Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: Involvement in cancer invasion and metastasis

Accepted Manuscript Title: Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: involvement in cancer invasion and metastasis Author: Marco Cordani, Raffaella Pacchiana, Giovanna Butera, Gabriella D'Orazi, Aldo Scarpa, Massimo Donadelli PII: DOI: Reference:

S0304-3835(16)30207-5 http://dx.doi.org/doi: 10.1016/j.canlet.2016.03.046 CAN 12846

To appear in:

Cancer Letters

Received date: Revised date: Accepted date:

29-2-2016 29-3-2016 30-3-2016

Please cite this article as: Marco Cordani, Raffaella Pacchiana, Giovanna Butera, Gabriella D'Orazi, Aldo Scarpa, Massimo Donadelli, Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: involvement in cancer invasion and metastasis, Cancer Letters (2016), http://dx.doi.org/doi: 10.1016/j.canlet.2016.03.046. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cordani et al.

Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: involvement in cancer invasion and metastasis Marco Cordani1, Raffaella Pacchiana1, Giovanna Butera1, Gabriella D’Orazi2, Aldo Scarpa3, Massimo Donadelli1*

1

Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona,

Verona, Italy 2

Unit of Cellular Networks and Therapeutic Targets, Department of Research, Advanced Diagnostic, and

Technological Innovation, Regina Elena National Cancer Institute – IRCCS, Rome, Italy 3

Applied Research on Cancer Centre (ARC-Net) and Department of Diagnostics and Public Health,

University of Verona, Verona, Italy

*Corresponding author: Massimo Donadelli, PhD Department of Neuroscience, Biomedicine and Movement, Biochemistry Section, University of Verona. Strada Le Grazie 8, 37134 Verona, Italy phone: +39 045 8027281; fax: +39 045 8027170; e-mail: [email protected]

Running title: Mutant p53 regulates cancer secretome Keywords: mutant p53; cancer; secretome; microenvironment; cytokines.

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Cordani et al. Highlights    

Mutp53 contributes to extracellular matrix remodeling Mutp53 induces the secretion of pro-inflammatory, immunomodulatory cytokines Mutp53 stimulates secretion of lactate and induces extracellular acidification Mutp53 regulates the crosstalk between cancer and stromal cells

Abstract An ever-increasing number of studies highlight the role of mutant p53 proteins in the alteration of cancer cell secretome and in the modification of tumour microenvironment, sustaining an invasive phenotype of cancer cell. The knowledge of the molecular mechanisms underlying the interplay between mutant p53 proteins and the microenvironment is becoming fundamental for the identification of both efficient anticancer therapeutic strategies and novel serum biomarkers. In this review, we summarize the novel findings concerning the regulation of secreted molecules by cancer cells bearing mutant TP53 gene. In particular, we highlight data from available literature suggesting that mutant p53 proteins are able to : i) alter the secretion of enzymes involved in the modulation of extracellular matrix components; ii) alter the secretion of inflammatory cytokines; iii) increase the extracellular acidification; and iv) regulate the crosstalk between cancer and stromal cells.

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Cordani et al. 1. Introduction The vast majority of neoplastic diseases are solid tumours of epithelial origin, including lung, breast, prostate, colon, pancreas and ovary, which severely threaten human health [1]. Despite in recent years significant advances have been moved in the development of both diagnostic and therapeutic tools the ultimate effectiveness of cancer treatments still remains limited. Overall, this can be explained by various mechanisms including: i) innate or acquired cancer resistance to therapies due, for instance, to oncogenic gene mutations; ii) pharmacokinetic and bio-distribution hitches; and iii) the development of an adverse tumour microenvironment as the consequence of the crosstalk between cancer and stromal cells. This last feature is typical of solid cancers and it could play a key role in the mechanisms of tumour relapse and resistance to therapies. Indeed, it is reported that solid tumours take advantage of a co-evolution of neoplastic and stromal cells and that the extracellular matrix (ECM) plays a dynamic role in cancer invasion. All this complex cancermicroenvironment system is also strongly influenced by impaired vascularization and by interaction with the immune system cells [2]. Thus, in addition to various pathological events, such as genetic mutations, epigenetic modifications, genomic instability and induction of oxidative stress, tumorigenesis and cancer progression may also strongly depend on extrinsic factors secreted by cancer cells themselves or by stromal cells. Those secreted factors, i.e. cytokines/chemokines, proteases, growth and angiogenic factors, as well as other molecules having an oncogenic effect, may regulate the crosstalk between stroma-cancer cells and tumour microenvironment. Several biological pathways, such as Smad, PI3K, Jak/Stat, NF-kB, MAPK, CXCR2 and IL-1 have been indicated as able to orchestrate this complex crosstalk system [3-10]. However, despite the scientific research studies on the tumour microenvironment and the identification of secretory molecules are recently improved, the functional involvement of this multifaceted three-dimensional system remains to be deciphered in light of the clinical outcome of cancer therapy.

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Cordani et al. In recent years, relevant scientific evidence demonstrated a key role of mutant p53 (mutp53) in altering the secretion of proteins and signaling molecules. In this review, we highlight the recent findings that reveal the interplay between cancer cells bearing mutp53 and the surrounding tumour microenvironment. In particular, we will focus on the role of mutp53 proteins in cancer invasion and metastasis through: i) modulation of the extracellular matrix (ECM) components; ii) secretion of pro-inflammatory, immunomodulatory interleukins and cytokines; iii) modification of the extracellular pH; and iv) regulation of the crosstalk between tumour and stromal cells. These findings are summarized in Figure 1.

2. Alteration of p53 function is one of the most common events in cancer TP53 tumour suppressor gene encodes for a DNA-binding transcription factor that, upon activation, regulates sequence-specific target genes involved in cell growth inhibition, senescence and apoptosis, providing powerful intrinsic defence against cancer [11]. TP53 is a commonly mutated gene in human cancers, indicating that the presence of a functional p53 pathway is incompatible with neoplastic cell growth [12]. Despite the frequency of TP53 gene mutations (mainly missense mutations) can largely vary depending on the tumour type, they occur over 60% of all human tumours representing one of the most frequently mutated gene in cancer disease [13, 14]. These mutations often cause conformational changes in the mutp53 proteins leading to their inability to bind DNA and to regulate the transcription of wild-type (wt) p53 target genes [15]. This determines both the loss-of-function (LOF) of the tumour suppressor wtp53 protein and the acquisition of mutp53-driven oncogenic gain-of-function (GOF), promoting proliferation and aggressiveness of cancers through the stimulation of cell cycle, genomic instability, chemoresistance, invasion, metastasis and the counteraction of apoptosis and cellular senescence (Figure 2) [16-20]. Most of TP53 mutations induce protein conformational changes mostly stabilizing mutp53 proteins which accumulate at high levels in cancer cells [21]. Overall, the 4 Page 4 of 23

Cordani et al. oncogenic function of mutp53 proteins is carried out by mutp53 ability to directly interact with some transcription factors or repressors, thus altering the expression of several mRNA and microRNAs having a key role in the regulation of tumour progression [22-24].

3. Mutp53 contributes to extracellular matrix remodelling The role of mutp53 in ECM remodelling is multifaceted. Cancer metastasis is a leading cause of death in cancer patients and this phenomenon involves a plethora of events resulting in the ECM degradation which allows the tumour cells to invade the surrounding tissue and generate metastasis [25]. Matrix metalloproteinases (MMPs), in particular MMP-2 and MMP-9 [26], are secreted or transmembrane enzymes known to play a key role in the cancer invasion process by degrading multiple elements of the ECM, including laminin, collagen, and fibrous proteins [27]. On the opposite, tissue inhibitors of metalloproteinases (TIMPs) comprise a multifunctional protein family that regulates the activity of MMPs after their secretion into the extracellular microenvironment. This family consists of 4 members (TIMP 1–4) with activities in cellular processes such as cell differentiation, proliferation, and apoptosis [28]. In this regard, Loging and Reisman showed that two different mutp53 proteins are able to repress the transcription of TIMP-3 which is crucial in the ECM turnover and construction [29]. TIMP-3 expression is both inhibited or absent in many tumours [30, 31] contributing to increased activity of secreted MMPs in the ECM and, subsequently, to tumour invasion and metastasis. As mentioned above, mutp53 proteins are highly expressed in many human cancers, therefore, their ability to repress TIMP-3 highlights one possible mechanism by which GOF mutp53 proteins contribute to the metastatic potential of cancer cells. Intriguingly, Toschi et al. showed that, in human melanoma cells carrying mutp53 proteins, the re-introduction of wtp53 overcomes the mutp53 GOF and reduces cancer cell invasion into the ECM due to inhibition of secreted MMP-2 [32]. This observation is consistent with the well-known role of MMP-2 as the predominant basal membrane degrading type IV collagenase in human 5 Page 5 of 23

Cordani et al. melanoma therefore contributing to the invasive phenotype [33]. In addition, these findings highlight the role of an intact wtp53 pathway to halt metastasis through different mechanisms involving ECM remodelling. Recent studies have also emphasized the contribution of a subset of proteins, known as ECM matricellular proteins, to enhance pro-carcinogenic interactions between cancer cells and ECM within the tumour microenvironment [34]. Among them, periostin (POSTN) has been reported as important in the dissemination of cancer cells. In particular, Wong et al. observed a reduction in esophageal squamous cell carcinoma (ESCC) growth showing that POSTN has a role on the invasion of esophageal epithelial cells transformed into mesenchymal ECM [35]. Intriguingly, the authors showed that POSTN-mediated capability to stimulate cancer invasion occurs through its cooperation with mutp53-R175H protein leading to the induction of STAT1 signaling in the esophageal tumor microenvironment. In addition, ablation of POSTN expression and reduction of its accumulation in the ECM decrease tumour growth, cancer proliferation and invasion [35]. Of note, the functional interplay between MMPs and mutp53 proteins has been implicated in a plethora of other abnormal physio-pathological conditions other than in cancer progression and metastasis. For instance, in recent years, several genes known to be activated by mutp53 proteins have been discovered to play a crucial role in the pathogenesis of rheumatoid arthritis (RA). Among them, hMMP-13, a gene codifying for a collagenase enzyme involved in the degradation of type IV collagen in ECM, has been strongly linked to the expression and function of mutp53 proteins [36]. The authors showed that mutp53 proteins cause a dysregulation of hMMP-13 and hMMP-1 gene expression and of other MMP-related genes in the RA degeneration, suggesting a direct interaction between mutp53 and the MMP gene promoters [36]. Finally, Petignat et al. showed that both MMP2 and mutp53 protein levels are increased in hydatidiform mole (HM) a type of gestational trophoblastic disease, as compared with normal placenta, indicating a possible interplay of mutp53 proteins/MMP also in the uncontrolled trophoblastic proliferation observed in HM [37]. Overall, these findings highlight a potential role of mutp53 in cancer invasion and metastasis, as well as in 6 Page 6 of 23

Cordani et al. other pathological conditions, through ECM components remodelling thus representing a mutp53related hot topic to be deeply investigated.

4. Mutp53 induces the secretion of pro-inflammatory and immunomodulatory cytokines A great number of epidemiologic and experimental studies have addressed the topic that many neoplastic diseases are characterized by a relevant inflammatory component. Thus, the crosstalk between inflammatory and tumour cells has been demonstrated to be pivotal for cancer development and, for that reason, inflammation is considered one of the hallmarks of cancer [38]. The major players that are recruited into the tumour microenvironment when the inflammation occurs are represented by inflammatory cells and by several biochemical inflammatory mediators, including cytokines, chemokines, interleukins, and enzymes which deeply influence tumour development and progression. Chemokines are inflammatory effectors which belong to the wide family of cytokines and can be classified into CC, CXC, XC, and CX3C based on their biochemical and functional features [39]. Usually, during the inflammatory process, chemokines can be induced by other cytokines and they are secreted by tumour or stromal cells in order to regulate the directional migration of leukocytes towards the inflammatory site [40]. Many studies have well established that chemokines have pro-oncogenic effects. Indeed, they can promote tumour cell growth, tumour invasion and metastasis in several cancer types [40-42]. It has been reported that chemokines can increase the metastatic potential of cancer cells by mediating their directional migration to specific distal sites, similarly to the way they control leukocyte migration [43]. In addition, they can induce the expression of MMPs and collagenases to degrade ECM components [44, 45]. It has been recently reported that wtp53 inhibits both angiogenesis and cell motility by mechanistically repress the transcription of CXC chemokines; specifically, chemokines CXCL12 [46], CXCL4 [47], CXCL5 and CXCL8 [48] have been found to be down-regulated by wtp53. These findings underscore how impairment of wtp53 function might induce a pro-inflammatory 7 Page 7 of 23

Cordani et al. phenotype through de-repression of chemokines therefore contributing to cancer invasion and metastasis. Indeed, mutp53 proteins, contrarily to the wild-type counterpart, enhance cancer cell motility by upregulating the expression of CXCL5, CXCL8 and CXCL12 through the NF-Bdependent pathway, highlighting a further molecular mechanism by which mutp53 proteins exert their oncogenic activity [48]. Indeed, NF-B family members have a pivotal role in immunity and inflammation and it has been reported that they are key transcriptional regulators of chemokines expression [8, 49, 50]. Furthermore, recent studies showed that mutp53 proteins induce a proinflammatory phenotype through both activation of NF-B pathway [51] and induction of NFkappaB2 gene expression [52, 53]. Interestingly, Yan and Chen unveiled an alternative mechanism by which mutp53 proteins upregulate chemokine expression, showing that they can directly bind and activate the CXCL1 or GRO1 promoters in SW480 colon cancer cells thus resulting in enhancement of mutp53 oncogenic potential [54]. These studies support the existence of different mechanisms utilized by mutp53 proteins to modulate the expression of inflammatory chemokines to sustain the inflammatory status of tumour microenvironment thus contributing to promote tumour invasion and metastasis. Interleukin-1 (IL-1), a cytokine usually secreted by both stromal cells and infiltrating leukocytes during inflammatory and immune response, has been proposed to have pleiotropic effects in cancer. It has been reported that a sustained chronic inflammatory status in tumour microenvironment can promote malignant transformation, tumour growth, invasion and metastasis by several mechanisms [55]. However, IL-1 can also act as tumour suppressor by limiting tumour growth [56]. Intriguingly, Ubertini et al. have analyzed the cytokines expression in various cancer cell lines carrying different mutp53 proteins identifying the secreted interleukin-1 receptor antagonist (sIL-1Ra) as a novel mutp53 repressed target gene [24]. sIL-1Ra is a specific antagonist of the IL-1 pro-inflammatory cytokine which can bind to both type I and type II IL-1 receptors without transmitting any stimulating signals, thus representing a physiological inhibitor of IL-1 8 Page 8 of 23

Cordani et al. [57]. They reported that mutp53 proteins, but not wild-type p53, suppress the expression of sIL-1Ra supporting a pro-inflammatory tumour microenvironment which promotes tumour malignancy. Mechanistically, they observed that mutp53 proteins bind to the sIL-1Ra promoter recruiting the transcriptional co-repressor MAFF by protein-protein interaction [24]. This finding sustains the existence of a functional link between sIL-1Ra and mutp53 proteins underscoring the effect of a pro-inflammatory phenotype in cancer progression (Figure 3). This suggests that the pharmacological inhibition of IL-1 might represent a promising therapeutic strategy in tumours bearing mutant TP53 gene.

5. Mutp53 stimulates secretion of lactate and extracellular acidification Cancer is a complex disease characterized by dramatic metabolic alterations, and the Warburg effect, also called aerobic glycolysis, is the best known metabolic shift that occurs in cancer cells [38, 58]. During this phenomenon, even under physiological oxygen concentrations, tumour cells adopt glycolysis for their energy requirements and undergo to both high rate glucose uptake and lactate production, as compared with normal cells [59, 60]. Many studies highlighted that the Warburg effect significantly contributes to tumorigenesis and that its impairment could be considered as a therapeutic anticancer opportunity [61, 62]. High lactate levels, frequently found in cancer cells, result from the transformation of pyruvate by lactate dehydrogenase (LDH), and such metabolic condition has been related to increased metastatic potential, tumour recurrence, and lower prognosis in cancer patients [63, 64]. Since cancer cells-secreted lactate usually functions as inflammatory mediator, that acts on a plethora of processes regulating various biochemical properties of tumour microenvironment, it is widely accepted that tumour-derived lactate is able to induce inflammation and immune deficiency. In particular, it has been demonstrated that lactate secretion can: i) increase IL-17A production by both T-cells and macrophages, promoting chronic inflammation in tumour microenvironment [65]; ii) inhibit the activation of dendritic cells during 9 Page 9 of 23

Cordani et al. antigen-specific autologous T-cell stimulation [66]; iii) inhibit both monocyte migration and cytokine release [67]; and iv) contribute to angiogenesis through both NF-κB-mediated induction of IL-8 [68] and hypoxia-inducible transcription factor-1 (HIF-1)-mediated induction of vascular endothelial growth factor (VEGF) [69]. Considerably, lactate produced by high glycolytic rate cancer cells can also stimulate tumour cell motility, tumour invasion and migration, through a cascade of events consequent to extracellular acidification and pH decrease. It has been reported that lactate accumulation in cancer cells upregulates several transport mechanisms, including the Na+/H+ exchanger (NHE) [70], the H+-lactate co-transporter, monocarboxylate transporter (MCT) and the H+-ATPase (H+ pump) which sustain the efflux of protons into the extracellular space [71]. Overall, growing evidence confirm that acidic microenvironment increases tumour malignancy by promoting proliferation, chemoresistance, and invasion [72, 73]. In various cancer types, low extracellular pH can induce expression and activity of many proteases, including MMP-2, MMP-9, cathepsin-B and cathepsin-L, enabling cancer cells to invade the surrounding tissue and generate metastasis [74-76]. Recently, it has been clearly demonstrated that mutp53 proteins stimulate the Warburg effect, in both cultured cells and mutp53 knock-in mice [77], contrarily to wtp53 which is known to repress glycolysis and the Warburg effect through transcriptional regulation of genes involved in energy metabolism, including SCO2, TIGAR, GLS2 and Parkin [78-80]. This metabolic-related oncogenic function of mutp53 proteins mostly occurs by promoting glucose transporter 1 (GLUT1) translocation to the plasma membrane through activation of RhoA/ROCK signaling, thus resulting in increased cancer cells glucose uptake and, consequently, in increased glycolytic rate and lactate production [77]. Overall, these findings finally establish that mutp53 proteins play a crucial role in the promotion of Warburg effect in cancer cells that, through both stimulation of lactate production and reduction of extracellular pH, makes the tumour microenvironment suitable to cancer cell invasion and tumour dissemination. Therefore, counteracting specific bio-elements involved in tumour microenvironment, acidification might be considered a valuable therapeutic strategy against 10 Page 10 of 23

Cordani et al. cancer cells bearing TP53 gene mutations in order to prevent the metastatic process, the main cause of death in cancer patients.

6. Mutp53 regulates the crosstalk between cancer and stromal cells Tumours are heterogeneous diseases not only for the aberrant cancer cells genetic profile but also for the differences between their relative microenvironments, including the presence and activation of stromal cells and their biochemical and mechanical properties. Thus, tumour microenvironment constantly changes during cancer progression in response to various oncogenic signals [38], and the reciprocal interaction between tumour cells and the surrounding stroma strongly influences both cancer progression and patient prognosis [81]. Several studies attribute to mutp53 proteins also a critical role in tumour-stroma interaction [82]. Addadi et al. clearly showed that mutp53 proteins exert an indirect oncogenic effect when expressed by stromal cells, providing a selective advantage to adjacent cancer cells. They observed that the expression of mutp53-R172H protein in mouse embryonic fibroblasts (MEFs) promoted tumour growth of PC3 epithelial cancer cells significantly more than p53-KO MEFs [83]. Interestingly, the same authors and others revealed that the expression of mutp53-R172H in MEFs or the re-introduction of the human hotspot mutp53-R175H protein in p53-null MEFs increased the secretion of the oncogenic chemokines SDF-1/CXCL12 and CXCL1, proposing a novel mechanism by which stromal mutp53 may promote tumour growth [46, 84]. Cancer angiogenesis is a hallmark of cancer and is critical for tumour growth, proliferation and metastasis. It is characterized by the formation of abnormal, tortuous, and poorly organized vessels with altered permeability within the tumour tissue [38, 85, 86]. The promotion of tumour angiogenesis stimulating the release of pro-angiogenic soluble mediators in tumour stroma has been also ascribed to mutp53 proteins. Fontemaggi et al. have established that mutp53 proteins lead an opposite effect on tumour angiogenesis compared to wild-type p53, directing the transcriptional 11 Page 11 of 23

Cordani et al. activity of E2F1 on the regulatory region of ID4, a member of the ID family proteins having a role in neo-vascularization [87, 88]. They found that ID4 participates in the post-transcriptional stabilization of pro-angiogenic cytokines IL8 and GRO-α, resulting in increased cancer cells angiogenic potential [87, 89, 90]. A number of studies has also addressed the role of mutp53 proteins in the induction of the pro-angiogenic extracellular mediator VEGF in order to sustain angiogenesis and cancer growth; in this regard, a significant direct correlation between mutp53 proteins and VEGF expression has been observed in human breast cancer [91]. Moreover, it has been reported that the exogenous expression of mutp53 proteins in NIH3T3 fibroblasts is able to induce VEGF production [92]. Intriguingly, Narendran et al. have shown that the expression of mutp53 proteins in bone marrow stromal cells are able to increase both the expression and the secretion of VEGF in tumour stroma, supporting the growth of leukemic cells through both paracrine and/or autocrine mechanisms [93]. Cancer

associated

fibroblasts

(CAFs),

the

major

constituent

of

the

tumour

microenvironment, are a sub-population of stromal cells that lie adjacently to tumour cells [94]. Since they are a source of classical growth factors as well as of other extra-cellular modulators, CAFs assume a critical role in the regulation of cancer cells migration, proliferation and invasion [95-99]. Recently, the interplay between mutp53-carrying cancer cells and CAFs has been deeply investigated and is summarized in Figure 4. Madar et al. have unveiled the molecular mechanisms underlying this reciprocal interaction showing that CAFs can secrete interferon-β (IFN-β) in response to the presence of mutp53-carrying cancer cells [100]. IFN-β is a secreted cytokine with a dual effect on cancer cells: it can either directly inhibit tumour growth when released by the tumour microenvironment [101] or promote tumour escape from the immune system by contributing to oncogenic Ras transformation [102]. It has been observed that CAFs-induced IFN-β response was attenuated by mutp53 through SOCS1-mediated inhibition of STAT1 phosphorylation. In turn, IFNβ is able to antagonize the oncogenic function of mutp53 proteins by downregulating its mRNA

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Cordani et al. stabilizer WIG1 [100]. Overall, these findings underline the emerging role for mutp53 proteins in the modulation of tumour microenvironment through the triggering of the cancer-stroma crosstalk.

7. Mutp53 and secreted biomarkers Currently, only few studies attribute a role of mutp53 proteins on the release of molecules that could potentially serve as biomarkers secreted by tumour cells. Prostate-specific antigen (PSA) is a glycoprotein enzyme that belongs to the family of kallikreins, chymotrypsin-like proteins commonly used as serological or tissue tumour markers for the early detection of prostate cancer [103]. In mice models bearing prostate cancer, Downing et al. reported a strong correlation between the expression of mutp53 proteins and increased PSA serum levels which are generally associated with more aggressive cancer feature [104]. Moreover, it has been demonstrated that PSA is strongly repressed by wtp53, while, in contrast, mutp53 proteins are able to stimulate gene transcription and the secretion of PSA biomarker in cancer cells [105]. In that study, various p53 mutants (F134L, M237L, and R273H) have been introduced into LNCaP prostate cancer cells to inactivate endogenous wtp53. It has been observed that the exogenous expression of all mutp53 proteins in cancer cells is strongly related to enhanced levels of PSA mRNA as well as to increased secreted PSA protein and activity, as compared with wtp53–expressing cancer cells used as control. Furthermore, increased PSA serum levels have been found in mice bearing tumours derived from mutp53-expressing cells, compared with PSA serum levels in mice transplanted with wtp53expressing control cells. These results obtained both in vitro and in vivo strongly suggest that PSA levels may serve like a tissue specific indicator of wt or mutp53 expression in prostate cancer [104]. Others proteases, belonging to the kallikrein superfamily of serine proteases (for instance kallikrein-6), have been observed being up-regulated in the secretome of cancer cell lacking functional wtp53 [106]. These results should encourage further studies devoted to the discovery of additional mutp53-related serum biomarkers in order to provide novel tools for diagnosis, prognosis 13 Page 13 of 23

Cordani et al. and therapy for cancer patients bearing mutant TP53 gene.

8. Concluding remarks An increasing number of studies recently demonstrated the involvement of mutp53 proteins in the regulation of cancer cells-secreted proteins/enzymes/molecules that functionally alter tumour microenvironment. Mutp53 proteins trigger both the production and release of cytokines that stimulate an inflammatory cancer-associated microenvironment and concomitantly repress the immune system. Remarkably, stimulation of pro-inflammatory cytokines by mutp53 proteins is in stark contrast to the inhibitory functions of pro-inflammatory cytokines by wtp53 protein. Another crucial element supporting the motility of tumour cells and their metastatic potential is covered by extracellular pH decrease. This phenomenon is mainly related to the stimulation by mutp53 proteins of secreted lactate and implies a global alteration of mutp53-carrying cancer cells metabolism. The extracellular acidification is also involved in increased MMPs activity which, in turn, promotes ECM degradation favoring tumour invasion and cancer motility. Furthermore, mutp53 proteins can also stimulate VEGF secretion, which is conversely inhibited by wtp53. This is likely the main known event that promote pro-angiogenic feature associated to enhanced metastasis of mutp53bearing cancer cells. In this multifaceted context of mutp53-induced modulation of secretome and tumour microenvironment only few studies currently identified secreted biomarkers associated with the mutant TP53 gene in tumour cells. We believe that in addition to understanding the biological events that characterize the alteration of the tumour microenvironment, it is absolutely essential to focus some research efforts on the study of specific secreted biomarkers which may likely indicate the presence of GOF mutant p53 proteins in cancer patients. This would have an enormous relevance in the clinical practice providing oncologists with novel prognostic and diagnostic tools in order to improve and possibly personalize the therapeutic strategy for cancer patients carrying 14 Page 14 of 23

Cordani et al. mutant TP53 gene, already known to be resistant to conventional therapies.

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Cordani et al. Acknowledgments This work was supported by Italian Association for Cancer Research (AIRC) to A. Scarpa (AIRC 5permille grant n. 12182) and Joint Projects program 2015 from University of Verona to M. Donadelli (grant n. B12I15002320003).

Conflict of interest The authors declare that they have no conflicts of interest.

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Legends to Figures Fig. 1 Summary of the overall events associated to mutp53-driven cancer cell invasion and metastasis promoted by secreted mediators described in the text. Fig. 2 Oncogenic properties of mutp53 proteins. Mutations of TP53 gene determine the expression of mutant proteins which lose their tumor suppressor functions (Loss-Of-Function; LOF) and/or acquire new oncogenic roles (Gain-Of-Function; GOF) revealed in human cancers. Fig. 3 Mechanism induced by mutp53 addressed to trigger IL-1/IL-1R signal transduction in cancer cells. Mutp53 transcriptionally represses the expression of the secreted interleukin-1 receptor antagonist (sIL-1Ra) to allow the inflammatory cytokine IL-1 secreted by stromal cells to interact and stimulate IL-1R receptor on cancer cells thus promoting tumor malignancy. Fig. 4 Upon encounter with cancer cells, cancer associated fibroblasts (CAFs) activate the IFNβ signal transduction which limits cancer cell invasion and migration. Mutp53 moderates IFNβ pathway via SOCS1-mediated inhibition of STAT1 phosphorylation. On turn, IFNβ is able to reduce mutp53 levels by attenuating the expression of mutp53 mRNA stabilizer WIG1.

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