The nucleolus, an ally, and an enemy of cancer cells.

In response to stress, including oncogenic one, some nucleolar factors participate in the induction of pathways that counteract the signals leading to hyperproliferation, and thus, these pathways prevent impaired or transformed cells from spreading. The change of a hitherto operating cell program into pathways leading cells to the defined destinations takes place. The activation of these pathways often occurs upon ribosome biosynthesis disturbance, i.e., upon nucleolar stress (Fig.  5 ). Predominant and best-known pathways are those activated by p53 that regulates activity of many genes responsible for processes related to the cell life cycle (Menedez et al. 2009 ) both under normal and abnormal conditions. It was reported that a few hundred genes are differently expressed and a number of genes are downregulated in various tissues by p53 under irradiation stress (Tanikawa et al. 2017 ). Action of p53 is regulated by its cellular level. In normal cells, a low level of p53 is maintained by Mdm2, which directs p53 to the proteasomal degradation, whereas, under various stresses, including nucleolar one, p53 protects cells taking part in a stress response. Under such conditions, Mdm2 activity may be blocked by the nucleolar factors which results in the increased level of p53 and its stabilization in the nucleoplasm. Then, Mdm2-p53 pathways are activated to counteract the effects of stress, including oncogenic one. Certain proteins that are related to a nucleolus play an essential role in Mdm2 inactivation and, thus, in activation of these pathways. p53-mediated pathways lead to modifications of cell scenario such as cell cycle arrest, DNA repair, cellular metabolism modulation, apoptosis, or aging (Lazo 2017 ). As Mdm2 not only shows pro-oncogenic activity in the above-mentioned context, but it also plays a key role in tumor propagation and increase in metastatic potential when it is overexpressed (Venkatesan et al. 2018 ), and thus, it is another significant target in anticancer therapy (Carvajal et al. 2018 ; Zhou et al. 2018c ). It is thought that, in response to impairment of ribosome production, NPM plays an important role in the activation of pathways in which other nucleolar and ribosomal proteins besides p53 are involved. Most extracellular insults, including physical and chemical ones, result not only nucleolar stress but also in its oxidation. The change of redox status in a nucleolus leads to S-glutathionylation of NPM and releases it from a nucleolus to the nucleoplasm. It is probable that this typical NPM translocation during nucleolar stress is a prerequisite for p53 activation, so NPM is thought to be a molecule recognizing nucleolar stress. Therefore, nucleolar or ribosomal proteins alone are supposed to be insufficient to induce p53 pathways until NPM is translocated to nucleoplasm (Yang et al. 2016b ). As such, a function of NPM has been discovered very recently; the reports do not mention common NPM participation in activation of the p-53-dependent pathways in response to nucleolar stress. Thus, only other nucleolar factors involved in the activation of appropriate pathways are taken into consideration. There is a growing body of evidence that RPs help to cope with unwelcome cells through their participation in pathways with or without p53 in response to the disruption of nucleolar function and integrity (Liu et al. 2017b ; Russo and Russo 2017 ). Recent studies point to the fact that RPs exhibit certain regularity of action during either fight with or generation of cancers. Those RPs which mediate activation of the p53 pathways show negative deregulation in cancers and seem to be tumor suppressors, e.g., RPL5, RPL11, or RPL26, whilst other RPs, e.g., RPL36A or RPS2, are overexpressed in various cancers and are related to the increased cell proliferation; however, paralogs of these RPs can exert antagonistic effects as in the case of RPL26L1 (Guimaraes and Zavolan 2016 ). Although NCL is known to be able to stabilize p53 (Saxena et al. 2006 ), another mechanism has been proposed, different from that with RPs, in which the conformational changes of human NCL cause exposure and availability of its specific domains capable of binding Mdm2. This blocks Mdm2 ligase activity which prevents ubiquitination and degradation of p53, thereby resulting in its activation (Bhatt et al. 2012 ). The inhibition of casein kinase 1δ/ε (CK1δ/ε; yeast homologue Hrr25) activity results in lack of phosphorylation of Ltv1, a factor involved in the maturation of small ribosomal subunits (SRSs, 40Ss). This causes a permanent bond between this factor and pre-40Ss and prevents their maturation, and thus, the number of functional ribosomes decreases which leads to inhibition of cell proliferation and growth. Since CK1δ/ε overexpression is observed in several types of malignancies (Ghalei et al. 2015 ), development of its inhibitors might be a strategy to combat cancers, including breast cancer (Monastyrskyi et al. 2018 ). Another nucleolar protein, cellular senescence-inhibited gene (CSIG, also known as ribosomal L1 domain containing 1, RSL1D1), is involved in processes such as cellular senescence and apoptosis. In addition, it regulates PTEN activity and is responsible for nucleolar localization of some proteins, including NS (Ma et al. 2008 ; Xie et al. 2016 ). Furthermore, its crucial role in regulation of Mdm2-p53 pathways was revealed. Due to that, under nucleolar stress, CSIG translocates from a nucleolus to the nucleoplasm where it connects through its ribosomal L1 domain with RING finger domain of Mdm2 and impairs its ligase activity. This stops ubiquitin-depended degradation of p53, stabilizes it, and activates appropriate pathways, while, when CSIG is knocked down, p53 activation is impaired which abrogates cell cycle arrest in G1 phase. Moreover, the block of Mdm2 RING finger domain probably prevents self-ubiquitination and degradation of Mdm2 (Xie et al. 2016 ). 1A6/DRIM (human UTP20) is a protein belonging to U3 proteins (t-UTPs) coordinating i.a. rDNA transcription through cooperation with UBF-1 and its acetyl-transferase hALP at rDNA promoter gene. The lack of UBF acetylation resulting from siRNA-mediated knockdown of 1A6/DRIM activity impairs initiation of RNA polymerase I-directed transcription and ribosome production. Then, ribosomal stress leads to activation of p53 pathway and to cell cycle arrest. On the other hand, 1A6/DRIM upregulation, promoting cell proliferation, occurs in certain cancer types (Peng et al. 2010 ). The protein interacting with carboxyl terminus 1 (PICT-1) also known as the glioma tumor suppressor candidate region 2 (GLTSCR2) is a human nucleolar factor involved in ribosome biosynthesis, mainly in rDNA transcription and ribosomal subunits assembly. Disruption of PICT-1 functioning impairs ribosome production at various stages, thus leading to the activation of pathways directing cells towards concrete fates. PICT-1 is phosphorylated by ataxia–telangiectasia-mutated (ATM) kinase in a nucleolus which guides it to the proteasomal degradation in the nucleoplasm, among others in response to DNA damages. Decreased PICT-1 level elicits nucleolar stress followed by release of RPL11 from a nucleolus to the nucleoplasm, where RPL11 inhibits Mdm2 action and activates p53-mediated apoptotic pathway (Chen et al. 2016a ). Interestingly, PICT-1 overexpression causes the reduction of UBF phosphorylation which considerably limits RNA polymerase I recruitment to r-gene promoters and inhibits rDNA transcription. This inactivates the AKT/mTOR/p70S6K signaling pathway and induces p53-independent autophagy in response to serum stimulation in human glioblastoma and breast adenocarcinoma cells. In addition, PICT-1 cooperates with tumor suppressor PTEN in the cytoplasm which causes anticancer effects. Given that PICT-1 mediates inhibition of cell proliferation and promotion of apoptosis and autophagy, while PICT-1 knockdown acts conversely and additionally enhances malignant progression of some cancers, PICT-1 itself can be considered as a suppressor (Chen et al. 2016b ). Its suppressive role was additionally confirmed by the fact that disruption of PICT-1 expression was significantly correlated with pathogenesis and neoplastic progression of endometrial cancer (Yoshimoto et al. 2018 ). Therefore, PICT-1-dependent ribosome biosynthesis deregulation may be a strategy to fight cancer cells. Among RPs, those which are involved in late-assembling of large ribosomal subunits, mainly to RPL11 and RPL5, are attributed the most important role in homeostasis and p53 stabilization. They together with 5S rRNA (5S ribonucleoprotein particle, 5S RNP, rRNA in complex with proteins) inhibit Mdm2 activity and maintain the structural integrity of nucleoli (Nicolas et al. 2016 ). 5S RNP–Mdm2–p53 pathway may be activated upon impairment of ribosome production, including downregulated RPS19 synthesis. Such a response significantly delays the initiation of AML, while it is not efficient when the disease is already established (Jaako et al. 2016 ). Regulation of the activity of factors involved in start-up of particular pathways is complex, ambiguous, and can be carried out at several levels. It depends on their interaction with specific domains or with other factors as well as on a cell type and certain cellular settings, i.e., physiological condition of a cell, and impact of intracellular stimuli or of external environment. An exemplary situation occurs in human cells in the case of p53 regulation by two proteins related to a nucleolus, NCL and RPL26. In stress-free cells, NCL inhibits expression of p53 at translational level through binding to p53 mRNA, whereas under stress, NCL in combination with RPL26 facilitates translation of p53 which stabilizes it and p53-dependent pathways are consistently activated (Chen et al. 2012 ). Based on the above-mentioned examples, it could be supposed that affecting nucleolar factors could be a potential mechanism eliminating defective cells which is generally beneficial for an organism in most cases (see below). However, ribosomal or nucleolar perturbations can promote cancer in certain situations. Such a situation is observed in colorectal cancer cells that first are subjected to the ribosome-inactivating stress (RIS) and next to anticancer agents. RIS imposes chemoresistance, i.e., it restrains death of cancer cells and increases their chance for survival following anticancer drug administration which ultimately impairs the efficiency of chemotherapy (Oh et al. 2016 ).

A growing number of new data concerning nucleoli make this nuclear suborganelle less and less mysterious. Just over 2 decades ago, the nucleolus was solely perceived as a ribosome factory. Since then, it has been shown to be a plurifunctional structure involved in many crucial cellular processes, many of which also concern cancer cells. On one hand, ribosome biosynthesis itself and a number of nucleolar factors drive transformation and cancer development; on the other hand, vulnerability of cells to impairment of ribosome biogenesis may help to fight cancers. Moreover, changes of nucleolar morphology and of many nucleolar factors give valuable information in diagnosis of several cancer types. Participation of a rather small number of nucleolar and ribosomal proteins both in signaling pathways favoring carcinogenesis and in those that combat cancer cells has been confirmed so far. Attribution of new functions to proteins connected with nucleolus and ribosome biosynthesis whose roles are already known as well as functional decipherment of many new proteins that were identified to reside for longer or shorter time in nucleoli whose roles have not been specified yet, will undoubtedly take place in the near future. Moreover, development and introduction of new molecular methods increasing expression of nucleolus-associated molecules activating known pathways and discovering new pathways and nucleolar factors or factors interacting with nucleolar ones participating in destroying undesired cells as well as searching for new inhibitors targeting pathways leading to transformation and cancer development which can enhance the effect of the agents whose effect was disappointing are the challenges facing cancer researchers. Furthermore, search for and application of ways and factors enhancing responsiveness of cancer cells that acquired resistance to a given drug could make chemotherapy more effective. Moreover, most chemotherapeutic agents are usually toxic towards normal cells, causing side effects. Minimizing adverse effects considerably improves therapeutic efficacy and comfort of patients. That is why, it is very important to develop new approaches reducing undesired toxicity in non-malignant cells and to introduce them into the therapeutic course. Although the mechanisms of selective disruption of different stages of ribosome biosynthesis by different chemotherapeutics are well known, the exact mechanisms and factors contributing to selective elimination of cancer cells through drug-induced nucleolar stress which simultaneously do not harm healthy cells are not quite so well known yet. It was proved that intense ribosome biosynthesis, which is characteristic of many cancer types, is vulnerability of these cells that can be readily targeted. Indeed, cells with high rate of ribosome production are much more sensitive to perturbation of this process than those characterized by low rate of rRNA synthesis. Selective inhibitors of ribosome biosynthesis may cause cytotoxic effects leading to apoptosis of the cancer cells, while only cytostatic effects, i.e., transient cell cycle arrest of the healthy cells. As most cellular responses to inhibition of ribosome biosynthesis are mediated by p53, wild-type p53 and its high enough level during nucleolar stress in malignant cells is a prerequisite for the induction of apoptosis in them, whereas tumor cells with low rRNA synthesis rate should be additionally intervened with a drug that would rise p53 level enough to effectively address them toward death (Scala et al. 2016 ). Recently, rDNA has been recognized as a genome region of high sensitivity, especially in rapidly proliferating cells, and hence, inhibitors such as BMH-21, that may intercalate rDNA loci, selectively affect neoplastic cells but not normal ones (Wei et al. 2018 ). It is not clear which molecular factors, including signaling and receptor molecules, if any, can sense the changes of ribosome biosynthesis rates, thereby allowing discrimination between malignant and normal cell types on the basis of their response to perturbed ribosome production. It seems that non-tumor cells have a much higher threshold for activation and stabilization of p53 in response to nucleolar stress than cancer cells. Most probably, the higher sensitivity of malignant cells with high proliferative capacity to perturbed ribosome biosynthesis results from disregulated expression of both activating mutations of oncogenes, especially overexpression of c-Myc, and loss-of-function mutations in suppressors which might contribute to the loss of cell cycle checkpoints and ribosome biosynthesis control. Furthermore, some agents, in addition to target ribosome production, may also exert genotoxic effects in the form of damaged DNA. As signaling pathways controlling DNA reparation are often inefficient in proliferating cancer cells in contrast to normal ones, the former are much more sensitive to chemotherapy. Cyclotherapy seems to be an interesting cytoprotective strategy which could use induction of p53-mediated nucleolar stress response in the form of reversible cell cycle arrest triggered by specific perturbation of ribosome biosynthesis at certain stages in conjunction with cytotoxic treatment. This method might be used for selective killing of intensely dividing cancer cells that have lost p53 in p53-independent manner and for sparing proliferating non-malignant p53-proficient cells that become temporarily non-dividing ones. In addition, if any mutation associated with nucleolar functioning was a reason of cell transformation and cancer progression, then the product of mutated gene could be a specific target of the most promising modern chemotherapy, and personalized therapy/molecularly targeted therapy. Moreover, as malignant cells are characterized by deregulation of signal transduction and metabolic pathways, including those associated with cellular nutrition, the specific dietary regimen seems to be a promising strategy to promote differential effects in non-malignant and cancer cells. In this case, therapeutic effects are based mainly on the deficit of cellular energy which is utilized mainly for processes associated with intense proliferation of disregulated cancer cells. In addition to the fact that the cancer cells weakened by this deficit are notably sensitive to chemotherapeutic drugs, including ribosome biosynthesis-perturbing ones, oncogenes prevent the activation of stress resistance in them. Consequently, cancer cells are selectively killed, mainly by autophagy. On the contrary, normal starved cells halt cell cycle through insulin-like growth factor-1 and shift energy deficiency towards survival (Buono and Longo 2018 ; Cangemi et al. 2016 ; Lee et al. 2010 ). Although reports concerning therapeutic effects directly connecting diet and ribosome production are lacking, relationship between these two seems to be plausible. As ribosome biosynthesis is the most energy-consuming process in human cells, cancer cells with high ribosome production must generate sufficiently large amount of energy. Hence, it could be expected that insufficiency of energetic sources, especially glucose, caused by severe diet, could affect ribosome biosynthesis through energetic- and nutrient-related signaling pathways and activate pathways leading to the inhibition of growth and proliferation or to death of starved cells, especially malignant ones sensitive to perturbed ribosome biosynthesis. In general, different types of dietary approaches, including specific forms of fasting and starvation, were proved to greatly reduce chemotherapy-related adverse effects as well as selectively protect normal cells and simultaneously sensitize cancer cells to chemotherapy (Lee et al. 2012 ). Summing up, disregulated signaling, resulting from altered genetic programs, connecting energetic metabolism, nutrient availability, cell growth, and proliferation, as well as ribosome and protein biosynthesis determines high sensitivity of cancer cells to appropriate chemotherapeutics in comparison to normal cells. Hence, detailed recognition of differences, including pathways and their players under various stress conditions, between cancer and normal cells would allow for more precise hit in targeted cells which would reduce or even eliminate side effects.

Nucleoli are present in all eukaryotic cells with exception of those that have lost their nuclei during differentiation. Their main role, ribosome production, seems to be conservative in animals, plants, and yeasts. This process includes transcription of ribosomal DNA (rDNA), maturation of ribosomal RNA primary transcript (pre-rRNA), as well as assembly of ribosomal subunits and their transport to the cytoplasm (Olson and Dundr 2015 ; Raška et al. 2004 ; Stępiński 2014 ). In addition, they participate in many important cellular functions, including those responsible for health and disease (Núñez Villacís et al. 2018 ), and stress response and development. This concerns both animals (Lindström et al. 2018 ; Tsai and Pederson 2014 ; Tsekrekou et al. 2017 ) and plants (Kalinina et al. 2018 ; Ohbayashi and Sugiyama 2018 ). Proliferating cells, including intensely dividing cancer cells, must meet the requirement of appropriate mass and size before they divide which is associated with huge demand for proteins. Nucleoli have to supply great enough number of translational nanomachineries, ribosomes, to provide optimal efficiency of protein biosynthesis. Thus, nucleoli perform a key function in maintenance of homeostasis in cells, and they can directly influence cell cycle progression, cell growth, and proliferation (Mayer and Grummt 2006 ; Zhou et al. 2018a ). Interestingly, this concerns also plants in which certain nucleolar proteins are preferentially involved in promotion of leaf cell proliferation (Kojima et al. 2018 ). Nucleoli synthesize half of all cell transcript pool and produce ca. 2 million ribosomes during 15 h. To complete this task, a proliferating cell utilizes up to 80% of energetic and material resources (Schmidt 1999 ). Hence, ribosome biosynthesis must be precisely controlled and adjusted to cell needs to ensure rational management of these resources. For example, Sfp1 and Sch9, factors that regulate processes depending on accessibility of nutrients and probably delay cell cycle until cells reach right sizes, make close correlation between cell growth and ribosome biosynthesis possible. Although this refers to yeast cells, analogs of these factors were identified in animal cells (Lempiäinen and Shore 2009 ; Rudra and Warner 2004 ). A relationship between morphology and function of nucleoli and certain features of dividing cells was observed long time ago. The nucleolar size was one of the first parameters by means of which the intensity of cell growth and proliferation, mostly cancer cells, was estimated. Hypertrophy of nucleoli, manifested by their increase in size and number, is correlated with enhanced rate of cell proliferation and growth in cancer tissues (Donizy et al. 2017 ). A positive correlation between nucleolar size and intensity of ribosome biosynthesis was quite well proven in cells undergoing transformation as well as during further cancer development. Consistently, the enlarged nucleoli and increased cell proliferation are observed along with more intensive rDNA transcriptional activity which is frequently accompanied by enhanced expression of such factors involved in various stages of ribosome biosynthesis as upstream-binding factor (UBF), DNA topoisomerase I, fibrillarin, argyrophylic proteins (AgNOR proteins), including nucleolin (NCL) and nucleophosmin (NPM, B23), as well as small nucleolar RNAs (snoRNAs) and ribosomal proteins (RPs) of both small (RPS) and large (RPL) ribosomal subunits (Chang et al. 2016 ; Derenzini et al. 1998 ). However, big nucleoli are not exclusively characteristics of intensively proliferating cells producing ribosomes as enlarged nucleoli are also present in apoptotic leukemic granulocytic progenitors (Smetana et al. 2017 ) and in chilling-stressed plant cells (Stępiński 2009 ) and both show reduced nucleolar activity. It has also been suggested that increased aggressiveness of cancer cells is associated not only with augmented rRNA biosynthesis but also with activation of specific pathways of pre-rRNA maturation during which new post-transcriptional modifications are introduced into rRNA leading to increase in biosynthesis of ribosomes and to changes of their translational functionality (Belin et al. 2009 ). Since ribosome biosynthesis is a driving force for cancer cells, they are more susceptible to disruption of ribosome production than normal cells, and this susceptibility is used to combat them (Bywater et al. 2012 ). A growing body of evidence suggests that disorder of nucleoli is a conservative mechanism leading to developmental cell death at least in certain cell types of multicellular organisms, including animals and plants, as well as in eukaryotic protist Dictyostelium (Golstein 2017 ; Luciani et al. 2016 ). In this context, natural or intentional disruption of nucleolar morphology and/or functioning can provoke death of eukaryotic cells, including cancer ones. Carcinogenesis and cancer development in humans are often associated with increased activity of oncogenes on one hand and inactivation of suppressors on the other. A lot of protooncogenic factors such as AKT (protein kinase B), PI3K (phosphatidylinositol 3-kinase), Ras (a family of small GTP binding proteins), and c-Myc (a family of regulator gens and protooncogenes coding for transcriptional factors) take part in the regulation of various stages of ribosome biosynthesis in normal cells, whereas their deregulation leads to intensified ribosome production which may contribute to tumorigenesis (Devlin et al. 2013 ; Sriskanthadevan-Pirahas et al. 2018 ). Moreover, many other proteins or different RNAs, which are related to ribosome production, perform non-ribosomal functions in a nucleolus or are just sequestrated in it, can be engaged in malignancy, including transformation, cancer development, and metastasis. In this context, a nucleolus favors neoplasia (Fig.  1 ). On the other hand, a nucleolus can have anticancer activity when ribosome biosynthesis is impaired by any stressor, including intentional therapeutic action, which induces a nucleolar/ribosomal stress followed by a protective response. In this case, a nucleolus can mediate activation of pathways with or without p53, a suppressor transcriptional factor, by means of nucleolar or ribosomal proteins (Fig.  2 ). Fig. 1 Cancerous processes associated with nucleolar functioning. Deregulation, especially overexpression of such oncogenes as AKT, PI3K, Ras, or c-Myc, causes upregulation of ribosomal (RPs) and nucleolar proteins which translates to more intensive nucleolar functioning (gray arrow). Consequently, increased ribosome biosynthesis drives processes related to neoplasia, i.e., cell transformation, cancer development, or metastasis Cancerous processes associated with nucleolar functioning. Deregulation, especially overexpression of such oncogenes as AKT, PI3K, Ras, or c-Myc, causes upregulation of ribosomal (RPs) and nucleolar proteins which translates to more intensive nucleolar functioning (gray arrow). Consequently, increased ribosome biosynthesis drives processes related to neoplasia, i.e., cell transformation, cancer development, or metastasis Fig. 2 Disruption of ribosome biosynthesis can stop cancer cell development or even kill them. Inhibition of any stage of ribosome biosynthesis, i.e., rDNA transcription, pre-rRNA processing, ribosome assembly, RP biosynthesis, or transport of ribosomal particles with chemical or physical agents results in reduction of ribosome production which elicits nucleolar/ribosomal stress. Nucleolus responds to the stress by releasing nucleolar factors that mediate activation of pathways leading cells to the specific destinations such as cell cycle arrest, aging, autophagy, apoptosis, and cell differentiation, or to metabolism change Disruption of ribosome biosynthesis can stop cancer cell development or even kill them. Inhibition of any stage of ribosome biosynthesis, i.e., rDNA transcription, pre-rRNA processing, ribosome assembly, RP biosynthesis, or transport of ribosomal particles with chemical or physical agents results in reduction of ribosome production which elicits nucleolar/ribosomal stress. Nucleolus responds to the stress by releasing nucleolar factors that mediate activation of pathways leading cells to the specific destinations such as cell cycle arrest, aging, autophagy, apoptosis, and cell differentiation, or to metabolism change As a number of discovered nucleolar factors still grow as well as new functions of well-known nucleolar factors are revealed in relation to cancer biology, this review summarizes the previous and latest knowledge concerning this issue.

Optimal functioning of cancer cells depends on intense protein biosynthesis. Since the main stages associated with formation of translational machineries, a process driving cell growth and proliferation as well as ensuring survival of every cell type, take place in a nucleolus, it accounts at least partly for carcinogenesis. Given that intense ribosome production favors transformation and cancer development, it means that cancer cells are susceptible to disruption of this process. Due to that, the intentional disruption of nucleolar functioning, which triggers nucleolar response followed by activation of appropriate pathways guiding damaged or dangerous cells, including transformed ones, to death, became an attractive anticancer strategy. Nucleolar dysfunction can be caused by the use of inhibitors, either in the form of chemical agents or RNAi, targeting any indispensable chain link at any stage of ribosome biogenesis, which would switch it off and trigger nucleolar stress. Therefore, every factor disturbing ribosome biogenesis and finally limiting cell growth and proliferation can be a potential tool in anticancer therapy (Fig.  2 ). For instance, targeted perturbation of RRP15 (ribosomal RNA processing protein) involved in nucleolar formation and ribosome biogenesis, including rDNA transcription and maturation of rRNA, interferes with nucleolar integrity and functioning and thereby induces nucleolar stress in human cells. This, depending on cellular p53 status, activates either RP–Mdm2–p53 pathway leading to the cell cycle arrest in p53-proficient non-transformed cells or ATR–ChkL–γH2Ax axis leading to the death of p53-deficient cancer cells (Dong et al. 2017 ). There are many well-known synthetic and natural drugs targeting nucleolar functions which are used to combat cancer cells (Quin et al. 2014 ; Stępiński 2016 ; Woods et al. 2015 ). Interestingly, proteomic analyses revealed that upon exposure to nucleolar stress-eliciting antitumor drugs, regardless of the fact whether they originate from plants as TBMS1 (saponin from Bolbostemma paniculatum ) (Lin et al. 2016b ) or are obtained by chemical synthesis as oxaliplatin (Ozdian et al. 2017 ), proteins in cancer cells, including RPs, exhibit different expression, some are upregulated, and the others are downregulated. Different proteins whose expression changes during stress as well as those nucleolar or ribosomal proteins which are released from nucleoli as a result of stress-induced nucleolar disintegration may contribute to the activation of pathways eliminating undesired cells. The mechanisms associated with activation of both pathways dependent on (Vlatkovic et al. 2014 ) and independent of p53 (Chen et al. 2016a ; Russo and Russo 2017 ) participate in the nucleolar surveillance response. Since more than half of human cancers are devoid of functional p53 (Soussi 2000 ), it is especially important to find the p53-independent anticancer pathways. Most available classic chemotherapeutics, including those which selectively impair nucleolar functioning and trigger nucleolar stress response, are cytostatics that reduce or inhibit cell proliferation, and thus, intensively dividing cells, both cancer and normal ones, are targets for such drugs. Thereby, chemotherapy may impair functioning of certain tissues which leads to various side effects (Chorawala et al. 2012 ; Liu et al. 2015a ). Therefore, on one hand, chemotherapy is beneficial, and on the other, it is a curse. Thus, as adverse effects often accompany chemotherapies, searching for strategies reducing them is very important. CX-5461 is mainly used in malignances of hematological origin. It selectively inhibits rDNA transcription without nucleolar disintegration and thereby induces nucleolar stress and then p53-mediated apoptosis. As this drug specifically alters the structure of rDNA which mimics DNA damage and activates DNA damage response (DDR), it can also activate p53-independent pathways leading to modulations of cell cycle checkpoints via nucleolar ATM/ATR signaling (Quin et al. 2016 ). CX-5461 seems to be one of the less harmful drugs for normal cells. In vivo, it selectively drives mice premalignant and malignant B cells to apoptosis, while it is non-genotoxic for wild-type B-cell population. Analogous results were observed in cells originating from human hematologic cancers (Bywater et al. 2012 ). In addition, admittedly, CX-5461 was shown to be selectively toxic to acute lymphoblastic leukemia cells, without serious side effects on normal bone marrow cells; however, its application in combination with other agents such as specific inhibitors of ATM/ATR pathway allows for its lower doses, potentially reducing its cytotoxicity (Negi and Brown 2015 ). Thus, the novel CX-5461-induced nucleolar stress response is proposed to enhance therapeutic efficiency not only in p53-inefficient aggressive c-Myc-driven lymphomas but also in solid tumors through the combinational use of CX-5461 and ATM/ATR signaling inhibitors (Quin et al. 2016 ). A new mechanism was revealed in which c-Myc-stimulated ribosome upregulation makes c-Myc-driven multiple myeloma cells more susceptible to nucleolar dysfunction. In this mechanism, CX-5461 elicits nucleolar stress and frees RPL5 from ribosomes, which mediates decrease in c-Myc level leading to the suppression of c-Myc-targeted gene expression. This inhibits growth and guides cells mainly to p53-independent apoptosis. In this case, the essential and novel possibility consist in the fact that antitumor action of CX-5461 can be applied not only in multiple myeloma but also in other c-Myc-driven cancers that additionally can be drug-resistant (Lee at al. 2017b ). Interestingly, the use of CX-5461 to affect rDNA transcription and ribosome biogenesis not only in nucleolus but also in mitochondrion seems to be a possible strategy to combat c-Myc-driven cancers (Rossetti et al. 2018 ). Targeting mTORC1 directly or indirectly is a promising anticancer approach (Mahoney et al. 2018 ), especially when mTORC1 signaling inhibitors are applied in combination with other drugs much better therapeutic efficacy is achieved. Temsirolimus, mTORC1 inhibitor, coupled with RG7388, an Mdm2 inhibitor, is effective therapy for p53-proficient neuroblastoma (Moreno-Smith et al. 2017 ). Devlin et al. ( 2016 ) evoked an interesting effect by application of the combined inhibition of rDNA transcription with CX-5461 and of PI3K-AKT-mTORC1-dependent ribosome and protein biosynthesis signaling with everolimus, mTORC1 inhibitor. In tests on mice, twice higher survival was achieved by the activation of two independent pathways triggering apoptotic tumor cell death, one with p53 and the other with BMF, a pro-apoptotic protein that was activated after the PI3K-AKT-mTORC1 pathway was inhibited. This seems to be a promising method to fight human c-Myc-driven B lymphoma (Devlin et al. 2016 ). 5-FU-resistant hepatocellular hepatoma become more sensitive to the drug when it operates in combination with mTORC1/2 inhibitors (Zhang et al. 2018 ). The examples quoted above prove that using at least two ways to disrupt the ribosome biosynthesis network can provide a more effective therapeutic tool to combat malignancies, including those in which c-Myc plays an essential role (Ni et al. 2018 ). Human ribosome-free RPL3, accumulated as a result of its upregulation and release from ribosomes upon nucleolar stress, seems to be the critical participant in the response of lung and colon cancer cell lines lacking p53 to Act D, a transcription blocker that selectively inhibits rDNA transcription at low concentrations. Consistently, RPL3-mediated positive regulation of p21 expression via ERK (extracellular-signal-regulated kinase) activation results in promotion of apoptosis as well as reduction of proliferation and migration of these cancer cells (Russo et al. 2016a ). In a similar experimental system, the use of 5-FU, DNA, and RNA synthesis inhibitor in combination with RPL3 overexpression considerably enhances sensitivity of 5-FU-resistant lung and colon cancer cells to this drug, and thereby, it strongly increases cytotoxic effect by means of the mitochondrial apoptotic cell response which prevents cell migration and invasion. Elicitation of such an effect involves RPL3-mediated negative regulation of two molecular targets in p53-null cells treated with 5-FU: (1) CBS (cystathionine-β-synthase), a factor enhancing energetics, proliferation, and migration of cancer cells and inflammation, and (2) NFҡB, a factor activated in many solid tumors linking cancer and inflammation. Since, in these cases, the success of therapy depends on RPL3 status; the treatment with Act D or 5-FU does not trigger the above-mentioned responses and makes drug chemotherapy ineffective when RPL3 expression is low. Thus, the development of agents increasing expression of this protein could make the fight with cancers more efficient (Pagliara et al. 2016 ; Russo et al. 2016b ). Furthermore, RPL3 was reported to be an essential factor in acquiring multidrug resistance (MDR) of p53-deprived lung cancer cells through controlling the cellular redox status. In this scenario, 5-FU induces the resistance to multiple drugs that elicit nucleolar stress. RPL3 downregulation increases MDR, while restoration of its appropriate level re-sensitizes the cells resistant to nucleolar stress-inducing drugs by the regulation of oxidative stress responsive genes. Hence, maintenance of adequate RPL3 level seems to be crucial for anticancer therapeutic success, i.e., in response of p53-mutated lung cancer cells that acquired drug resistance (Russo et al. 2017 ). From a large number of RPs (33 of a small subunit and 47 of a large subunit), the involvement of RPL3 in activation of the anticancer pathways as a response to nucleolar stress induced by inhibitors of ribosome biogenesis seems to be well grounded in the case of p53-null cells as well as of those with acquired chemoresistance. 5-FU is commonly used in many solid tumors; however, it exerts cytotoxic effects by its active metabolite, 5-fluorodeoxyuridine monophosphate, not only in cancer but also in healthy cells causing numerous adverse effects (Thomas et al. 2016 ). Interestingly, polymorphic abnormality of genes responsible for 5-FU metabolism significantly enhances the toxicity of 5-FU-based therapy (Shahrokni et al. 2009a , b ). Platinum-derived agents, including oxaliplatin, are among anticancer chemicals targeting nucleolar function. Although oxaliplatin shows high antitumor cytotoxic activity, it is also responsible for adverse effects, mainly neurotoxicity (Argyriou 2015 ; Chukyo et al. 2018 ; Noh et al. 2018 ; Pereira et al. 2018 ). It is reported that oxaliplatin-derived neurotoxicity results from sodium channel dysfunctions which cause superexcitability of nerve axons (Heide et al. 2018 ). In addition, oxalate, a metabolite of oxaliplatin, is probably involved in the early development of peripheral sensory neuropathy and oxalate-induced toxicity might be mediated by ROS (Pereira et al. 2018 ). Indeed, oxidative stress during which overproduction of ROS can modify cellular components, including DNA, is implicated in side effects accompanying chemotherapy. Dox, which affects ribosome biosynthesis, is one of the drugs with proved high pro-oxidant activity. Although it is an efficient anticancer agent with a broad spectrum of action from hematological cancers to solid tumors, its administration is restricted because of side effects. Cardiotoxicity is the major detriment upon application of Dox which significantly decreases antioxidant compounds in tissues (Dogan et al. 2014 ). Oxygen-derived free radicals damage nuclear and mitochondrial DNA which induces apoptosis of cardiomyocytes, thus antioxidative strategy should be introduced for cardioprotection (Mobaraki et al. 2017 ). It was reported that Dox and its metabolite, doxorubicinol, and impaired functioning of cardiac Ca 2+ signaling pathway, because they bind to its essential protein components. This mechanism, in addition to oxidative stress, certainly contributes to fatal cardiotoxic side effects (Hanna et al. 2014 ). Since the conventional chemotherapy is usually toxic to healthy cells, natural bioactive chemoprotective compounds with rare or negligible adverse effects are in the focus of interest. Oxaliplatin-induced neuropathic side effects could be mitigated by pre-treatment with AC591, a herbal medicine composed of certain neuroprotective herbs (Cheng et al. 2017 ; Noh et al. 2018 ). However, the combined treatment of Dox with a plant-originating antioxidant polyphenolic compound, cichoric acid, revealed that the latter reduced oxidative stress and apoptosis in human normal fibroblasts, so it can counteract Dox-induced cytotoxicity (Hajra et al. 2018 ; Jabłońska-Trypuć et al. 2018 ). Moreover, plant-derived small molecules such as indole-3-carbinol (Hajra et al. 2018 ), d -methionine (Lin et al. 2018a ), or vivartana (Gnanasekaran et al. 2017 ) were reported to protect certain tissue types against specific chemotherapy side effects. Natural components, administered alone or as complementation of standard anticancer agents, get more and more attractive in anticancer therapy because of their softer side effects. It was reported that plant-derived compounds such as sulforaphane, ursolic acid, and betulinic acid have cytostatic effect. These phytodrugs induce oxidant-based nucleolar stress in breast cancer both in cell lines from wild as well as mutated p53 types. Disruption of nucleolar function results in activation of pathways leading to p21-mediated inhibition of proliferation in both cell populations. Thus, plant drugs evoking ribotoxic stress may be new promising candidates in breast cancer therapy even in the cells deprived of functional p53 (Lewinska et al. 2017 ). Moreover, sulforaphane can influence epigenetic histone acetylation in breast cancer cells and thereby can restore specific gene expression which interferes with tumor growth (Gianfredi et al. 2017 ). It is also capable of eliminating pancreatic carcinoma cells via autophagy without toxic adverse events, but this activity involves participation of ROS (Naumann et al. 2011 ). In addition, a phytochemical, farnesiferol C (FC), reduces the levels of both c-Myc and RPL11, which co-regulate each other, as well as it attenuates the expression of survival genes leading to apoptosis in NSCLC. Furthermore, puromycin or Dox combined with FC enhance its cytotoxic effect (Jung et al. 2016 ). Although it was said in the previous paragraph that NPM was likely to be commonly implicated in nucleolar stress response, interestingly, specific C-terminal mutation of NPM (NPMmut), which is present in ca. one-third of patients with AML, does not induce nucleolar stress. Furthermore, it prevents neither Act D-induced nucleolar stress nor activation of the p53-dependent and independent pathways leading cells to death by apoptosis or necrosis, respectively. Since NPM status is not so important in the case of leukemias, AML patients with NPMmut may be expected to have a better outcome of therapy, which is associated with cellular localization of NPMmut (Brodská et al. 2016 ). It is worth noting that Act D in combination with inhibitors of histone deacetylase significantly enhances therapeutic effects (Brodská et al. 2016 ), whereas combined with other standard chemotherapeutic agents little contributes to therapeutic efficiency of these agents (Parsons-Doherty et al. 2014 ). However, although Act D is often successful in treatment of a variety of cancers and it is relatively well tolerated, treatment with Act D is restricted mainly by its toxicity at higher doses, but its application at lower doses in combination with other drugs to synergize therapeutic efficacy allows to minimize its genotoxicity toward normal tissues (Choong et al. 2009 ). Acrolein (Acr), as ubiquitous environmental contaminant also abundantly present in tobacco smoke, elicits dangerous diseases. Paradoxically, toxicity of Acr makes it a potential agent to be used for killing cancer cells regardless of p53 status. Acr preferably binds to nucleolar DNA causing oxidative damage of rDNA. This leads to nucleolar disintegration, thereby preventing rDNA transcription and ribosome biogenesis which induces RPL11-mediated apoptotic pathways as a response to nucleolar stress in human cancer cells with active and inactive p53 (Wang et al. 2016 ). Interestingly, some nucleolar factors, including RPs, apart from being diagnostic parameters, can be useful as indicators of the cell response to an inhibitor of a given pathway, i.e., applied chemotherapy. For example, phosphorylation status of RPS6 changes in response to the agents targeting any component of RTK (receptor tyrosine kinase)/K-RAS downstream pathways in gastric cancer cells. In addition to RAF/MEK/ERK pathway, also the axis with mTORC1 is responsive to MEK inhibitors which results in their lowered activity and thereby in reduced cell proliferation. The reduced phosphorylation of RPS6 is observed in these cases. This feature could be used prior to treatment to select those gastric cancer patients who are sensitive to MEK inhibition (Hirashita et al. 2016 ).