Ramón García-Escudero

Tumour-infiltrating Gr-1+ myeloid cells antagonize senescence in cancer

Aberrant activation of oncogenes or loss of tumour suppressor genes opposes malignant transformation by triggering a stable arrest in cell growth, which is termed cellular senescence. This process is finely tuned by both cell-autonomous and non-cell-autonomous mechanisms that regulate the entry of tumour cells to senescence. Whether tumour-infiltrating immune cells can oppose senescence is unknown. Here we show that at the onset of senescence, PTEN null prostate tumours in mice are massively infiltrated by a population of CD11b+Gr-1+ myeloid cells that protect a fraction of proliferating tumour cells from senescence, thus sustaining tumour growth. Mechanistically, we found that Gr-1+ cells antagonize senescence in a paracrine manner by interfering with the senescence-associated secretory phenotype of the tumour through the secretion of interleukin-1 receptor antagonist (IL-1RA). Strikingly, Pten-loss-induced cellular senescence was enhanced in vivo when Il1ra knockout myeloid cells were adoptively transferred to PTEN null mice. Therapeutically, docetaxel-induced senescence and efficacy were higher in PTEN null tumours when the percentage of tumour-infiltrating CD11b+Gr-1+ myeloid cells was reduced using an antagonist of CXC chemokine receptor 2 (CXCR2). Taken together, our findings identify a novel non-cell-autonomous network, established by innate immunity, that controls senescence evasion and chemoresistance. Targeting this network provides novel opportunities for cancer therapy.

Enhancing Chemotherapy Efficacy in Pten-Deficient Prostate Tumors by Activating the Senescence-Associated Antitumor Immunity

Prosenescence therapy has recently emerged as a novel therapeutic approach for treating cancer. However, this concept is challenged by conflicting evidence showing that the senescence-associated secretory phenotype (SASP) of senescent tumor cells can have pro- as well as antitumorigenic effects. Herein, we report that, in Pten-null senescent tumors, activation of the Jak2/Stat3 pathway establishes an immunosuppressive tumor microenvironment that contributes to tumor growth and chemoresistance. Activation of the Jak2/Stat3 pathway in Pten-null tumors is sustained by the downregulation of the protein tyrosine phosphatase PTPN11/SHP2, providing evidence for the existence of a novel PTEN/SHP2 axis. Importantly, treatment with docetaxel in combination with a JAK2 inhibitor reprograms the SASP and improves the efficacy of docetaxel-induced senescence by triggering a strong antitumor immune response in Pten-null tumors. Altogether, these data demonstrate that immune surveillance of senescent tumor cells can be suppressed in specific genetic backgrounds but also evoked by pharmacological treatments.

Molecular determinants of Akt-induced keratinocyte transformation

The PI3K/PTEN/Akt signaling pathway has emerged in recent years as a main player in human cancers, increasing proliferation and decreasing apoptosis of transformed cells, and thus becoming a potential target for therapeutic intervention. Our previous data have demonstrated that Akt-mediated signaling is of a key relevance in the mouse skin carcinogenesis system, one of the best-known models of experimental carcinogenesis. Here, we investigated the involvement of several pathways as mediators of Akt-induced increased proliferation and tumorigenesis in keratinocytes. Tumors produced by subcutaneous injection of Akt-transformed keratinocytes showed increased Foxo3a phosphorylation, but no major alterations in p21Cip1/WAF1, p27Kip1 or mdm2 expression and/or localization. In contrast, we found increased expression and nuclear localization of ΔNp63, β-catenin and Lef1. Concomitantly, we also found increased expression of c-myc and CycD1, targets of the β-catenin/Tcf pathway. Such increase is associated with increased phosphorylation and stabilization of c-myc protein as well as increased translation of c-myc and CycD1 due to mTOR activation. Using immunohistochemistry approaches in samples of oral dysplasias and human head and neck squamous cell carcinomas, we confirmed that increased Akt activation significantly correlates with increased ΔNp63 and CycD expression, c-myc phosphorylation and nuclear accumulation of β-catenin. Collectively, these results demonstrate that Akt is able to transform keratinocytes by specific mechanisms involving transcriptional and post-transcriptional processes.

Deregulated Activity of Akt in Epithelial Basal Cells Induces Spontaneous Tumors and Heightened Sensitivity to Skin Carcinogenesis

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age. Furthermore, both wtAkt and myrAkt transgenic lines displayed heightened sensitivity to the epidermal proliferative effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and heightened sensitivity to two-stage skin carcinogenesis. Finally, enhanced susceptibility to two-stage carcinogenesis correlated with a more sustained proliferative response following treatment with TPA as well as sustained alterations in Akt downstream signaling pathways and elevations in cell cycle regulatory proteins. Collectively, the data provide direct support for an important role for Akt signaling in epithelial carcinogenesis in vivo, especially during the tumor promotion stage.

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

Human papillomaviruses (HPV) have been implicated in the development of nonmelanoma skin cancer (NMSC). The molecular mechanisms by which these viruses contribute towards NMSC are poorly understood. We have used an in vitro skin-equivalent model generated by transducing primary adult human epidermal keratinocytes with retroviruses expressing HPV genes to investigate the mechanisms of viral transformation. In this model, keratinocytes expressing HPV genes are seeded onto a mesenchyme composed of deepidermalized human dermis that had been repopulated with primary dermal fibroblasts. Expression of the HPV8 E7 gene caused both an enhancement of terminal differentiation and hyperproliferation, but most strikingly, the acquisition of the ability to migrate and invade through the underlying dermis. The basement membrane integrity was disrupted in a time-dependent manner in areas of invading keratinocytes, as evidenced by immunostaining of its protein components collagen types VII, IV, and laminin 5. This was accompanied by the overexpression of extracellular matrix metalloproteinases MMP-1, MMP-8, and MT-1-MMP. These results suggest that the cutaneous HPV type 8 that is frequently found in NMSC of epidermodysplasia verruciformis patients may actively promote an invasive keratinocyte phenotype. These findings also highlight the importance of epithelial-extracellular matrix-mesenchymal interactions that are required to support cell invasion.

The Rho Exchange Factors Vav2 and Vav3 Control a Lung Metastasis–Specific Transcriptional Program in Breast Cancer Cells

Two Vav isoforms could be targeted to prevent breast tumors from metastasizing to the lung. Metastatic Route to the Lung Many individuals with cancer die from secondary tumors or metastases that spread through blood or lymph vessels to other tissues from the primary tumor site. The members of the Rho family of guanosine triphosphatases (GTPases) promote tumor growth and metastasis and are activated by guanine nucleotide exchange factors (GEFs). Rho GEFs are attractive pharmacological targets because they have potentially druggable catalytic activities and more restricted distribution patterns than Rho proteins. Citterio et al. found that the mRNA abundance of the GEFs Vav2 and Vav3 was increased in certain breast cancer subtypes in patient samples. Mice implanted with breast cancer cells in which Vav2 and Vav3 had been silenced developed slowly growing breast tumors and did not develop lung metastases. Vav2- and Vav3-deficient breast cancer cells showed an altered transcriptional profile, leading the authors to further analyze the role of select target genes encoding proteins that could be pharmacologically inhibited, such as the enzyme cyclooxygenase-2. When implanted into mice, breast cancer cells with deficiencies in individual Vav target genes showed defects in proliferation, angiogenesis, the ability to enter or exit blood vessels during metastasis, and the ability to colonize the lung. When applied to human breast cancer data sets, the changes in the abundance of a subset of mRNAs from the Vav transcriptome generated a gene signature that accurately predicted if patients survived and were free of detectable lung metastasis. These results identify possible targets for treating breast cancer and preventing secondary lung metastases and provide a potential prognostic tool for clinicians. The guanosine triphosphatases of the Rho and Rac subfamilies regulate protumorigenic pathways and are activated by guanine nucleotide exchange factors (Rho GEFs), which could be potential targets for anticancer therapies. We report that two Rho GEFs, Vav2 and Vav3, play synergistic roles in breast cancer by sustaining tumor growth, neoangiogenesis, and many of the steps involved in lung-specific metastasis. The involvement of Vav proteins in these processes did not correlate with Rac1 and RhoA activity or cell migration, implying the presence of additional biological programs. Microarray analyses revealed that Vav2 and Vav3 controlled a vast transcriptional program in breast cancer cells through mechanisms that were shared between the two proteins, isoform-specific or synergistic. Furthermore, the abundance of Vav-regulated transcripts was modulated by Rac1-dependent and Rac1-independent pathways. This transcriptome encoded therapeutically targetable proteins that played nonredundant roles in primary tumorigenesis and lung-specific metastasis, such as integrin-linked kinase (Ilk), the transforming growth factor–β family ligand inhibin βA, cyclooxygenase-2, and the epithelial cell adhesion molecule Tacstd2. It also contained gene signatures that predicted disease outcome in breast cancer patients. These results identify possible targets for treating breast cancer and lung metastases and provide a potential diagnostic tool for clinical use.

Role of HPV E6 proteins in preventing UVB-induced release of pro-apoptotic factors from the mitochondria

Apoptotic elimination of UV-damaged cells from the epidermis is an important step in preventing both the emergence and expansion of cells with carcinogenic potential. A pivotal event in apoptosis is the release of apoptogenic factors from the mitochondria, although the mechanisms by which the different proteins are released are not fully understood. Here we demonstrate that UV radiation induced the mitochondrial to nuclear translocation of apoptosis inducing factor (AIF) in normal skin. The human papillomavirus (HPV) E6 protein prevented release of AIF and other apoptotic factors such as cytochrome c and Omi from mitochondria of UV-damaged primary epidermal keratinocytes and preserved mitochondrial integrity. shRNA silencing of Bak, a target for E6-mediated proteolysis, demonstrated the requirement of Bak for UV-induced AIF release and mitochondrial fragmentation. Furthermore, screening non-melanoma skin cancer biopsies revealed an inverse correlation between HPV status and AIF nuclear translocation. Our results indicate that the E6 activity towards Bak is a key factor that promotes survival of HPV-infected cells that facilitates tumor development.

EMT and induction of miR-21 mediate metastasis development in Trp53-deficient tumours

Missense mutations in TP53 gene promote metastasis in human tumours. However, little is known about the complete loss of function of p53 in tumour metastasis. Here we show that squamous cell carcinomas generated by the specific ablation of Trp53 gene in mouse epidermis are highly metastatic. Biochemical and genome-wide mRNA and miRNA analyses demonstrated that metastases are associated with the early induction of epithelial-mesenchymal transition (EMT) and deregulated miRNA expression in primary tumours. Increased expression of miR-21 was observed in undifferentiated, prometastatic mouse tumours and in human tumours characterized by p53 mutations and distant metastasis. The augmented expression of miR-21, mediated by active mTOR and Stat3 signalling, conferred increased invasive properties to mouse keratinocytes in vitro and in vivo, whereas blockade of miR-21 in a metastatic spindle cell line inhibits metastasis development. Collectively these data identify novel molecular mechanisms leading to metastasis in vivo originated by p53 loss in epithelia.

Spontaneous squamous cell carcinoma induced by the somatic inactivation of retinoblastoma and Trp53 tumor suppressors.

Squamous cell carcinomas (SCC) represent the most aggressive type of nonmelanoma skin cancer. Although little is known about the causal alterations of SCCs, in organ-transplanted patients the E7 and E6 oncogenes of human papillomavirus, targeting the p53- and pRb-dependent pathways, have been widely involved. Here, we report the functional consequences of the simultaneous elimination of Trp53 and retinoblastoma (Rb) genes in epidermis using Cre-loxP system. Loss of p53, but not pRb, produces spontaneous tumor development, indicating that p53 is the predominant tumor suppressor acting in mouse epidermis. Although the simultaneous inactivation of pRb and p53 does not aggravate the phenotype observed in Rb-deficient epidermis in terms of proliferation and/or differentiation, spontaneous SCC development is severely accelerated in doubly deficient mice. The tumors are aggressive and undifferentiated and display a hair follicle origin. Detailed analysis indicates that the acceleration is mediated by premature activation of the epidermal growth factor receptor/Akt pathway, resulting in increased proliferation in normal and dysplastic hair follicles and augmented tumor angiogenesis. The molecular characteristics of this model provide valuable tools to understand epidermal tumor formation and may ultimately contribute to the development of therapies for the treatment of aggressive squamous cancer.

UV-B irradiation stimulates the promoter activity of the high-risk, cutaneous human papillomavirus 5 and 8 in primary keratinocytes

Summary.Human papillomaviruses (HPV) have been implicated in the development of non-melanoma skin cancer (NMSC). HPV types 5 and 8 are strongly associated with NMSC in patients with the inherited disease Epidermodysplasia verruciformis (Ev). In these patients tumours arise predominantly on sun-exposed skin and consistently harbour HPV DNAs. To determine whether UV-B irradiation modulates the noncoding region (NCR) promoter activity of the Ev-HPV types 5, 8, 9, 14, 23, 24, and 25 we performed transient transfection assays with NCR luciferase reporter gene constructs in primary human epithelial keratinocytes (PHEKs) and in p53-null RTS3b cells. Each of the HPVs showed different basal NCR activity in both cell types and reacted differently upon UVB treatment and p53 cotransfection in RTS3b cells. The NCR of HPV5 and 8 were the only ones to be activated by UV-B in PHEKs. The stimulation of the NCR activity of the high-risk cutaneous HPV types 5 and 8 by UV-B irradiation may point to a role of this interaction in the development of NMSC.