Loxl3 Promotes Melanoma Progression and Dissemination Influencing Cell Plasticity and Survival

Malignant melanoma is the most lethal skin cancer due to its aggressive clinical behavior and therapeutic resistance. A comprehensive knowledge of the molecular mechanisms underlying melanoma progression is urgently needed to improve the survival of melanoma patients. Phenotypic plasticity of melanoma cells has emerged as a key process in melanomagenesis and therapy resistance. This phenotypic plasticity is sustained by an epithelial-to-mesenchymal (EMT)-like program that favors multiple intermediate states and allows adaptation to changing microenvironments along melanoma progression. Given the essential role of lysyl oxidase-like 3 (LOXL3) in human melanoma cell survival and its contribution to EMT, we generated mice with conditional melanocyte-specific targeting of Loxl3, concomitant to Braf activation and Pten deletion. Our results supported a key role of Loxl3 for melanoma progression, metastatic dissemination, and genomic stability, and supported its contribution to melanoma phenotypic plasticity by modulating the expression of several EMT transcription factors (EMT-TFs). Malignant melanoma is a highly aggressive tumor causing most skin cancer-related deaths. Understanding the fundamental mechanisms responsible for melanoma progression and therapeutic evasion is still an unmet need for melanoma patients. Progression of skin melanoma and its dissemination to local or distant organs relies on phenotypic plasticity of melanoma cells, orchestrated by EMT-TFs and microphthalmia-associated TF (MITF). Recently, melanoma phenotypic switching has been proposed to uphold context-dependent intermediate cell states benefitting malignancy. LOXL3 (lysyl oxidase-like 3) promotes EMT and has a key role in human melanoma cell survival and maintenance of genomic integrity. To further understand the role of Loxl3 in melanoma, we generated a conditional Loxl3-knockout (KO) melanoma mouse model in the context of BrafV600E-activating mutation and Pten loss. Melanocyte-Loxl3 deletion increased melanoma latency, decreased tumor growth, and reduced lymph node metastatic dissemination. Complementary in vitro and in vivo studies in mouse melanoma cells confirmed Loxl3's contribution to melanoma progression and metastasis, in part by modulating phenotypic switching through Snail1 and Prrx1 EMT-TFs. Importantly, a novel LOXL3-SNAIL1-PRRX1 axis was identified in human melanoma, plausibly relevant to melanoma cellular plasticity. These data reinforced the value of LOXL3 as a therapeutic target in melanoma

Lysyl oxidase-like 2 (LOXL2), a new regulator of cell polarity required for metastatic dissemination of basal-like breast carcinomas

Basal-like breast carcinoma is characterized by the expression of basal/ myoepithelial markers, undifferentiated phenotype, highly aggressive behaviour and frequent triple negative status (ESR , PR , Her2neu ). We have previously shown that epithelial–mesenchymal transition (EMT) occurs in basal-like breast tumours and identified Lysyl-oxidase-like 2 (LOXL2) as an EMT player and poor prognosis marker in squamous cell carcinomas. We now show that LOXL2 mRNA is overexpressed in basal-like human breast carcinomas. Breast carcinoma cell lines with basal-like phenotype show a specific cytoplasmic/perinuclear LOXL2 expression, and this subcellular distribution is significantly associated with distant metastatic incidence in basal-like breast carcinomas. LOXL2 silencing in basal-like carcinoma cells induces a mesenchymal-epithelial transition (MET) associated with a decrease of tumourigenicity and suppression of metastatic potential. Mechanistic studies indicate that LOXL2 maintains the mesenchymal phenotype of basal-like carcinoma cells by a novel mechanism involving transcriptional downregulation of Lgl2 and claudin1 and disorganization of cell polarity and tight junction complexes. Therefore, intracellular LOXL2 is a new candidate marker of basal-like carcinomas and a target to block metastatic dissemination of this aggressive breast tumour subtypeThis work was supported by grants from the Spanish Ministry of Science and Innovation, MICINN, (SAF2007-53061; SAF2010-21143; Consolider Ingenio CSD2007/00017, to AC; SAF2007-63075; SAF2010-20175 to GM-B); Fundacion Mutua Madrileña (2007, 2009 to AC and GM-B); Instituto de Salud Carlos III (ISCIII) (PI 080971 to JP), and Junta de Andalucıa (PI-0384/2007; PI 080971, P07-CVI- 03100 to JP). FS and A Martı´n are recipients of JAE-pre and JAE-postdoc contracts from the Spanish Research Council (CSIC), respectively; MAC is founded by the RETICS (ISCIII)

PAI-1 and functional blockade of SNAI1 in breast cancer cell migration

12 pages, 5 figures.-- PMID: 19055748 [PubMed].-- et al.[Introduction]: Snail, a family of transcriptional repressors implicated in cell movement, has been correlated with tumour invasion. The Plasminogen Activation (PA) system, including urokinase plasminogen activator (uPA), its receptor and its inhibitor, plasminogen activator inhibitor type 1(PAI-1), also plays a key role in cancer invasion and metastasis, either through proteolytic degradation or by non-proteolytic modulation of cell adhesion and migration. Thus, Snail and the PA system are both over-expressed in cancer and influence this process. In this study we aimed to determine if the activity of SNAI1 (a member of the Snail family) is correlated with expression of the PA system components and how this correlation can influence tumoural cell migration.[Methods]: We compared the invasive breast cancer cell-line MDA-MB-231 expressing SNAI1 (MDA-mock) with its derived clone expressing a dominant-negative form of SNAI1 (SNAI1-DN). Expression of PA system mRNAs was analysed by cDNA microarrays and real-time quantitative RT-PCR. Wound healing assays were used to determine cell migration. PAI-1 distribution was assessed by immunostaining.[Results]: We demonstrated by both cDNA microarrays and realtime quantitative RT-PCR that the functional blockade of SNAI1 induces a significant decrease of PAI-1 and uPA transcripts. After performing an in vitro wound-healing assay, we observed that SNAI1-DN cells migrate more slowly than MDA-mock cells and in a more collective manner. The blockade of SNAI1 activity resulted in the redistribution of PAI-1 in SNAI1-DN cells decorating large lamellipodia, which are commonly found structures in these cells.[Conclusions]: In the absence of functional SNAI1, the expression of PAI-1 transcripts is decreased, although the protein is redistributed at the leading edge of migrating cells in a manner comparable with that seen in normal epithelial cells.This work was supported by the CNRS ACI Program "Complexité du vivant" (grant # 050009DR11) and by the Evry Genopole grant "Aide à l'acquisition d'équipement semi-lourd" 2007 and 2008.Peer reviewe

Study of breast cancer incidence in patients of lymphangioleiomyomatosis

Molecular evidence has linked the pathophysiology of lymphangioleiomyomatosis (LAM) to that of metastatic breast cancer. Following on this observation, we assessed the association between LAM and subsequent breast cancer. An epidemiological study was carried out using three LAM country cohorts, from Japan, Spain, and the United Kingdom. The number of incident breast cancer cases observed in these cohorts was compared with the number expected on the basis of the country-specific incidence rates for the period 2000–2014. Immunohistochemical studies and exome sequence analysis were performed in two and one tumors, respectively. All cohorts revealed breast cancer standardized incidence ratios (SIRs) ≥ 2.25. The combined analysis of all cases or restricted to pre-menopausal age groups revealed significantly higher incidence of breast cancer: SIR = 2.81, 95 % confidence interval (CI) = 1.32–5.57, P = 0.009; and SIR = 4.88, 95 % CI = 2.29–9.99, P = 0.0007, respectively. Immunohistochemical analyses showed positivity for known markers of lung metastatic potential. This study suggests the existence of increased breast cancer risk among LAM patients. Prospective studies may be warranted to corroborate this result, which may be particularly relevant for pre-menopausal women with LAM. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10549-016-3737-8) contains supplementary material, which is available to authorized users

LOXL2 in epithelial cell plasticity and tumor progression

Several members of the lysyl oxidase family have recently emerged as important regulators of tumor progression. Among them, LOXL2 has been shown to be involved in tumor progression and metastasis of several tumor types, including breast carcinomas. Secreted LOXL2 participates in the remodeling of the extracellular matrix of the tumor microenvironment, in a similar fashion to prototypical lysyl oxidase. In addition, new intracellular functions of LOXL2 have been described, such as its involvement in the regulation of the epithelial-to-mesenchymal transition, epithelial cell polarity and differentiation mediated by transcriptional repression mechanisms. Importantly, intracellular (perinuclear) expression of LOXL2 is associated with poor prognosis and distant metastasis of specific tumor types, such as larynx squamous cell carcinoma and basal breast carcinomas. These recent findings open new avenues for the therapeutic utility of LOXL2. © 2012 Future Medicine Ltd.Research in the laboratories of A Cano and G Moreno-Bueno is supported by grants from the Ministry of Innovation and Science (MICINN) (SAF2007-63051; SAF2010-21143; SAF2010-20175; Consolider-Ingenio CDS2007-0017), Comunidad de Madrid (ReCaRe S2010/BMD-2303) and by the AECC Scientific Foundation 2011 (PG Santamaría).Peer Reviewe

Contribution of Epithelial Plasticity to Therapy Resistance

Therapy resistance is responsible for tumour recurrence and represents one of the major challenges in present oncology. Significant advances have been made in the understanding of the mechanisms underlying resistance to conventional and targeted therapies improving the clinical management of relapsed patients. Unfortunately, in too many cases, resistance reappears leading to a fatal outcome. The recent introduction of immunotherapy regimes has provided an unprecedented success in the treatment of specific cancer types; however, a good percentage of patients do not respond to immune-based treatments or ultimately become resistant. Cellular plasticity, cancer cell stemness and tumour heterogeneity have emerged as important determinants of treatment resistance. Epithelial-to-mesenchymal transition (EMT) is associated with resistance in many different cellular and preclinical models, although little evidence derives directly from clinical samples. The recognition of the presence in tumours of intermediate hybrid epithelial/mesenchymal states as the most likely manifestation of epithelial plasticity and their potential link to stemness and tumour heterogeneity, provide new clues to understanding resistance and could be exploited in the search for anti-resistance strategies. Here, recent evidence linking EMT/epithelial plasticity to resistance against conventional, targeted and immune therapy are summarized. In addition, future perspectives for related clinical approaches are also discussed

EMT: Present and future in clinical oncology

Epithelial/mesenchymal transition (EMT) has emerged as a key regulator of metastasis by facilitating tumor cell invasion and dissemination to distant organs. Recent evidences support that the reverse mesenchymal/epithelial transition (MET) is required for metastatic outgrowth; moreover, the existence of hybrid epithelial/mesenchymal (E/M) phenotypes is increasingly being reported in different tumor contexts. The accumulated data strongly support that plasticity between epithelial and mesenchymal states underlies the dissemination and metastatic potential of carcinoma cells. However, the translation into the clinics of EMT and epithelial plasticity processes presents enormous challenges and still remains a controversial issue. In this review, we will evaluate current evidences for translational applicability of EMT and depict an overview of the most recent EMT in vivo models, EMT marker analyses in human samples as well as potential EMT therapeutic approaches and ongoing clinical trials. We foresee that standardized analyses of EMT markers in solid and liquid tumor biopsies in addition to innovative tools targeting the E/M states will become promising strategies for future translation to the clinical setting.Work in our laboratories is supported by grants from the Spanish Ministerio de Economía y Competitividad SAF2013-44739-R and SAF2016-76504-R (AC and FP), the Spanish Instituto de Salud Carlos III [RETIC-RD12/0036/0007, CIBERONC-CB16/12/00295 (AC); PI13/00132, PI16/00134 (GMB)] (all of them partly supported from EUFEDER funds), and Worldwide Cancer Research (WWCR, formerly AICR) WWCR 16-0295 (AC, PGS, and FP). PGS was funded by a postdoctoral contract from the Fundacion Científica AECC and presently from WWCR 16-0295.Peer reviewe

Lysyl oxidase–like protein LOXL2 promotes lung metastasis of breast cancer

The lysyl oxidase-like protein LOXL2 has been suggested to contribute to tumor progression and metastasis, but in vivo evidence has been lacking. Here we provide functional evidence that LOXL2 is a key driver of breast cancer metastasis in two conditional transgenic mouse models of PyMT-induced breast cancer. LOXL2 ablation in mammary tumor cells dramatically decreased lung metastasis, whereas LOXL2 overexpression promoted metastatic tumor growth. LOXL2 depletion or overexpression in tumor cells does not affect extracellular matrix stiffness or organization in primary and metastatic tumors, implying a function for LOXL2 independent of its conventional role in extracellular matrix remodeling. In support of this likelihood, cellular and molecular analyses revealed an association of LOXL2 action with elevated levels of the EMT regulatory transcription factor Snail1 and expression of several cytokines that promote premetastatic niche formation. Taken together, our findings established a pathophysiologic role and new function for LOXL2 in breast cancer metastasis.This work has been supported by grants from the Spanish Ministry of Economy and Innovation SAF2010-21143 (to A. Cano, F. Portillo), SAF2013-44739-R (to A. Cano, P.G. Santamaría, F. Portillo), SAF2016-76504-R (to A. Cano, C. Lopez-Menendez, P.G. Santamaría, F. Portillo), and CONSOLIDER-INGENIO 2010 CSD2007-00017 (to A. Cano), the Spanish Instituto de Salud Carlos III [(RETIC-RD12/0036/0007 to A. Cano, G. Moreno-Bueno, P.G. Santamaria, F. Portillo and CIBERONC to A. Cano, G. Moreno-Bueno, F. Portillo, R. Lopez-Lopez, L. Muinelo-Romay, PI13/00132 to G. Moreno-Bueno (all partly supported by FEDER funds)]; Worldwide Cancer Research (formerly AICR) 12-1057 to A. Cano, A. Martin, P.G. Santamaría; Worldwide Cancer Research 16-0295 to A. Cano, F. Portillo, P.G. Santamaría; Comunidad de Madrid (S2010/BMD-2303 to A. Cano, G. Moreno-Bueno); AECC-2011 to G. Moreno-Bueno; and TV3-Telemarato 2013 to G. Moreno-Bueno. A. Martin, F. Salvador, and A. Vazquez-Naharro were funded by the JAE-CSIC program, S2010/BMD-2303, SAF2013-44739-R, and Worldwide Cancer Research 12-1057 grants; P.G. Santamaría by a contract from Fundacion AECC (Spain) and, presently, by Worldwide Cancer Research 16-0295 grant; V. Santos by CSD2007-00017, Worldwide Cancer Research 12-1057 and SAF2013-44739-R grants, and S. Morales by RETIC-RD12/0036/0007 grant.Peer reviewe