Integration of Chronobiological Concepts for NSCLC Management

*Christian Focan, Anne-Catherine Davin, Maryam Bourhaba and Marie-Pascale Graas*

### **Abstract**

The authors reviewed pertinent experimental and clinical data allowing to consider the interest of taking into account the temporal dimension ('circadian') for prevention and management of the majority of cancers, i.e., non-small cell lung carcinoma (NSCLC). The universal importance of circadian rhythms has been acknowledged in animal or human situations regarding carcinogenesis and cancer promotion; cell kinetics, apoptosis, molecular genetics, as well as DNA repair mechanisms, platinum resistance…; molecular targets (i.e., epidermal growth factor reception-EGFR); and all lymphoid and immunology machinery components. Also chronotolerance to all chemotherapeutic agents useful for treating human lung cancer has also been evidenced. A few randomized clinical chronotherapy trials were performed in human NSCLC. One limited trial has shown apparent chronoefficiency, while in another one, chronotolerance to 5-fluorouracil and a platinum derivative were confirmed. The limited improvement of outcome in human NSCLC, even through the use of targeted and biological therapies (such as tyrosine-kinase (TKI) or vascular-endothelialgrowth-factor (VEGFR) inhibitors; immunotherapy), allows to consider launching specific trials in human NSCLC aiming at either restoring a normal circadian structure of the host or taking into account circadian variations of specific targets. By now unfortunately, no targeted or immunotherapy trials have been launched considering temporal dimension.

**Keywords:** circadian rhythms NSCLC review

## **1. Introduction**

#### **1.1 Circadian timing system**

Life is structured in space but also in time. Biological rhythms have been documented in all processes involved in the malignant transformation of cells as well as in the cellular proliferation of both healthy and tumor tissues [1–5]. All physiological functions expressed their metabolic or specific activities according to a circadian variation [1–5]. This is the case not only for actively dividing tissues but also for all other tissues, such as the myocardium, central nervous system, or organs involved in the metabolization, detoxification and excretion of drugs (kidney, liver) [3–5].

Recent advances identify critical molecular events that rhythmically control drug metabolism and detoxification, cell cycle, molecular targets, deoxyribonucleic acid (DNA) repair, apoptosis, and angiogenesis [3–5]. The coordination of these processes along the 24-h period is ensured by the circadian timing system (CTS) whose hierarchical organization determines chronotherapeutic effects [3–5]. The CTS coordinates physiology and cellular functions over a 24-h period (**Figures 1** and **2**). This circadian physiology is generated or controlled by a central pacemaker, the suprachiasmatic nuclei (SCN) in the hypothalamus. The SCN generate circadian physiology through diffusible signals, including transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), prokineticin-2, cardiotrophin-like cytokine, and neuroanatomic sympathetic and parasympathetic pathways [1–8].

A dozen specific clock genes constitute the core of the molecular clock in mammals [3–6]. These genes are involved in transcriptional and posttranscriptional activation and inhibition regulatory loops that result in the generation of the circadian oscillation in all physiological systems and individual mammalian cells [3–5]. In particular, the circadian locomotor output cycles kaput-brain and muscle ARNT-like protein-1 (CLOCK-BMAL1) or NPAS2-BMAL1 protein dimers play a key role in the molecular clock through the activation of transcriptional clock genes period's (Per's), cryptochrome (Cry's), and Reverb's [3–5, 9] (**Figures 1** and **2**).

Proper circadian regulation is essential for the well-being of the organism, and disruption of circadian rhythms is associated with pathological conditions including cancer [1, 5, 10, 11]. In mammals, the core clock genes, Per1 and Per2, are key regulators of circadian rhythms in central clock, in the hypothalamus, and in peripheral tissues [9–13]. Recent findings revealed molecular links between Per

#### **Figure 1.**

*Schematic view of the circadian timing system (CTS). The suprachiasmatic nucleus (SCN) is a biological clock located at the floor of the hypothalamus. Its period (cycle duration) is calibrated by the alternation of light (L) and darkness (D) through the rhythmic melatonin secretion by the pineal gland. The SCN controls or coordinates circadian rhythms in the body. Abbreviations: PVN, paraventricular nucleus; NPY, neuropeptide Y; TGF-alpha, transforming growth factor α; EGF, epidermal growth factor; Σ, sympathetic (after Levi et al. [3]; adapted).*

**75**

*Integration of Chronobiological Concepts for NSCLC Management*

genes and cellular components that control fundamental cellular processes such as cell division and DNA damage [9–13]. New data also shed light on mechanisms by which circadian oscillators operate in peripheral organs to influence tissuedependent metabolic and hormonal pathways [4, 5]. Circadian cycles are linked to basic cellular functions, as well as to tissue-specific processes through the control of gene expression and protein interactions. By controlling global networks such as chromatin remolding and protein families, which themselves regulate a broad range of cellular functions, circadian regulation impinges upon almost all major physi-

*Schematic representation of the molecular clock and the pathways involved into the control of relevant drug metabolism, cell cycle, DNA repair, and apoptosis in mammalian tissues. The protein dimer BMAL1-CLOCK or BMAL1-NPAS2 (a CLOCK homolog) plays an essential role in the rhythmic transcription of clock-*

In 2002, we performed an overview of accessible experimental and clinical data allowing to believe in possible improvement in NSCLC management through chronobiological considerations. Here we will update our previous review with experimental

and clinical recent contributions considering only circadian rhythmicity [14]. It is to be emphasized that we were unable to find any study on that subject using new biological alternatives such as targeted therapies or immunotherapy

Studies performed by Hashimoto et al. on a murine model with circadianvarying lung tumor induction through timed single- or split-dose irradiation have

Cancer development associated to circadian disturbances both in damaged (target) and undamaged tissues and systems [1, 3–5] has been described some years

ological pathways including immunological ones [4, 5].

**1.2 Aim of this review**

*controlled genes. After Levi et al. [3]; modified.*

approaches.

**Figure 2.**

**2. Carcinogenesis**

already been reviewed [14, 15].

*DOI: http://dx.doi.org/10.5772/intechopen.85710*

#### **Figure 2.**

*Chronobiology - The Science of Biological Time Structure*

Recent advances identify critical molecular events that rhythmically control drug metabolism and detoxification, cell cycle, molecular targets, deoxyribonucleic acid (DNA) repair, apoptosis, and angiogenesis [3–5]. The coordination of these processes along the 24-h period is ensured by the circadian timing system (CTS) whose hierarchical organization determines chronotherapeutic effects [3–5]. The CTS coordinates physiology and cellular functions over a 24-h period (**Figures 1** and **2**). This circadian physiology is generated or controlled by a central pacemaker, the suprachiasmatic nuclei (SCN) in the hypothalamus. The SCN generate circadian physiology through diffusible signals, including transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), prokineticin-2, cardiotrophin-like cytokine, and neuroanatomic sympathetic and parasympathetic pathways [1–8]. A dozen specific clock genes constitute the core of the molecular clock in mammals [3–6]. These genes are involved in transcriptional and posttranscriptional activation and inhibition regulatory loops that result in the generation of the circadian oscillation in all physiological systems and individual mammalian cells [3–5]. In particular, the circadian locomotor output cycles kaput-brain and muscle ARNT-like protein-1 (CLOCK-BMAL1) or NPAS2-BMAL1 protein dimers play a key role in the molecular clock through the activation of transcriptional clock genes period's (Per's), cryptochrome (Cry's), and Reverb's [3–5, 9] (**Figures 1** and **2**). Proper circadian regulation is essential for the well-being of the organism, and disruption of circadian rhythms is associated with pathological conditions including cancer [1, 5, 10, 11]. In mammals, the core clock genes, Per1 and Per2, are key regulators of circadian rhythms in central clock, in the hypothalamus, and in peripheral tissues [9–13]. Recent findings revealed molecular links between Per

*Schematic view of the circadian timing system (CTS). The suprachiasmatic nucleus (SCN) is a biological clock located at the floor of the hypothalamus. Its period (cycle duration) is calibrated by the alternation of light (L) and darkness (D) through the rhythmic melatonin secretion by the pineal gland. The SCN controls or coordinates circadian rhythms in the body. Abbreviations: PVN, paraventricular nucleus; NPY, neuropeptide Y; TGF-alpha, transforming growth factor α; EGF, epidermal growth factor; Σ, sympathetic (after Levi et al. [3]; adapted).*

**74**

**Figure 1.**

*Schematic representation of the molecular clock and the pathways involved into the control of relevant drug metabolism, cell cycle, DNA repair, and apoptosis in mammalian tissues. The protein dimer BMAL1-CLOCK or BMAL1-NPAS2 (a CLOCK homolog) plays an essential role in the rhythmic transcription of clockcontrolled genes. After Levi et al. [3]; modified.*

genes and cellular components that control fundamental cellular processes such as cell division and DNA damage [9–13]. New data also shed light on mechanisms by which circadian oscillators operate in peripheral organs to influence tissuedependent metabolic and hormonal pathways [4, 5]. Circadian cycles are linked to basic cellular functions, as well as to tissue-specific processes through the control of gene expression and protein interactions. By controlling global networks such as chromatin remolding and protein families, which themselves regulate a broad range of cellular functions, circadian regulation impinges upon almost all major physiological pathways including immunological ones [4, 5].

#### **1.2 Aim of this review**

In 2002, we performed an overview of accessible experimental and clinical data allowing to believe in possible improvement in NSCLC management through chronobiological considerations. Here we will update our previous review with experimental and clinical recent contributions considering only circadian rhythmicity [14].

It is to be emphasized that we were unable to find any study on that subject using new biological alternatives such as targeted therapies or immunotherapy approaches.

#### **2. Carcinogenesis**

Studies performed by Hashimoto et al. on a murine model with circadianvarying lung tumor induction through timed single- or split-dose irradiation have already been reviewed [14, 15].

Cancer development associated to circadian disturbances both in damaged (target) and undamaged tissues and systems [1, 3–5] has been described some years ago. Experimentally, the importance of deregulation of circadian rhythms before the development of liver cancer in rats exposed to diethyl-nitrosamine, a carcinogen that can also induce lung tumors, was reported by Filipsky et al. [16].

Molecular insights illustrate how dysregulation of circadian rhythms might influence the susceptibility to cancer development and provide further support for the emerging role of circadian genes in tumor suppression [12]. Silencing of tumor suppressor genes, such as the Per1—clock core gene, resulting from epigenetic alterations may occur early in lung cancer tumorigenesis [9, 17].

These observations allow considering Per1 gene as a potential target for chemoprevention.

Epidemiologic studies in human beings have evidenced a probable relationship between altered circadian rhythms and tumorigenesis. A high incidence of cancer has been observed in long-term shift workers such as flight attendants, nurses, or industrial workers [18–20]. Recent studies have also suggested that alterations of sleep quality were susceptible to enhance the risk of various cancers including lung cancers [21, 22]. Disruption of melatonin circadian rhythms (peak at night after dim-lighting) could partially explain such observations [23]. However, a large epidemiologic on thousands of Chinese female textile workers apparently failed to confirm an increased risk of lung carcinoma [24].

On the other hand, sport practice and regular physical activity, which are known to facilitate and maintain circadian general activities, may have an inverse effect, thus minimizing the risk of developing a lung cancer [25].

#### **3. Cell kinetics and molecular biology**

#### **3.1 Cell kinetics**

Hashimoto et al. recently reported that DNA synthesis activity in the normal lung was low but higher during the night [15]. Also a large number of experimental animal studies document circadian rhythm in cell proliferation in spontaneous or transplanted tumors, growing in ascitic fluid or solid phases [1, 2]. Precisely, Burns et al. studied alterations of DNA synthesis rhythmicity in selected organs of mice (i.e., bone marrow) bearing a transplanted Lewis lung carcinoma (LLC) [26].

Colombo et al. [27] reported day/night differences of spontaneous apoptosis in two different murine tumors, one of these being a lung one, in addition to circadian rhythms of division, peaks of apoptosis matching with mitoses valleys [27].

In human, circadian rhythmicity of cell proliferation has been reported for squamous cell carcinomas of the lung, as those of the skin and cervix [1, 2, 14, 28]. Various mechanisms responsible for the deregulation of the cell cycle and enhanced susceptibility to oncogenesis through activation of cell proliferation and cancer promotion have been identified. For example, in NSCLC, overexpression of cyclin D1, and mutation of p16 leading to a shortened and accelerated G1-phase and permanent phosphorylation (and inactivation) of pRb are known; in addition, mutations of p53 (with further impaired apoptosis) or pRb have been observed both in NSCLC and SCLC [29].

#### **3.2 Molecular biology**

#### *3.2.1 Clock genes and circadian regulation*

Tissues such as the liver, pituitary, and kidney but also the lung exhibit robust circadian rhythmicity in cultures [3–5]. Circadian timers are important for lung functions; for example, there is a well-documented link between diurnal variations

**77**

*Integration of Chronobiological Concepts for NSCLC Management*

in lung physiology (i.e., airway narrowing and inflammation) and nocturnal asthma [30]. Gibbs et al. have studied the cellular localization of core clock genes in both mouse and human organotypic lung slices [30]; they also established the effects of glucocorticoids on pulmonary clock [30]. They were able to demonstrate a marked circadian rhythm in PER2 expression that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins and glucocorticoid receptor on the epithelial cells lining the bronchioles (Clara cells and type II pneumocyte cells) in both mouse and human lung tissues [30]. Selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating these cells to be critical for maintaining coherent circadian oscillations in the lung tissue. The coexpression of glucocorticoid receptor and core clock components establishes them as a likely interface between humoral

suprachiasmatic nucleus output and circadian lung physiology [30].

between circadian epigenetic regulation and cancer development.

Hori et al. working on experimental Sato lung tumor were able to correlate biological time of greatest tumor growth and highest tissue blood flow (during

*3.2.2 Circadian regulation of tumor blood flow and angiogenesis*

Clock genes or proteins PER1 and PER2 have been linked to DNA damage response pathways in a series of studies, involving among others Lewis lung carcinoma (LLC) cells [3, 6, 9, 11–13]. Overexpression of either PER1 or PER2 in cancer cells inhibits their neoplastic growth both in vitro and in vivo and increases their apoptotic rate [13]. Also high expression of circadian gene mPer2 is able to diminish radiosensitivity of LLC and EMT6 cells with decreased expression of bax and p53 and increased expression of c-myc and bcl-2 [12, 13, 31]. This type of observations illustrates that the circadian system is involved in the protection and restoration of tumor cells, i.e., those of LC, against environmental detriments, such as gamma irradiation [13]. The gene, mPer2, might be considered as an inhibitor of tumor radiotherapy effects [32]. Downregulation of Per1 or Per2 enhanced tumor growth (i.e., of LLC cells) in vitro [12, 13, 31, 33]. Thus Per1 and Per2 exert their tumor suppressor functions in a circadian time-dependent manner [9, 31–33]. Also downregulation of Per1 or Per2 increases tumor growth only at given specific times of the day [12]. These optimal times may be shifted in tumors that have mutant period genes [12]. Overexpression of Per1 makes human cancer cells sensitive to DNA damageincluded apoptosis; in contrast, inhibition of Per1 in similarly treated cells blunted apoptosis [9]. The apoptotic phenotype was associated with altered expression of key cell cycle regulators. In addition, Per1 interacted with the checkpoint proteins ATM and Chk2. Ectopic expression of Per1 in human NSCLC cell lines led to significant growth reduction [6, 9]. Per1 m-RNA expression was high in the normal lung and downregulated in a large panel of tumor samples from NSCLC patient samples as well as in lung cancer cell lines [3–6, 8, 11]. In addition, Gery et al. showed that ectopic or forced expression of Per1 in NSCLC cell lines led to growth inhibition, G2M cell cycle arrest, apoptosis, and reduced clonogenic potential [6, 17]. The influence of Per1 on cell cycle and apoptosis seems to be p53-status independent [5, 13]. Timeless (TIM) a homolog of a drosophila circadian rhythm gene has circadian properties in exploration in mammals [34]. Precisely its expression is enhanced in lung cancer cell lines where its knockdown was related to the induction of apoptosis, suppression of proliferation, and clonogenic growth [34]. In surgically resected specimens from 88 consecutive patients, high TIM protein levels as gauged by immunohistochemistry (IHC) correlated with poor overall survival [34, 35]. Taken together those results support clearly the hypothesis that circadian rhythm disruption plays an important role in lung tumorigenesis, as well as a link

*DOI: http://dx.doi.org/10.5772/intechopen.85710*

*Chronobiology - The Science of Biological Time Structure*

confirm an increased risk of lung carcinoma [24].

**3. Cell kinetics and molecular biology**

thus minimizing the risk of developing a lung cancer [25].

chemoprevention.

**3.1 Cell kinetics**

**3.2 Molecular biology**

*3.2.1 Clock genes and circadian regulation*

ago. Experimentally, the importance of deregulation of circadian rhythms before the development of liver cancer in rats exposed to diethyl-nitrosamine, a carcinogen

Molecular insights illustrate how dysregulation of circadian rhythms might influence the susceptibility to cancer development and provide further support for the emerging role of circadian genes in tumor suppression [12]. Silencing of tumor suppressor genes, such as the Per1—clock core gene, resulting from epigenetic

These observations allow considering Per1 gene as a potential target for

Epidemiologic studies in human beings have evidenced a probable relationship between altered circadian rhythms and tumorigenesis. A high incidence of cancer has been observed in long-term shift workers such as flight attendants, nurses, or industrial workers [18–20]. Recent studies have also suggested that alterations of sleep quality were susceptible to enhance the risk of various cancers including lung cancers [21, 22]. Disruption of melatonin circadian rhythms (peak at night after dim-lighting) could partially explain such observations [23]. However, a large epidemiologic on thousands of Chinese female textile workers apparently failed to

On the other hand, sport practice and regular physical activity, which are known to facilitate and maintain circadian general activities, may have an inverse effect,

Hashimoto et al. recently reported that DNA synthesis activity in the normal lung was low but higher during the night [15]. Also a large number of experimental animal studies document circadian rhythm in cell proliferation in spontaneous or transplanted tumors, growing in ascitic fluid or solid phases [1, 2]. Precisely, Burns et al. studied alterations of DNA synthesis rhythmicity in selected organs of mice (i.e., bone marrow) bearing a transplanted Lewis lung carcinoma (LLC) [26].

Colombo et al. [27] reported day/night differences of spontaneous apoptosis in two different murine tumors, one of these being a lung one, in addition to circadian

In human, circadian rhythmicity of cell proliferation has been reported for squamous cell carcinomas of the lung, as those of the skin and cervix [1, 2, 14, 28]. Various mechanisms responsible for the deregulation of the cell cycle and enhanced susceptibility to oncogenesis through activation of cell proliferation and cancer promotion have been identified. For example, in NSCLC, overexpression of cyclin D1, and mutation of p16 leading to a shortened and accelerated G1-phase and permanent phosphorylation (and inactivation) of pRb are known; in addition, mutations of p53 (with further impaired apoptosis) or pRb have been observed both in NSCLC and SCLC [29].

Tissues such as the liver, pituitary, and kidney but also the lung exhibit robust circadian rhythmicity in cultures [3–5]. Circadian timers are important for lung functions; for example, there is a well-documented link between diurnal variations

rhythms of division, peaks of apoptosis matching with mitoses valleys [27].

that can also induce lung tumors, was reported by Filipsky et al. [16].

alterations may occur early in lung cancer tumorigenesis [9, 17].

**76**

in lung physiology (i.e., airway narrowing and inflammation) and nocturnal asthma [30]. Gibbs et al. have studied the cellular localization of core clock genes in both mouse and human organotypic lung slices [30]; they also established the effects of glucocorticoids on pulmonary clock [30]. They were able to demonstrate a marked circadian rhythm in PER2 expression that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins and glucocorticoid receptor on the epithelial cells lining the bronchioles (Clara cells and type II pneumocyte cells) in both mouse and human lung tissues [30]. Selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating these cells to be critical for maintaining coherent circadian oscillations in the lung tissue. The coexpression of glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology [30].

Clock genes or proteins PER1 and PER2 have been linked to DNA damage response pathways in a series of studies, involving among others Lewis lung carcinoma (LLC) cells [3, 6, 9, 11–13]. Overexpression of either PER1 or PER2 in cancer cells inhibits their neoplastic growth both in vitro and in vivo and increases their apoptotic rate [13]. Also high expression of circadian gene mPer2 is able to diminish radiosensitivity of LLC and EMT6 cells with decreased expression of bax and p53 and increased expression of c-myc and bcl-2 [12, 13, 31]. This type of observations illustrates that the circadian system is involved in the protection and restoration of tumor cells, i.e., those of LC, against environmental detriments, such as gamma irradiation [13]. The gene, mPer2, might be considered as an inhibitor of tumor radiotherapy effects [32].

Downregulation of Per1 or Per2 enhanced tumor growth (i.e., of LLC cells) in vitro [12, 13, 31, 33]. Thus Per1 and Per2 exert their tumor suppressor functions in a circadian time-dependent manner [9, 31–33]. Also downregulation of Per1 or Per2 increases tumor growth only at given specific times of the day [12]. These optimal times may be shifted in tumors that have mutant period genes [12].

Overexpression of Per1 makes human cancer cells sensitive to DNA damageincluded apoptosis; in contrast, inhibition of Per1 in similarly treated cells blunted apoptosis [9]. The apoptotic phenotype was associated with altered expression of key cell cycle regulators. In addition, Per1 interacted with the checkpoint proteins ATM and Chk2. Ectopic expression of Per1 in human NSCLC cell lines led to significant growth reduction [6, 9]. Per1 m-RNA expression was high in the normal lung and downregulated in a large panel of tumor samples from NSCLC patient samples as well as in lung cancer cell lines [3–6, 8, 11]. In addition, Gery et al. showed that ectopic or forced expression of Per1 in NSCLC cell lines led to growth inhibition, G2M cell cycle arrest, apoptosis, and reduced clonogenic potential [6, 17]. The influence of Per1 on cell cycle and apoptosis seems to be p53-status independent [5, 13].

Timeless (TIM) a homolog of a drosophila circadian rhythm gene has circadian properties in exploration in mammals [34]. Precisely its expression is enhanced in lung cancer cell lines where its knockdown was related to the induction of apoptosis, suppression of proliferation, and clonogenic growth [34]. In surgically resected specimens from 88 consecutive patients, high TIM protein levels as gauged by immunohistochemistry (IHC) correlated with poor overall survival [34, 35].

Taken together those results support clearly the hypothesis that circadian rhythm disruption plays an important role in lung tumorigenesis, as well as a link between circadian epigenetic regulation and cancer development.

#### *3.2.2 Circadian regulation of tumor blood flow and angiogenesis*

Hori et al. working on experimental Sato lung tumor were able to correlate biological time of greatest tumor growth and highest tissue blood flow (during dark span) [36]. This finding strongly suggests that tumor tissue blood flow has a determining influence on tumor proliferative activity and that tumor growth is influenced by circadian variation in tumor tissue blood flow [36].

These results were confirmed by Blumenthal et al. who also showed that the blood flow rhythm may differ between tumor and normal tissues, thus creating a window of opportunity when tumors could be targeted with a therapeutic agent such as vascular endothelial growth factor (VEGF) inhibitors [37].

The molecular mechanism regulating circadian expression of VEGF in tumor cells (Lewis lung carcinoma cells among others) has been investigated by Koyanagi et al. [38]. They found that the expression of VEGF in hypoxic tumor cells was affected by the circadian organization of molecular clockwork. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop in which clock and BMAL1 are positive regulators and period (Per) and cryptochrome (Cry) genes whose expression in the implanted tumor cells showed also a circadian oscillation act as negative ones. The levels of VEGF m-ribonucleic acid (RNA) in tumor cells implanted in mice rose substantially in response to hypoxia, but the levels fluctuated rhythmically in a circadian fashion. These findings support the notion that monitoring of circadian rhythm in VEGF production may be useful for choosing the most appropriate time of day (i.e., when VEGF production is increased) for administrating antiangiogenic agents [38].

In order to identify possible mechanisms underlying tumor progression related to circadian disrhythmicity, Yasumina et al. injected epidermoid HeLa cells in nude mice exposed to a 24-h light cycle (L/L) or to a "normal" 12-h light/dark cycle (L/D) [39]. A significant increase in tumor volume in the L/L group compared with the L/D group was observed. In addition, tumor microvessels and stroma were strongly increased in L/L mice but were not associated to an increase in the production of VEGF. DNA microarray analysis showed enhanced expression of WNT10A (wingless gene 10A). WNT10A could stimulate growth of both microvascular endothelial cells and fibroblasts in tumors from light-stressed mice, along with marked increases in angio-/stromagenesis [39]. Thus, WNT10A may be a novel angio-/ stromagenic growth factor. These findings also suggest that circadian disruption induces the progression of malignant tumors via a WNT signaling pathway in models involving tumor cells similar to that encountered in human NSCLC [39].

#### *3.2.3 Circadian regulation of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) pathways*

Binding parameters and constant dissociation of EGFR circadian variations, peaking late in dark span, were related to DNA synthesis activity variations in actively dividing mouse tissues [40]. EGFR by itself was found to be capable of phase shifting the prominent circadian rhythm of DNA synthesis, i.e., in the esophagus [40, 41]. Phosphorylation of the cyclic monophosphate (cAMP) response element-binding protein (CREB) and SER-133-phospho-CREB (PCREB) is a transcriptional factor that may regulate circadian cell rhythmicity [41]. Concentrations of EGF (and nerve growth factor (NGF)) were monitored in mouse saliva [42]; both growth factors exhibited identical diurnal variations, peaking between 12:00 and 20:00 h. Otherwise, the effect of EGF injections on cell kinetics of mouse tongue epithelium appeared to be time-dependent [41, 42].

Of interest, some studies were also performed in humans. Salivary (on the contrary to urinary or plasmatic) EGF level followed an apparent diurnal rhythm related to feed [43]. It was markedly reduced in the case of oral inflammation or head and neck cancer; in those situations, the capacity of oral mucosal defense could therefore be impaired [43].

**79**

matory disease [49].

*Integration of Chronobiological Concepts for NSCLC Management*

EGFR is present in the majority of tumor cells in head and neck cancer [43]. Though circadian rhythmicities in cell proliferation and clock genes have been demonstrated in oral mucosal [45] and in related cancers [1, 2], so far, no study has dealt with the search for circadian variability in EGFR expression in squamous cell carcinomas. However, a circadian rhythm in plasmatic EGF level (with an acrophase around 14:20, a peak-to-trough interval of 26% and a superimposed 12-h

As stated earlier, the circadian axis comprises a central clock (SCN; paraventricular areas) and a downstream network of hypothalamic relay station that modulates

Communication between the clock and these hypothalamic signaling centers is

frequency) has been reported in metastatic breast cancer patients [45].

arousal, feeding, and sleeping behavior among others [4, 46] (**Figure 2**).

mediated in part by diffusible substances that include ligands of the EGFR [4–8, 44, 46] (**Figures 1** and **2**). A significant functional role for EGFR in the suprachiasmatic nucleus is suggested by recent findings showing that epidermal growth factor receptor and its ligand TGF-α are highly expressed in the suprachiasmatic nucleus. Also EGFR activation induces behavioral and physiological effects, strengthening the notion that EGFR can modulate suprachiasmatic nucleus neural function and behavior [4–8, 44, 46]. Furthermore, Vadigepalli et al. confirmed that gene expression response to EGFR is circadian time dependent [8]; this response includes several genes encoding different neuropeptide receptors, ion channels, and kinases. In order to hypothesize the transcription factors underlying the EGFR response, different circadian time-dependent gene expression groups were analyzed for enriched transcriptional regulatory elements in the promoters. Results indicate that several transcription factors such as Elk 1 and cAMP-responsive element-binding protein/activating transcription factor family, known to be "input points" to the core clock network, are playing a role. Taken together, these results indicate that EGFR has a circadian time-dependent neuromodulatory function in

Diurnal variation in immune and inflammatory function is evident in the

Disruption of the circadian clockwork in macrophages (primary effector cells of the innate immune system) by conditional targeting of a key clock gene (bmal1) removed all temporal gating of endotoxin-induced cytokine response in cultured cells and in vivo. The loss of circadian gating was coincidental with suppressed REV-ERBα expression. This work demonstrates that the macrophage clockwork provides temporal gating of systemic responses to endotoxin and identifies REV-ERBα as the key link between the clock and immune function. REV-ERBα may therefore represent a unique therapeutic target in human inflam-

Also mechanistically, Bmal1 deficiency in macrophages increases pyruvate kinase M2 (PKM2) expression and lactate production, which is required for expression of the immune checkpoint protein PD-L1 (programmed cell death-ligand 1) in a STAT1 dependent manner (signal transducer and activator of transcription 1). Consequently, targeted ablation of PKM2 in myeloid cells or administration of anti-PD-L1-neutralizing

Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, controls fundamental aspects of the immune response [47–49]. Examples include the BMAL1-CLOCK heterodimerregulating Toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppress-

*DOI: http://dx.doi.org/10.5772/intechopen.85710*

the suprachiasmatic nucleus [7, 8].

*3.2.4 Circadian regulation of immune pathways*

ing the induction of interleukin-6 (IL-6) [49].

physiology and pathology of animals and humans [47, 48].

#### *Integration of Chronobiological Concepts for NSCLC Management DOI: http://dx.doi.org/10.5772/intechopen.85710*

*Chronobiology - The Science of Biological Time Structure*

dark span) [36]. This finding strongly suggests that tumor tissue blood flow has a determining influence on tumor proliferative activity and that tumor growth is

These results were confirmed by Blumenthal et al. who also showed that the blood flow rhythm may differ between tumor and normal tissues, thus creating a window of opportunity when tumors could be targeted with a therapeutic agent

The molecular mechanism regulating circadian expression of VEGF in tumor cells (Lewis lung carcinoma cells among others) has been investigated by Koyanagi et al. [38]. They found that the expression of VEGF in hypoxic tumor cells was affected by the circadian organization of molecular clockwork. The core circadian oscillator is composed of an autoregulatory transcription-translation feedback loop in which clock and BMAL1 are positive regulators and period (Per) and cryptochrome (Cry) genes whose expression in the implanted tumor cells showed also a circadian oscillation act as negative ones. The levels of VEGF m-ribonucleic acid (RNA) in tumor cells implanted in mice rose substantially in response to hypoxia, but the levels fluctuated rhythmically in a circadian fashion. These findings support the notion that monitoring of circadian rhythm in VEGF production may be useful for choosing the most appropriate time of day (i.e., when VEGF production

In order to identify possible mechanisms underlying tumor progression related to circadian disrhythmicity, Yasumina et al. injected epidermoid HeLa cells in nude mice exposed to a 24-h light cycle (L/L) or to a "normal" 12-h light/dark cycle (L/D) [39]. A significant increase in tumor volume in the L/L group compared with the L/D group was observed. In addition, tumor microvessels and stroma were strongly increased in L/L mice but were not associated to an increase in the production of VEGF. DNA microarray analysis showed enhanced expression of WNT10A (wingless gene 10A). WNT10A could stimulate growth of both microvascular endothelial

cells and fibroblasts in tumors from light-stressed mice, along with marked increases in angio-/stromagenesis [39]. Thus, WNT10A may be a novel angio-/ stromagenic growth factor. These findings also suggest that circadian disruption induces the progression of malignant tumors via a WNT signaling pathway in models involving tumor cells similar to that encountered in human NSCLC [39].

*3.2.3 Circadian regulation of epidermal growth factor (EGF) and epidermal* 

Binding parameters and constant dissociation of EGFR circadian variations, peaking late in dark span, were related to DNA synthesis activity variations in actively dividing mouse tissues [40]. EGFR by itself was found to be capable of phase shifting the prominent circadian rhythm of DNA synthesis, i.e., in the esophagus [40, 41]. Phosphorylation of the cyclic monophosphate (cAMP) response element-binding protein (CREB) and SER-133-phospho-CREB (PCREB) is a transcriptional factor that may regulate circadian cell rhythmicity [41].

Concentrations of EGF (and nerve growth factor (NGF)) were monitored in mouse saliva [42]; both growth factors exhibited identical diurnal variations, peaking between 12:00 and 20:00 h. Otherwise, the effect of EGF injections on cell kinetics

Of interest, some studies were also performed in humans. Salivary (on the contrary to urinary or plasmatic) EGF level followed an apparent diurnal rhythm related to feed [43]. It was markedly reduced in the case of oral inflammation or head and neck cancer; in those situations, the capacity of oral mucosal defense

of mouse tongue epithelium appeared to be time-dependent [41, 42].

*growth factor receptor (EGFR) pathways*

influenced by circadian variation in tumor tissue blood flow [36].

such as vascular endothelial growth factor (VEGF) inhibitors [37].

is increased) for administrating antiangiogenic agents [38].

**78**

could therefore be impaired [43].

EGFR is present in the majority of tumor cells in head and neck cancer [43]. Though circadian rhythmicities in cell proliferation and clock genes have been demonstrated in oral mucosal [45] and in related cancers [1, 2], so far, no study has dealt with the search for circadian variability in EGFR expression in squamous cell carcinomas. However, a circadian rhythm in plasmatic EGF level (with an acrophase around 14:20, a peak-to-trough interval of 26% and a superimposed 12-h frequency) has been reported in metastatic breast cancer patients [45].

As stated earlier, the circadian axis comprises a central clock (SCN; paraventricular areas) and a downstream network of hypothalamic relay station that modulates arousal, feeding, and sleeping behavior among others [4, 46] (**Figure 2**).

Communication between the clock and these hypothalamic signaling centers is mediated in part by diffusible substances that include ligands of the EGFR [4–8, 44, 46] (**Figures 1** and **2**). A significant functional role for EGFR in the suprachiasmatic nucleus is suggested by recent findings showing that epidermal growth factor receptor and its ligand TGF-α are highly expressed in the suprachiasmatic nucleus. Also EGFR activation induces behavioral and physiological effects, strengthening the notion that EGFR can modulate suprachiasmatic nucleus neural function and behavior [4–8, 44, 46]. Furthermore, Vadigepalli et al. confirmed that gene expression response to EGFR is circadian time dependent [8]; this response includes several genes encoding different neuropeptide receptors, ion channels, and kinases. In order to hypothesize the transcription factors underlying the EGFR response, different circadian time-dependent gene expression groups were analyzed for enriched transcriptional regulatory elements in the promoters. Results indicate that several transcription factors such as Elk 1 and cAMP-responsive element-binding protein/activating transcription factor family, known to be "input points" to the core clock network, are playing a role. Taken together, these results indicate that EGFR has a circadian time-dependent neuromodulatory function in the suprachiasmatic nucleus [7, 8].

#### *3.2.4 Circadian regulation of immune pathways*

Diurnal variation in immune and inflammatory function is evident in the physiology and pathology of animals and humans [47, 48].

Studies highlight the extent to which the molecular clock, most notably the core clock proteins BMAL1, CLOCK, and REV-ERBα, controls fundamental aspects of the immune response [47–49]. Examples include the BMAL1-CLOCK heterodimerregulating Toll-like receptor 9 (TLR9) expression and repressing expression of the inflammatory monocyte chemokine ligand (CCL2) as well as REV-ERBα suppressing the induction of interleukin-6 (IL-6) [49].

Disruption of the circadian clockwork in macrophages (primary effector cells of the innate immune system) by conditional targeting of a key clock gene (bmal1) removed all temporal gating of endotoxin-induced cytokine response in cultured cells and in vivo. The loss of circadian gating was coincidental with suppressed REV-ERBα expression. This work demonstrates that the macrophage clockwork provides temporal gating of systemic responses to endotoxin and identifies REV-ERBα as the key link between the clock and immune function. REV-ERBα may therefore represent a unique therapeutic target in human inflammatory disease [49].

Also mechanistically, Bmal1 deficiency in macrophages increases pyruvate kinase M2 (PKM2) expression and lactate production, which is required for expression of the immune checkpoint protein PD-L1 (programmed cell death-ligand 1) in a STAT1 dependent manner (signal transducer and activator of transcription 1). Consequently, targeted ablation of PKM2 in myeloid cells or administration of anti-PD-L1-neutralizing antibody or supplementation with recombinant interleukin-7 (IL-7) facilitates microbial clearance, inhibits T cell apoptosis, reduces multiple organ dysfunction, and reduces septic death in Bmal1-deficient mice [47–49].

### **4. Chronopharmacology of anticancer drugs active against human NSCLC**

#### **4.1 Animal data**

Circadian variation in pharmacokinetics (PK) has been observed in rodents for all the drugs routinely administered to LC patients, i.e., pyrimidine derivatives, anthracyclines, vinca-alkaloïds (vinorelbine), topoisomerase inhibitors, taxanes, platinum derivatives, gemcitabine, other antimetabolites, etc. [1, 3–5]. These chrono-PK were expressed though circadian-varying metabolization, detoxification, excretion, and also maximal concentration (CMax) or area under the curve (AUC) [1, 3–5].

Chronotolerance has been observed in rodent studies long time ago for any chemotherapy agents routinely used for NSCLC patients [1–5]. As a recent example, best tolerance and chronoefficacy of gemcitabine alone or in combination with cisplatin were observed with best antitumor efficacy when both drugs were given around their least toxic time schedule, respectively, 11 and 15 hours after light onset (HALO) in animal facility [50]. In an older study, Flentje et al. had also documented the circadian chronoefficacy of cyclophosphamide (CPA) in LLC [51].

Chronotolerance to an experimental radioimmunotherapy with 131 I-anticarcinoembryonic antigen (CEA) IgG was reported [52]. A 30% increase in maximum tolerated dose was possible when the drug was given at the trough of the bone marrow division activity (around 9 HALO) [52].

Clock, as a member of histone acetyltransferases, controls acetylation of histone 4 required for repair of DNA double-strand breaks thanks to several repair genes such as excision repair cross-complementing group 1 (ERCC1) or activator protein 1 (AP1) [6]. Expression of histone acetyltransferase genes is associated with cisplatin resistance [6, 53]. Histone acetyltransferase inhibition (i.e., by vorinostat) may increase carboplatin and paclitaxel activity in NSCLC cells [54]. The acetyl-CoAbinding motive is found in clock and shows sequence similarity with MYST members, i.e., Tip 60. Tip 60 which is overexpressed in human epidermoid cisplatin-resistant cancer cells [53] exerts a control regulation on several genes implicated in DNA repair (i.e., ERCC1 and AP1) [53].

Furthermore, the promoter region of the Tip 60 gene contains several E-boxes, and its expression is regulated by the E-box-binding circadian transcription factor clock! Thus, clock and Tip 60 regulate not only transcription but also DNA repair, through periodic (diurnal) histone acetylation in cell populations that can be found in human NSCLC [53].

Finally, of interest, diurnal-varying pharmacokinetics of erlotinib (a largely used tyrosine kinase inhibitor (TKI) for treating human NSCLC) has been reported both in xenograft-bearing nude mice [55] and in Lewis tumor-bearing mice. [56]. Circadian rhythm plays a critical role in the pharmacokinetics of erlotinib in mice, and the mechanisms may be attributed to gene expression rhythms of drug-metabolizing enzymes in liver tissues [56]. The inhibitory effect of erlotinib on phosphorylation of EGFR, AKT (type of serine/threonine protein kinase, also called protein kinase B), and mitogen-activated protein kinase (MAPK) varies with its administration time. The results indicate that the antitumor effect of erlotinib is more potent when the drug is administered when the activities of EGFR and its downstream factors increase [55, 56].

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**Figure 3.**

*Integration of Chronobiological Concepts for NSCLC Management*

In human as well, pharmacokinetics of some major drugs used in NSCLC have been reported to be circadian varying [1–5, 28, 44]. Circadian variation of plasma 5-fluorouracil (5 FU) concentration has been repeatedly observed when the drug is infused for a few days at a constant rate [57, 58]. This was reported when the drug was given either as a single agent or with a platinum derivative [58]. Similarly, plasmatic concentration of vindesine, a semisynthetic vinca-alkaloïd derived from vinblastine, exhibit circadian variation with peak between 9 am and 3 pm, when infused at a constant rate for 48 h [59]. Also the fixation of platinum ion to plasma proteins was shown to be circadian varying with an acrophase during late afternoon [60]. More recent assessment of circadian variability of cisplatin pharmacokinetics confirmed that cisplatin clearance was 1.38- and 1. 22-fold higher for total and unbound drug with administration at 06:00 pm vs.

Host chronotolerance to anticancer drugs used in NSCLC patients has also been observed in clinical practice. Pyrimidine derivatives such as 5 FU are less toxic when infused during nighttime sleep [1, 3–5, 57, 58]. Also platinum derivatives such as cisplatin, carboplatin, and oxaliplatin are better tolerated between 3 and 6 pm while anthracyclines are less toxic in the morning [1, 3–5, 57, 58]. The first reported chronotherapy randomized trial, based on diurnal cell kinetics, treating mostly NSCLC patients, compared a 40-h sequential chemotherapy beginning either at 10 am or at 10 pm [28]. In this study, patients who received the sequential chronotherapy from 10 am experienced significantly greater

Focan et al. also reported on host chronotolerance of 124 chemotherapy-naïve advanced NSCLC patients, receiving randomly etoposide for 3 days either at 6 am

*Programs of ambulatory chronotherapy with 5 FU, folinic acid (leucovorin-LV), and carboplatin (CBDCA). The reference schedule is compared to two others with varying peaks (−8 h; +8 h). Daily delivery is automatically repeated by a chrono-programmable pump (Melodie) five times every 21 days (FFC5\_16).*

*DOI: http://dx.doi.org/10.5772/intechopen.85710*

**5. Human beings**

06:00 am [61].

granulocyte toxicity [14, 28].
