Gender medicine and oncology: report and consensus of an ESMO workshop.

BACKGROUND: The importance of sex and gender as modulators of disease biology and treatment outcomes is well known in other disciplines of medicine, such as cardiology, but remains an undervalued issue in oncology. Considering the increasing evidence for their relevance, European Society for Medical Oncology decided to address this topic and organized a multidisciplinary workshop in Lausanne, Switzerland, on 30 November and 1 December 2018. DESIGN: Twenty invited faculty members and 40 selected physicians/scientists participated. Relevant content was presented by faculty members on the basis of a literature review conducted by each speaker. Following a moderated consensus session, the final consensus statements are reported here. RESULTS: Clinically relevant sex differences include tumour biology, immune system activity, body composition and drug disposition and effects. The main differences between male and female cells are sex chromosomes and the level of sexual hormones they are exposed to. They influence both local and systemic determinants of carcinogenesis. Their effect on carcinogenesis in non-reproductive organs is largely unknown. Recent evidence also suggests differences in tumour biology and molecular markers. Regarding body composition, the difference in metabolically active, fat-free body mass is one of the most prominent: in a man and a woman of equal weight and height, it accounts for 80% of the man's and 65% of the woman's body mass, and is not taken into account in body-surface area based dosing of chemotherapy. CONCLUSION: Sex differences in cancer biology and treatment deserve more attention and systematic investigation. Interventional clinical trials evaluating sex-specific dosing regimens are necessary to improve the balance between efficacy and toxicity for drugs with significant pharmacokinetic differences. Especially in diseases or disease subgroups with significant differences in epidemiology or outcomes, men and women with non-sex-related cancers should be considered as biologically distinct groups of patients, for whom specific treatment approaches merit consideration.

The p63 target HBP1 is required for skin differentiation and stratification.

Genetic experiments established that p63 is crucial for the development and maintenance of pluristratified epithelia. In the RNA interference (RNAi) screening for targets of p63 in keratinocytes, we identified the transcription factor, High Mobility Group (HMG) box protein 1 (HBP1). HBP1 is an HMG-containing repressor transiently induced during differentiation of several cell lineages. We investigated the relationship between the two factors: using RNAi, overexpression, chromatin immunoprecipitations and transient transfections with reporter constructs, we established that HBP1 is directly repressed by p63. This was further confirmed in vivo by evaluating expression in p63 knockout mice and in transgenics expressing p63 in basal keratinocytes. Consistent with these findings, expression of HBP1 increases upon differentiation of primary keratinocytes and HaCaT cells in culture, and it is higher in the upper layers of human skin. Inactivation of HBP1 by RNAi prevents differentiation of keratinocytes and stratification of organotypic skin cultures. Finally, we analyzed the keratinocyte transcriptomes after HBP1 RNAi; in addition to repression of growth-promoting genes, unexpected activation of differentiation genes was uncovered, coexisting with repression of other genes involved in epithelial cornification. Our data indicate that suppression of HBP1 is part of the growth-promoting strategy of p63 in the lower layers of epidermis and that HBP1 temporally coordinates expression of genes involved in stratification, leading to the formation of the skin barrier.

Notch tumor suppressor function.

Cancer development results from deregulated control of stem cell populations and alterations in their surrounding environment. Notch signaling is an important form of direct cell-cell communication involved in cell fate determination, stem cell potential and lineage commitment. The biological function of this pathway is critically context dependent. Here we review the pro-differentiation role and tumor suppressing function of this pathway, as revealed by loss-of-function in keratinocytes and skin, downstream of p53 and in cross-connection with other determinants of stem cell potential and/or tumor formation, such as p63 and Rho/CDC42 effectors. The possibility that Notch signaling elicits a duality of signals, involved in growth/differentiation control and cell survival will be discussed, in the context of novel approaches for cancer therapy.

Cross talk among calcineurin, Sp1/Sp3, and NFAT in control of p21(WAF1/CIP1) expression in keratinocyte differentiation.

Calcium functions as a trigger for the switch between epithelial cell growth and differentiation. We report here that the calcium/calmodulin-dependent phosphatase calcineurin is involved in this process. Treatment of primary mouse keratinocytes with cyclosporin A, an inhibitor of calcineurin activity, suppresses the expression of terminal differentiation markers and of p21(WAF1/Cip1) and p27(KIP1), two cyclin-dependent kinase inhibitors that are usually induced with differentiation. In parallel with down-modulation of the endogenous genes, suppression of calcineurin function blocks induction of the promoters for the p21(WAF1/Cip1) and loricrin differentiation marker genes, whereas activity of these promoters is enhanced by calcineurin overexpression. The calcineurin- responsive region of the p21 promoter maps to a 78-bp Sp1/Sp3-binding sequence next to the TATA box, and calcineurin induces activity of the p21 promoter through Sp1/Sp3-dependent transcription. We find that the endogenous NFAT-1 and -2 transcription factors, major downstream targets of calcineurin, associate with Sp1 in keratinocytes in a calcineurin-dependent manner, and calcineurin up-regulates Sp1/Sp3-dependent transcription and p21 promoter activity in synergism with NFAT1/2. Thus, our study reveals an important role for calcineurin in control of keratinocyte differentiation and p21 expression, and points to a so-far-unsuspected interconnection among this phosphatase, NFATs, and Sp1/Sp3-dependent transcription.

Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation.

The role of Notch signaling in growth/differentiation control of mammalian epithelial cells is still poorly defined. We show that keratinocyte-specific deletion of the Notch1 gene results in marked epidermal hyperplasia and deregulated expression of multiple differentiation markers. In differentiating primary keratinocytes in vitro endogenous Notch1 is required for induction of p21WAF1/Cip1 expression, and activated Notch1 causes growth suppression by inducing p21WAF1/Cip1 expression. Activated Notch1 also induces expression of 'early' differentiation markers, while suppressing the late markers. Induction of p21WAF1/Cip1 expression and early differentiation markers occur through two different mechanisms. The RBP-Jkappa protein binds directly to the endogenous p21 promoter and p21 expression is induced specifically by activated Notch1 through RBP-Jkappa-dependent transcription. Expression of early differentiation markers is RBP-Jkappa-independent and can be induced by both activated Notch1 and Notch2, as well as the highly conserved ankyrin repeat domain of the Notch1 cytoplasmic region. Thus, Notch signaling triggers two distinct pathways leading to keratinocyte growth arrest and differentiation.

A PKC-eta/Fyn-dependent pathway leading to keratinocyte growth arrest and differentiation.

Growth control of epithelial cells differs substantially from other cell types. Activation of Fyn, a Src kinase family member, is required for normal keratinocyte differentiation. We report that increased Fyn activity by itself suppresses growth of keratinocytes, but not dermal fibroblasts, through downmodulation of EGF receptor (EGFR) signaling. Protein kinase C-eta has also been implicated in keratinocyte growth/differentiation control. We show that growth suppression of keratinocytes by PKC-eta depends mostly on Fyn. PKC-eta activity is both necessary and sufficient for Fyn activation, PKC-eta and Fyn are found in association, and recombinant PKC-eta directly activates Fyn. Thus, our findings reveal a direct cross talk between PKC-eta and Fyn, which presides over the decision between keratinocyte (epithelial) cell growth and differentiation.

Defective neurogenesis in citron kinase knockout mice by altered cytokinesis and massive apoptosis.

Citron-kinase (Citron-K) has been proposed by in vitro studies as a crucial effector of Rho in regulation of cytokinesis. To further investigate in vivo its biologic functions, we have inactivated Citron-K gene in mice by homologous recombination. Citron-K-/- mice grow at slower rates, are severely ataxic, and die before adulthood as a consequence of fatal seizures. Their brains display defective neurogenesis, with depletion of specific neuronal populations. These abnormalities arise during development of the central nervous system due to altered cytokinesis and massive apoptosis. Our results indicate that Citron-K is essential for cytokinesis in vivo but only in specific neuronal precursors. Moreover, they suggest a novel molecular mechanism for a subset of human malformative syndromes of the CNS.

Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.

The regression phase of the hair cycle (catagen) is an apoptosis-driven process accompanied by terminal differentiation, proteolysis, and matrix remodeling. As an inhibitor of keratinocyte proliferation and inductor of keratinocyte apoptosis, transforming growth factor beta1 (TGF-beta1) has been proposed to play an important role in catagen regulation. This is suggested, for example, by maximal expression of TGF-beta1 and its receptors during late anagen and the onset of catagen of the hair cycle. We examined the potential involvement of TGF-beta1 in catagen control. We compared the first spontaneous entry of hair follicles into catagen between TGF-beta1 null mice and age-matched wild-type littermates, and assessed the effects of TGF-beta1 injection on murine anagen hair follicles in vivo. At day 18 p.p., hair follicles in TGF-beta1 -/- mice were still in early catagen, whereas hair follicles of +/+ littermates had already entered the subsequent resting phase (telogen). TGF-beta1-/- mice displayed more Ki-67-positive cells and fewer apoptotic cells than comparable catagen follicles from +/+ mice. In contrast, injection of TGF-beta1 into the back skin of mice induced premature catagen development. In addition, the number of proliferating follicle keratinocytes was reduced and the number of TUNEL + cells was increased in the TGF-beta1-treated mice compared to controls. Double visualization of TGF-beta type II receptor (TGFRII) and TUNEL reactivity revealed colocalization of apoptotic nuclei and TGFRII in catagen follicles. These data strongly support that TGF-beta1 ranks among the elusive endogenous regulators of catagen induction in vivo, possibly via the inhibition of keratinocyte proliferation and induction of apoptosis. Thus, TGF-betaRII agonists and antagonists may provide useful therapeutic tools for human hair growth disorders based on premature or retarded catagen development (effluvium, alopecia, hirsutism).

The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis.

Hair follicle development serves as an excellent model to study control of organ morphogenesis. Three specific isoforms of TGF-beta exist which exhibit a distinct pattern of expression during hair follicle morphogenesis. To clarify the still elusive role of these factors in hair follicle development, we have used a combined genetic and functional approach: analysis of hair follicle development in mice with disruptions of the TGF-beta1, 2, and 3 genes was coupled with a direct functional test of the effect of added purified factors on fetal hair follicle development in skin organ cultures. TGF-beta2 null mice exhibited a profound delay of hair follicle morphogenesis, with a 50% reduced number of hair follicles. In contrast to hair follicle development, growth and differentiation of interfollicular keratinocytes proceeded unimpaired. Unlike TGF-beta2-/- mice, mice with a disruption of the TGF-beta1 gene showed slightly advanced hair follicle formation, while lack of the TGF-beta3 gene did not have any effects. Treatment of wild-type, embryonic skin explants (E14.5 or E15.5) with TGF-beta2 protein in either soluble form or slow release beads induced hair follicle development and epidermal hyperplasia, while similar TGF-beta1 treatment exerted suppressive effects. Thus, the TGF-beta2 isoform plays a specific role, not shared by the other TGF-beta isoforms, as an inducer of hair follicle morphogenesis and is both required and sufficient to promote this process.

p21(WAF1/Cip1) functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential.

p21(WAF1/Cip1) is one of the best characterized downstream targets of p53, and the growth suppressing function of this cyclin-dependent kinase inhibitor is well established. However, whether p21 exerts a tumor-suppressing function of its own remains to be established. We report here that, similarly to loss of p53, disruption of the p21(WAF1/Cip1) gene results in a markedly increased susceptibility to chemically induced skin carcinoma formation, whereas the number of papillomas is reduced. Previous evidence indicates that malignant versus benign keratinocyte tumor formation is likely to involve distinct target-cell populations with a different commitment to differentiation. In parallel with the increased susceptibility to carcinoma formation, loss of p21(WAF1/Cip1) was found to promote keratinocyte subpopulations with increased growth/differentiation potential, including clonal growth capability, reversible commitment to differentiation, and capability to generate all types of terminally differentiated keratinocytes present in vivo, not only in the interfollicular epidermis but also in hair follicles. Thus, these findings have revealed a function of p21 as a suppressor of malignant but not benign skin-tumor formation and a determinant of the growth/differentiation potential of keratinocyte subpopulations.

TGF-beta3, but not TGF-beta1, protects keratinocytes against 12-O-tetradecanoylphorbol-13-acetate-induced cell death in vitro and in vivo.

We have examined the role that individual TGF-beta isoforms, and in particular TGF-beta3, play in control of epidermal homeostasis. Mice with a knockout mutation of the TGF-beta3 gene die a few hours after birth. A full-thickness skin grafting approach was used to investigate the postnatal development and homeostatic control of the skin of these mice. Grafted skin of mice with a disruption of the TGF-beta3 gene developed similarly to grafts of wild type and TGF-beta1 knockout animals. However, a strikingly different response was observed after acute treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). When exposed to TPA, the grafted skin of wild type and TGF-beta1 knockout mice underwent a hyperplastic response similar to that of normal mouse skin. In marked contrast, TPA treatment of TGF-beta3 knockout grafts induced widespread areas of keratinocyte cell death. Analysis of cultured keratinocytes treated with purified TGF-beta isoforms revealed that TGF-beta3 plays a direct and specific function in protecting keratinocytes against TPA-induced cell death. The protective function of TGF-beta3 on TPA-induced cell death was not because of general suppression of the signaling pathways triggered by this agent, as ERK1/2 activation occurred to a similar if not greater extent in TGF-beta3-treated versus control keratinocytes. Instead, TGF-beta3 treatment led to a significant reduction in TPA-induced c-Jun N-terminal kinase activity, which was associated and possibly explained by specific counteracting effects of TGF-beta3 on TPA-induced disruption of keratinocyte focal adhesions.

Signal transduction pathways controlling the switch between keratinocyte growth and differentiation.

Self-renewing epithelia are characterized by a high turnover rate and a fine balance between growth and differentiation. Such a balance is influenced by many exogenous factors, including gradients of diffusible molecules, cell/substrate adhesion contacts, and direct cell-cell communication. The inter-connection between these various extracellular signals and underlying intracellular pathways is clearly of great interest. Primary keratinocytes of either human or murine origin provide an ideal experimental system to elucidate early signaling events involved in the control of epithelial differentiation. Relative to established cell lines, use of a primary system eliminates the possibility of alterations in critical regulatory events which may occur during prolonged propagation in culture. Primary keratinocytes are easily grown in large numbers, and their differentiation can be induced under well-defined culture conditions. The ensuing rapid and homogeneous response is amenable to careful biochemical analysis. Gene transfer technology (transient transfections, adenoviral and retroviral vectors), together with the use of keratinocytes derived from gene knockout and transgenic mice, makes it possible to assess the specific contribution of individual genes to the control of the differentiation process. This review focuses on the significant progress that has been made over the last few years in our understanding of the specific signals that trigger keratinocyte differentiation, the underlying signaling pathways, and how they impinge on specific transcription and cell-cycle control mechanisms associated with the onset of keratinocyte differentiation. Recent developments and future directions in this important area of research will be highlighted.

Citron rho-interacting kinase, a novel tissue-specific ser/thr kinase encompassing the Rho-Rac-binding protein Citron.

We have identified a novel serine/threonine kinase belonging to the myotonic dystrophy kinase family. The kinase can be produced in at least two different isoforms: a approximately 240-kDa protein (Citron Rho-interacting kinase, CRIK), in which the kinase domain is followed by the sequence of Citron, a previously identified Rho/Rac binding protein; a approximately 54-kDa protein (CRIK-short kinase (SK)), which consists mostly of the kinase domain. CRIK and CRIK-SK proteins are capable of phosphorylating exogenous substrates as well as of autophosphorylation, when tested by in vitro kinase assays after expression into COS7 cells. CRIK kinase activity is increased severalfold by coexpression of costitutively active Rho, while active Rac has more limited effects. Kinase activity of endogenous CRIK is indicated by in vitro kinase assays after immunoprecipitation with antibodies recognizing the Citron moiety of the protein. When expressed in keratinocytes, full-length CRIK, but not CRIK-SK, localizes into corpuscular cytoplasmic structures and elicits recruitment of actin into these structures. The previously reported Rho-associated kinases ROCK I and II are ubiquitously expressed. In contrast, CRIK exhibits a restricted pattern of expression, suggesting that this kinase may fulfill a more specialized function in specific cell types.

In vivo antitumor effect of retrovirus-mediated gene transfer of the adenovirus E1a gene.

The adenovirus E1a gene has been shown to be associated with high sensitivity to DNA-damaging agents and a decrease in the tumorigenicity of some human malignant cell lines. We have analyzed the tumorigenicity of the murine epidermoid carcinoma cell lines MSC11A5 and HaCa4, which have constitutive E1a expression, after the concomitant injection of retrovirus E1a producer cells with the carcinoma cells and even after the intratumoral injection of the E1a producer cells. The level of E1a expression was studied by Western blotting. Tumors induced by carcinoma cell lines expressing E1a showed greater latencies and less tumorigenicity. In the spindle cell carcinomas MSC11A5, E1a gene expression partially blocked tumorigenicity. Similar results were obtained after the concomitant injection of the carcinoma cells and the retrovirus E1a producer cells. Intratumoral injection of retrovirus E1a producer cells was associated with a significant delay of tumorigenicity. By transfection with different E1a mutants Ntd1598, d1922/947, and d1787N, we observed that only the mutant that has complete CR2 domains is associated with the decrease in tumorigenicity. According to these results, we conclude that, at least in these carcinoma cell lines, E1a expression exerts a significant antitumor effect in vivo that is mediated by the CR2 region of E1a gene. We propose that injection of retrovirus E1a producer cells may be a novel therapeutic approach in cancer.

Differentiation-specific increase in ALA-induced protoporphyrin IX accumulation in primary mouse keratinocytes.

A treatment regimen that takes advantage of the induction of intracellular porphyrins such as protoporphyrin IX (PPIX) by exposure to exogenous 5-amino-laevulinic acid (ALA) followed by localized exposure to visible light represents a promising new approach to photodynamic therapy (PDT). Acting upon the suggestion that the effectiveness of ALA-dependent PDT may depend upon the state of cellular differentiation, we investigated the effect of terminal differentiation upon ALA-induced synthesis of and the subsequent phototoxicity attributable to PPIX in primary mouse keratinocytes. Induction of keratinocyte differentiation augmented intracellular PPIX accumulation in cells treated with ALA. These elevated PPIX levels resulted in an enhanced lethal photodynamic sensitization of differentiated cells. The differentiation-dependent increase in cellular PPIX levels resulted from several factors including: (a) increased ALA uptake, (b) enhanced PPIX production and (c) decreased PPIX export into the culture media. Simultaneously, steady-state levels of coproporphyrinogen oxidase mRNA increased but aminolaevulinic acid dehydratase mRNA levels remained unchanged. From experiments using 12-o-tetradecanoylphorbol-13-acetate, transforming growth factor beta 1 and calcimycin we demonstrated that the increase in PPIX concentration in terminally differentiating keratinocytes is calcium- and differentiation specific. Stimulation of the haem synthetic capacity is seen in primary keratinocytes, but not in PAM 212 cells that fail to undergo differentiation. Interestingly, increased PPIX formation and elevated coproporphyrinogen oxidase mRNA levels are not limited to differentiating keratinocytes; these were also elevated in the C2C12 myoblast and the PC12 adrenal cell lines upon induction of differentiation. Overall, the therapeutic implications of these results are that the effectiveness of ALA-dependent PDT depends on the differentiation status of the cell and that this may enable selective targeting of several tissue types.

Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control.

The cyclin-dependent kinase inhibitor p21(Cip1/WAF1) has been implicated as an inducer of differentiation. However, although expression of p21 is increased in postmitotic cells immediately adjacent to the proliferative compartment, its expression is decreased in cells further along the differentiation program. Expression of the p21 protein was decreased in terminally differentiated primary keratinocytes of mice, and this occurred by a proteasome-dependent pathway. Forced expression of p21 in these cells inhibited the expression of markers of terminal differentiation at both the protein and messenger RNA levels. These inhibitory effects on differentiation were not observed with a carboxyl-terminal truncation mutant or with the unrelated cyclin-dependent kinase inhibitor p16(INK4a), although all these molecules exerted similar inhibition of cell growth. These findings reveal an inhibitory role of p21 in the late stages of differentiation that does not result from the effects of p21 on the cell cycle.

Primary mouse keratinocyte cultures contain hair follicle progenitor cells with multiple differentiation potential.

The interfollicular epidermis contains a single type of terminally differentiated keratinocytes, whereas hair follicles are composed of a minimum of six or seven distinct types. Whether or not these various populations of terminally differentiated keratinocytes originate from one or more progenitor cells has not been established. A related and important question is whether keratinocyte progenitor cells with a pluripotent potential, able to form not only epidermis but also hair follicles, can be maintained in vitro for any period of time. We have addressed these questions using skin reconstitution assays with admixed populations of genetically labeled, cultured keratinocytes. Examination of reconstituted epidermis and hair follicles showed that neither was composed of a random mixture of differently labeled keratinocytes, as would be predicted if they originated from a random reassociation of cells. Instead, the reconstituted interfollicular epidermis contained distinct columnar units, comprising all the overlying layers and most likely derived from a single progenitor cell. In contrast, hair follicles were found to be composed of cells of multiple origin, with each population showing a striking localization to a separate concentric region. The vast majority of reconstituted follicles appeared to derive from a minimum of two or, in a significant fraction of cases, three progenitor cells, one for the generation of the shaft (cuticle, cortex, and medulla), one for the inner root sheath, and the third for the outer root sheath. The general implications of these findings for epidermis and hair follicle formation and for keratinocyte stem cell cultivation are discussed.

Involvement of the Sp3 transcription factor in induction of p21Cip1/WAF1 in keratinocyte differentiation.

The cyclin-dependent kinase inhibitor p21 is induced in several in vitro terminal differentiation systems as well as in differentiating tissues in vivo. To determine the mechanism responsible for p21 induction during differentiation of mouse primary keratinocytes, we performed a deletion analysis of the p21 promoter. The minimal region of the p21 promoter required for its induction in keratinocyte differentiation consists of a contiguous stretch of 78 base pairs, which contains a GC-rich region as well as the TATA box. We determined that transcription factors Sp1 and Sp3, present in primary keratinocyte nuclear extracts, bind the GC region concomitantly. Expression studies established that both Sp1 and Sp3 activate the p21 promoter, but showed that only Sp3 overexpression enhances promoter inducibility during differentiation. Furthermore, disruption of the GC-rich region dramatically decreases transcription factor binding as well as promoter activity and inducibility upon differentiation. The overexpression of either Sp1 or Sp3 restores the basal activity of the disrupted promoter, but only Sp3 can restore its inducibility. These findings show that both Sp1 and Sp3 can contribute to the basal activity of the p21 promoter, and establish Sp3 as a specific transcription factor involved in the induction of p21 promoter during keratinocyte differentiation.

The absence of p21Cip1/WAF1 alters keratinocyte growth and differentiation and promotes ras-tumor progression.

p21Cip1/WAF1 was the first cyclin-dependent kinase (CDK) inhibitor to be identified, as a mediator of p53 in DNA damage-induced growth arrest, cell senescence, and direct CDK regulation. p21 may also play an important role in differentiation-associated growth arrest, as its expression is augmented in many terminally differentiating cells. A general involvement of p21 in growth/differentiation control and tumor suppression has been questioned, as mice lacking p21 undergo a normal development, harbor no gross alterations in any of their organs, and exhibit no increase in spontaneous tumor development. However, a significant imbalance between growth and differentiation could be unmasked under conditions where normal homeostatic mechanisms are impaired. We report here that primary keratinocytes derived from p21 knockout mice, transformed with a ras oncogene, and injected subcutaneously into nude mice exhibit a very aggressive tumorigenic behavior, which is not observed with wild-type control keratinocytes nor with keratinocytes with a disruption of the closely related p27 gene. p21 knockout keratinocytes tested under well-defined in vitro conditions show a significantly increased proliferative potential, which is also observed but to a lesser extent with p27 knockout cells. More profound differences were found in the differentiation behavior of p21 versus p27 knockout keratinocytes, with p21 (but not p27) deficiency causing a drastic down-modulation of differentiation markers linked with the late stages of the keratinocyte terminal differentiation program. Thus, our results reveal a so far undetected role of p21 in tumor suppression, demonstrate that this function is specific as it cannot be attributed to the closely related p27 molecule, and point to an essential involvement of p21 in terminal differentiation control, which may account for its role in tumor suppression.