in the genes including histones, histone variants, and histone chaperones, which impair epigenetic regulations, have been revealed. Mutated histones can contribute to the tumorigenesis or initiation of the cancer; hence mutated histones are termed as “onco-histones.” Mutations in the histone can lead to the disruption of the nucleosome structure, hence disturbing the higher-order chromatin architecture. Onco-histones have been associated with several pediatric cancers (Mohammad and Helin 2017). However, in this chapter we will discuss the role of histone epigenetics that is beyond genetics, in different types of cancer. In a normal mammalian cell, epigenetic regulations occur in a regular manner, and it maintains the homeostasis of the physiological processes. Altered epigenetic regulations have a direct link to the deregulation in the cellular processes leading to diseases like cancer. Previously cancer was mainly studied from the aspect of the genetic disease, but in the recent decade, the role of epigenetics in contributing to the initiation as well as progression of the cancer has been well studied. These important outcomes were so evident that in the recently updated hallmarks of cancer “nonmutational epigenetic reprogramming” was considered to have the potential that could facilitate the acquisition of hallmark capabilities in cancer cells (Hanahan 2022). Hence, studying and understanding histone epigenetics and its different components have become absolutely essential in the context of cancer.

Histone Epigenetics

There are three layers in histone epigenetics: histone modifications mediated by histone-modifying enzymes, histone variants, and histone chaperones. Post-translational modifications of histones have been extensively studied. These modifications not only maintain normal cellular physiological functions but also retain the repressive chromatin state and, hence upon deregulation, contribute to cancer (Audia and Campbell 2016). The enzymes associated with different modifications including methyl transferase and acetyl transferase have been significantly implicated in cancer and especially their role in therapeutic interventions (Audia and Campbell 2016). In addition to the canonical histones, regulatory histone variants exist that have been implicated in transcriptional regulation. Recent advances have demonstrated the significant involvement of histone H3 and H2A variants in tumor biology (Vardabasso et al. 2014). The incorporation or removal of the histone variants or the canonical histones is mediated by a group of protein molecules, known as histone chaperones. This class of epigenetic factor is the least studied and hence remain significantly less explored in understanding their role in regular cellular phenomena or diseased state (Nandy et al. 2020). In this chapter, we will specifically discuss histone chaperones implicated in different types of cancer.

Histone Chaperones

Histone chaperones are fundamental protein molecules that aid in histone metabolism. They basically shield the histones from any non-specific interaction. This cluster of molecules could recruit or evict histone in a replication-dependent or replication-independent manner. They associate with varied molecular phenomena including replication, repair, and transcription (Hammond et al. 2017). Recent studies have demonstrated the role of histone chaperones in cancer malignancy or even metastasis (Nandy et al. 2020). We will highlight the role of all those histone chaperones which have been implicated in different types of cancer.

HIRA

Histone cell cycle regulator A (HIRA) is encoded by a gene within a region of human chromosome 22. HIRA forms a complex comprising HIRA, ubinuclein-1 (UBN1), and calcineurin-binding protein cabin-1 (CABIN1) that eventually cooperate with another histone chaperone anti-silencing factor 1A (ASF1A) to mediate H3.3-specific binding in a DNA synthesis-independent manner at a transcriptionally active region of the chromatin (Ray-Gallet et al. 2002). Unlike the other histone chaperones, HIRA has been rarely associated with cancer. Rather, it is involved in senescence of cancer cells. HIRA interact with cyclin-dependent kinase 2 (CDK2), the expression of which inhibits S-phase advancement and induces cellular senescence that arrests proliferation and suppresses tumorigenesis (Hall et al. 2001). In senescent cells, HIRA is crucial in maintaining the histone H4K16-acetylated mark at promoters of active genes, indicating that HIRA regulates a dynamic chromatin landscape in senescent cells subsequently suppressing neoplasia (Rai et al. 2014). In contrast to anti-cancer activity of HIRA, few recent reports are pointing out the role of HIRA in cancer. Prohibitin (PHB) associates with HIRA complex and stabilizes all the components of HIRA in breast cancer cells (Zhu et al. 2017). Overexpression of PHB in breast cancer cell leads to the increased nuclear PHB that results in the HIRA-mediated incorporation of H3.3 at promoter regions of the mesenchymal markers involved in epithelial-to-mesenchymal transition (EMT), the cellular transition responsible for cancer metastasis of solid tumors. Increased expression of these genes induces EMT associated with breast cancer metastasis (Huang et al. 2020). Among the hematological malignancies, chronic myeloid leukemia (CML) patient sample expresses increased HIRA in comparison to normal healthy individuals, and upon loss of HIRA, the CML cells are induced toward differentiation over proliferation (Majumder et al. 2019).

The most interesting and at the same time a point of concern is the role of HIRA in incomplete compensation in cancer cells. In multiple epithelial and cancer cell lines, with EMT induction, chromatin accessibility increased reproducibly, accompanied by a general decrease in the number of histones integrated into chromatin. The loss is for histone H3.1 and its associated chaperone chromatin assembly factor 1 (CAF1). This loss in the histone level is replenished by gap-filling histone H3.3 and hence an increase in HIRA expression. The enrichment of H3.3 specifically occurs at the promoters of EMT-related genes and hence stimulating cancer metastasis (Gomes et al. 2019). Recent studies have suggested that an imperfect compensation between H3 and H4 chaperones favors induction in tumor progression by instigating an alternative lengthening of telomere (ALT) pathway. In normal somatic cells, due to the absence of telomerase, the cells undergo telomere shortening at the repeats and thus have a limited replicated life, whereas cancer cells evade this phenomenon either due to high telomerase activity or involving alternative strategies. Those 10–15% of tumors had recurrent mutation in a chromatin remodeler ATRX which along with another histone chaperone, DAXX (will be discussed later along with ATRX), is key in the deposition of histone H3.3–H4 at the repetitive units. Mutation in ATRX leads to loss in histone recruitment, which is then compensated by HIRA, and thereby the cancer cells could maintain the telomere length and survive safely (Hoang et al. 2020). So, indirectly, HIRA contributes in cancer cell survival. However, this character of HIRA could be targeted for anti-cancer therapy.

DAXX

Death domain-associated protein 6 (DAXX) in association with the chromatin remodeler alpha-thalassemia/mental retardation, X-linked syndrome (ATRX), like HIRA, is associated with replication-independent incorporation of histone H3.3/H4 in the chromatin (Drané et al. 2010). But, on the contrary to HIRA, this deposition is at the telomeres and pericentrin heterochromatin (Ray-Gallet and Almouzni 2022). DAXX-ATRX plays a significant role in the ALT pathway, as discussed above. DAXX upregulation has been associated with poor survival in metastatic high-grade serous carcinoma along with prostate, ovarian and gastric cancer while reduced level has been detected in lung cancer patients (Davidson et al. 2020; Wen et al. 2022). In glioblastoma, interaction of tumor suppressor PTEN with DAXX resulted in limited incorporation of H3.3 at the promoter regions of the tumor-promoting genes. Inhibition of the DAXX in PTEN-null mice leads to the suppression of intracranial tumor and improves survival of mice with glioma (Benitez et al. 2017).

DEK

Histone chaperone DEK incorporates histone H3.3 at telomeres and regulatory elements in a DNA-dependent manner and is also responsible for maintaining heterochromatin integrity along with heterochromatin protein 1-alpha (HP1α) (Kappes et al. 2011). Human DEK gets phosphorylated by casein kinase (CK2). Phosphorylation of DEK is important for normal hematopoiesis (Sawatsubashi et al. 2010). Upregulation in DEK is prevalent in glioblastoma, melanoma, colorectal carcinoma, and bladder carcinoma. DEK can also promote epithelial transformation, can inhibit senescence and apoptosis, and hence could induce carcinogenesis. Amplification, copy number increase, or chromosomal translocation contributes to the oncogenic character of DEK protein. Increase in the DEK level associates with poor prognosis in many cancers (de Albuquerque Oliveira et al. 2018). By triggering M2 tumor-associated macrophage polarization, DEK expression in breast cancer cells generates a potentially immunosuppressive tumor microenvironment (Pease et al. 2020).

CAF1

Chromatin assembly factor 1 (CAF1) deposits histone H3.1/2 throughout the genome in a DNA-dependent manner. CAF1 is upregulated in cervical cancer, cutaneous lymphoma, and oral squamous carcinoma (Sykaras et al. 2021; Yang et al. 2020a; Mascolo et al. 2021). This increase in CAF1 expression is associated with proliferation and metastasis. Interestingly, a recent study demonstrated that EMT inducers deregulate HCs, and there is a loss in histone H3.1 and also its chaperone, CAF1, which implies an opposing effect of CAF1 in tumor progression (Gomes et al. 2019). Upregulation of CAF1 associates with high histological tumor grade, metastasis, recurrence, and poor outcome (Sykaras et al. 2021). CHAF1B is a p60 subunit of CAF1 complex that includes CHAF1A, p150 subunit of CAF1, and retinoblastoma-binding protein 4 (RBBP4). CHAF1B is upregulated in acute myeloid leukemia (AML) with chromosome 21 trisomy as well as in the mixed-lineage leukemia (MLL) rearrangement. CHAF1B inhibits the expression of the myeloid-specific genes by binding to the respective promoter regions of the gene followed by their inhibition (Volk et al. 2018). Specifically, it was shown that CAF1-mediated nucleosome assembly competes with the transcription factors and blocks the differentiation of the leukemic cells.

ASF1

Anti-silencing factor 1 (ASF1) has two human paralogue genes, ASF1A and ASF1B, and associates with recycling of histones and chaperoning of histone H3.1/2–H4 and H3.3/H4 to CAF1 and HIRA, respectively. ASF1 is elevated in a variety of cancers including breast, cervical, gastric, lung, skin, and prostate cancers (Corpet et al. 2011; Liu et al. 2020; Zhang et al. 2022; Feng et al. 2021; Shi et al. 2021; Carrion et al. 2021). Increased expression of ASF1 corresponds to increased proliferation of cancer cells resulting in poor prognosis of the disease. In breast cancer, ASF1B correlates with increased disease progression and metastasis (Corpet et al. 2011). Loss in ASF1A led to growth arrest and senescence in prostate and hepatocellular carcinoma (HCC) cells having wild-type p53 (Wu et al. 2019). High levels of ASF1A promote proliferation and invasion of gastro-intestinal cancer cells mediated by increase in cMYC (a proto-oncogene), cyclin D1, and LGR5 (leucine-rich repeat containing G protein-coupled receptor 5) level (Liang et al. 2017). ASF1B is highly expressed in pancreatic cancer patients and associates with poor prognosis. Downregulation of ASF1B in pancreatic cells leads to increased caspase-3 and caspase-9 activation along with PARP1 [poly(ADP-ribose) polymerase 1] cleavage that sensitizes cells to cisplatin (Kim et al. 2022).

NAP1L

The human nucleosome assembly proteins 1-like protein (NAP1L) is involved in histone transport, nucleosome assembly, and eviction of histones from the nucleosome (Le et al. 2019). Among other family members, NAP1L1 has been most widely studied in the context of cancer. NAP1L associates with histone H2A–H2B dimers. Multiple forms of human malignancies, including colorectal cancer, hepatocellular cancer, lung adenocarcinoma, ovarian cancer, and renal cancer, have been identified to express NAP1L1 at high levels (Aydin et al. 2020; Le et al. 2019; Nagashio et al. 2020; Zhu et al. 2022; Zhai et al. 2018). High expression of NAP1L1 associates with poor prognosis and poor survival. Uncontrolled proliferation, increased metastasis, reduced differentiation, and enhanced aggressive behavior constitute the outcomes of NAP1L1 overexpression in cancer cells. The elevated level, to some extent in a few cancers, offers chemo-resistance, and it also serves as a prognostic marker. Regulation at different levels including cell cycle or signaling contributes to its role in cancer.

FACT

Facilitates chromatin transcription (FACT) [consisting of two subunits, SPT16 (suppressor of Ty 16 homolog) and SSRP1 (structure-specific recognition protein 1)] evicts both the H2A–H2B dimer and (H3–H4)2 tetramer, positioning them for re-deposition following the passage of RNA or DNA polymerases. FACT is involved in three major cellular phenomena including replication, transcription, and DNA repair. Triple-negative breast cancer, lung cancer, hepatocellular carcinoma, glioblastoma, and pediatric neuroblastoma express higher FACT levels corresponding to their aggressive behavior and poor clinical outcome (Gurova et al. 2018). The levels of both FACT subunits increased gradually in human and mouse cells undergoing various stages of transformation. Notably, artificial interventions that increased or lowered FACT levels improved or decreased, respectively, the effectiveness of oncogene-induced transformation (Garcia et al. 2013). FACT expression in tumor cells instigates the oncogenes to drive transformation. Tumor cells rely on FACT to a greater extent than normal cells and that FACT may play a crucial role in sustaining the malignant features of tumor cells. Increased sensitivity to chromatin instability is connected with the increased demand for FACT in tumor cells. This preferential dependence of cancer cells on FACT has been exploited to properly characterize only one cancer therapeutics angle tested till date among all the histone chaperones (will be discussed in later section).

Nucleolin

Nucleolin (NCL) is a histone chaperone with FACT-like activity and can destabilize histone octamer and in turn modulate the nucleosome (Angelov et al. 2006). In endometrial carcinoma and prostate cancer, expression levels of nucleolin and nucleolin-associated genes are upregulated. NCL plays a key role in transformation of hyperplastic glands into cancer and metastasis. Hence it can be a potential therapeutic target in the near future (Barzilova et al. 2022; Firlej et al. 2022). NCL, till date, does not serve as a prognostic marker.

HJURP

Holliday junction recognition protein (HJURP) is responsible for the incorporation of centromere protein A (CENPA) at centromeres in addition to canonical histone H4. HJURP expression levels are significantly higher in colorectal cancer, prostate cancer, glioblastoma, breast cancer, and thymic epithelial carcinoma (Kang et al. 2020; Chen et al. 2019a; Serafim et al. 2020; Hu et al. 2010; Levidou et al. 2022). CENPA is one of the histone variants of H3 which plays a crucial role in centromere and kinetochore structure. Deregulation in the expression of CENPA or its activity can lead to the altered cell cycle leading to cancer. Stoichiometry between HJURP and CENPA is important for the normal cellular functioning. The enhanced HJURP level is associated with poor survival in patients (Hu et al. 2010). CENPA deposition at the centromere or modulation of yes-associated protein 1 by HJURP accounts for its role in increased proliferation in cancer cells (Mao et al. 2022). Thus, HJURP can serve as a prognostic marker, and specifically it serves as an independent prognostic marker for luminal A breast cancer subtype (Montes de Oca et al. 2015).

APLF

Aprataxin-PNK-like factor (APLF) is an interesting histone chaperone molecule which was discovered as a DNA repair factor of the NHEJ repair pathway (Mehrotra et al. 2011). It can recruit histone variant macroH2A, a repressive mark, and also can bind and assemble the histone octamer in a single step (Corbeski et al. 2022). Significant upregulation in APLF associates with breast cancer, bladder cancer, and glioblastoma (Majumder et al. 2018; Richter et al. 2019; Dong et al. 2020). The APLF level gets upregulated with an increase in invasive behavior of the breast cancer subtypes (Majumder et al. 2018). APLF regulates the genes implicated in EMT. Regulation by miRNAs is involved in APLF-mediated regulation of invasion of cancer cells (Richter et al. 2019). APLF is yet to be categorized as a prognostic marker.

NPM1

Nucleophosmin 1 (NPM1) preferably binds to histone H3 in a DNA-templated process. A typical chaperone that shuttles between cytoplasm and nucleus plays a crucial role in mRNA processing, chromatin remodeling, and embryogenesis. In addition to these activities, NPM1 is also associated with proliferative and growth-suppressive roles in the cell. It is abundantly expressed as a phosphoprotein and is involved in cancers of different histological origins. NPM1 could contribute to the diseased state of cancer either because of increased expression, mutation, or chromosomal translocation. Abnormalities in NPM1 associate with hematological malignancies, glioblastoma, and oral squamous cell carcinoma (Chen et al. 2020; Holmberg Olausson et al. 2015; Shandilya et al. 2009). Majority of the AML patients demonstrates mutation within NPM1 (Karimi Dermani et al. 2021). NPM1 undergoes post-translational modifications including acetylation, SUMOylation, and ubiquitination. Acetylated NPM1 is involved in transcriptional regulation and along with NPM1 causes increased expression of genes associated with proliferation, migration, and invasion in oral cancer (Senapati et al. 2022; Shandilya et al. 2009).

ANP32E

Acidic nuclear phosphoprotein 32 family member E (ANP32E) is a histone H2A.Z-specific chaperone. ANP32E is responsible for the eviction of histone H2A.Z variants, thereby causing a loss in chromatin accessibility resulting in reduction in transcription followed by gene expression (Murphy et al. 2020). ANP32E expression is increased in CML, gastric cancer, hepatocellular carcinoma, pediatric medulloblastoma, and pancreatic cancer (Yang et al. 2021; Zhu et al. 2022; Hupfer et al. 2021; Ma et al. 2021). The level of ANP32E dictates the proliferation vs differentiation of breast cancer cells (Ruff et al. 2021). An upregulated ANP32E expression corresponds to reduced patient survival, high recurrence risk, and poor sensitivity to immunotherapy. Increase in cell proliferation is a common observation in patient samples or cell lines having an increased level of ANP32. Regulation at the transcriptional, signaling, or structural protein levels contributes to the progression of the disease owing to the elevated ANP32E expression. Deletion of ANP32E could impair CML stem cell function and improved the tyrosine kinase inhibitor treatment in mouse model suggesting that ANP32E can be exploited as a therapeutic target for CML (Yang et al. 2021).

Table 1 summarizes the histone chaperones implicated in different types of cancer, and Fig. 1 depicts the alteration at the chromatin and cellular phenomena associated with change in expression of histone chaperones in cancer.

Table 1 Summary of different histone chaperones involved in variety of cancers
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Fig. 1
figure 1

Histone chaperone level dictate cellular machinery in cancer

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Usage in Therapeutics

Novel approach is to target epigenetic modifiers regulating expression of the genes without causing any permanent changes to the genetic material. As epigenetic changes are reversible and can be manipulated using specific inhibitors for epigenetic modifiers, it can be efficient approach to treat diseases including cancer. Genetic code in tumors is fixed, and it cannot be altered, but epigenetic modifications can be reprogrammed therapeutically by targeting epigenetic modifiers. The aim of epigenetic therapy is to re-modulate epigenetic silencing and reactivate various genes that promote apoptosis of malignant cells, differentiation, and growth arrest (Yoo and Jones 2006). Tyrosine kinase inhibitor-resistant cells, cancer stem cells, and normal wild-type cells show a unique epigenetic signature as well as DNA methylation pattern that determine their fate (Baylin and Jones 2016). Inhibitors regulating the activity of these epigenetic modifiers can serve as a novel and very effective treatment strategy for many cancers. Among the different factors in the histone epigenetics, histone-modifying enzymes have been extensively studied, and thereby a significant number of drugs targeting these enzymes have undergone clinical trials and few have been successfully used to treat cancer. Table 2 summarizes different clinical trials that have been conducted using inhibitors against the histone-modifying enzymes. On the other hand, histone chaperones have been rarely targeted. Apart from one report on the drug curaxin CBL0137 that targets histone chaperone FACT by binding to it and then sequestering FACT at chromatin. Trapping of FACT on chromatin allows p53-phosphorylation at serine 392 and suppression of NF-κB-dependent transcription (Gasparian et al. 2011; Chang et al. 2019). Administration of curaxin showed an increase in the apoptosis of tumor cells and suppression of cell proliferation. Clinical trial, #NCT01905228, at Phase I, curaxin was administered to the patients of diverse age group in order to decide the maximum tolerable dose and to decide dosage for the Phase II clinical trials. Clinical trial, #NCT02931110, is focused on deciding the safe range of the concentration of curaxin that can be administered to treat hematological malignancies. It is a Phase I trial targeting different categories of hematological malignancies including lymphoid as well as myeloid. Curaxin is being studied at clinical trial Phase I level for the treatment of melanoma or sarcoma (#NCT03727789). Future studies have been proposed to check efficacy of curaxin in combination therapy with ipilimumab and nivolumab to treat metastatic melanoma (#NCT05498792). Hence slowly but steadily, histone chaperones might be developed into potential candidates for targeting cancers of different origins.

Table 2 Clinical trials conducted targeting histone-modifying enzymes in cancer
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Conclusion

Histone chaperones are very selective toward the type of histones they deal with and thereby act as a guardian of the genome. Except for the histone chaperone HIRA, almost all are elevated in multiple cancerous states. This upregulation in general enhances proliferation, invasion, and aggressive behavior of cancer of solid tumors. In hematological malignancies, the balance shifts to proliferation than differentiation. They serve as excellent prognostic markers. An interesting facet is their role in imperfect compensation of histones such that upon the loss of one histone chaperone, another acts as a replacement and thereby overcomes the mechanism that inhibits EMT, associated with cancer invasion and migration, and ALT pathway that induce the state of cancer. Unlike histone-modifying enzymes, the potential of the histone chaperones for therapeutic intervention is yet to be exploited. With a role in cancer of varied histological origins, these molecules are waiting to be rediscovered in the context of cancer therapeutics.