LOX, but not LOXL2, promotes bone metastasis formation and bone destruction in triple-negative breast cancer.

The primary function of the lysyl oxidase (LOX) family, including LOX and its paralogue LOX-like (LOXL)-2, is to catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. LOX and LOXL2 are also facilitating breast cancer invasion and metastatic spread to visceral organs (lungs, liver) in vivo. Conversely, the contribution of LOX and LOXL2 to breast cancer bone metastasis remains scant. Here, using gene overexpression or silencing strategies, we investigated the role of LOX and LOXL2 on the formation of metastatic osteolytic lesions in animal models of triple negative breast cancer. In vivo, the extent of radiographic metastatic osteolytic lesions in animals injected with LOX-overexpressing [LOX(+)] tumor cells was 3-fold higher than that observed in animals bearing tumors silenced for LOX [LOX(-)]. By contrast, the extent of osteolytic lesions between LOXL2(+) and LOXL2(-) tumor-bearing animals did not differ, and was comparable to that observed with LOX(-) tumor-bearing animals. In situ, TRAP staining of bone tissue sections from the hind limbs of LOX(+) tumor-bearing animals was substantially increased compared to LOX(-), LOXL2(+) and LOXL2(-)-tumor-bearing animals, which was indicative of enhanced active-osteoclast resorption. In vitro, tumor-secreted LOX increased osteoclast differentiation induced by RANKL, whereas LOXL2 seemed to counteract LOX's pro-osteoclastic activity. Furthermore, LOX (but not LOXL2) overexpression in tumor cells induced a robust production of IL-6, the latter being a pro-osteoclastic cytokine. Based on these findings, we propose a model in which LOX and IL-6 secreted from tumor cells act in concert to enhance osteoclast-mediated bone resorption that, in turn, promotes metastatic bone destruction in vivo.

Lysyl Oxidase Is a Strong Determinant of Tumor Cell Colonization in Bone.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1 In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1 Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268-78. ©2016 AACR.

TD-60 is required for interphase cell cycle progression.

We previously identified TD-60 (RCC2) as a mitotic centromere-associated protein that is necessary for proper completion of mitosis. We now report that TD-60 is an essential regulator of cell cycle progression during interphase. siRNA suppression blocks progression of mammalian G1/S phase cells and progression of G2 cells into mitosis. Prolonged arrest occurs both in non-transformed cells and in transformed cells lacking functional p53. TD-60 associates with Rac1 and Arf6 and has recently been demonstrated to be an element of α5ß1 integrin and cortactin interactomes. These associations with known elements of cell cycle control, together with our data, suggest that TD-60 is an essential component of one or more signaling pathways that drive cell cycle progression. During mitosis, TD-60 is required for correct assembly of the mitotic spindle and activation of key mitotic proteins. In contrast, in interphase TD-60 promotes cell cycle progression through what must be distinct mechanisms. TD-60 thus appears to be one of the growing categories of proteins that "moonlight," or have more than one distinct cellular function.

The HIF-1-inducible lysyl oxidase activates HIF-1 via the Akt pathway in a positive regulation loop and synergizes with HIF-1 in promoting tumor cell growth.

Adaptation to hypoxia is a driving force for tumor progression that leads to therapy resistance and poor clinical outcome. Hypoxic responses are mainly mediated by hypoxia-inducible transcription factor-1 (HIF-1). One critical HIF-1 target mediating tumor progression is lysyl oxidase (LOX), which catalyzes cross-linking of collagens and elastin in the extracellular matrix, thereby regulating tissue tensile strength. Paradoxically, LOX has been reported to be both upregulated and downregulated in cancer cells, especially in colorectal cancer. Thus, we hypothesized that LOX might regulate expression of HIF-1 to create a self-timing regulatory circuit. Using human colorectal carcinoma cell lines in which HIF-1 and LOX expression could be modulated, we showed that LOX induction enhanced HIF-1 expression, whereas LOX silencing reduced it. Mechanistic investigations revealed that LOX activated the PI3K (phosphoinositide 3-kinase)-Akt signaling pathway, thereby upregulating HIF-1α protein synthesis in a manner requiring LOX-mediated hydrogen peroxide production. Consistent with these results, cancer cell proliferation was stimulated by secreted and active LOX in an HIF-1α-dependent fashion. Furthermore, nude mice xenograft assays established that HIF-1 potentiated LOX action on tumor growth in vivo. Taken together, these findings provide compelling evidence that LOX and HIF-1 act in synergy to foster tumor formation, and they suggest that HIF-1/LOX mutual regulation is a pivotal mechanism in the adaptation of tumor cells to hypoxia.

Morpholino knockdown of lysyl oxidase impairs zebrafish development, and reflects some aspects of copper metabolism disorders.

Lysyl oxidase (LOX), a copper-dependent amine oxidase known in mammals to catalyze the cross-linking of collagen and elastin in the extracellular matrix, is a member of a multigenic family. Eight genes encoding lysyl oxidase isoforms have been identified in zebrafish. Recent studies have revealed a critical role for two zebrafish lysyl oxidases-like in the formation of the notochord. We now present the role of Lox in zebrafish development. lox morpholino-mediated knockdown results in a mildly undulated notochord, truncated anterior-posterior axis, tail bending and smaller head. Analyses of morphants show a complete disorganization of muscle somites and neural defects, in accordance with the lox expression pattern. Lox inhibition also induces pigment defects and pharyngeal arch deformities consistent with neural crest dysfunction. Taken together, these data reveal a role for Lox in early morphogenesis, especially in muscle development and neurogenesis, and resume some aspects of physiopathology of copper metabolism.

The lysyl oxidase LOX is absent in basal and squamous cell carcinomas and its knockdown induces an invading phenotype in a skin equivalent model.

Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.

Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome.

Several viral proteins expressed by DNA or RNA transforming viruses have the particular property of binding via their C-terminal end to various cellular proteins with PDZ domains. This study is focused on the PDZ protein TIP-2/GIPC, which was originally identified in two-hybrid screens performed with two different baits: the human T-cell leukemia virus type 1 Tax oncoprotein and the regulator of G signaling RGS-GAIP. Further studies have shown that TIP-2/GIPC is also able to associate with the cytoplasmic domains of various transmembrane proteins. In this report we show that TIP-2/GIPC interacts with the E6 protein of human papillomavirus type 18 (HPV-18). This event triggers polyubiquitination and proteasome-mediated degradation of the cellular protein. In agreement with this observation, silencing of E6 by RNA interference in HeLa cells causes an increase in the intracellular TIP-2/GIPC level. This PDZ protein has been previously found to be involved in transforming growth factor beta (TGF-beta) signaling by favoring expression of the TGF-beta type III receptor at the cell membrane. In line with this activity of TIP-2/GIPC, we observed that depletion of this protein in HeLa cells hampers induction of the Id3 gene by TGF-beta treatment and also diminishes the antiproliferative effect of this cytokine. Conversely, silencing of E6 increases the expression of Id3 and blocks proliferation of HeLa cells. These results support the notion that HPV-18 E6 renders cells less sensitive to the cytostatic effect of TGF-beta by lowering the intracellular amount of TIP-2/GIPC.

The mammalian passenger protein TD-60 is an RCC1 family member with an essential role in prometaphase to metaphase progression.

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

Arrest of mammalian fibroblasts in G1 in response to actin inhibition is dependent on retinoblastoma pocket proteins but not on p53.

p53 and the retinoblastoma (RB) pocket proteins are central to the control of progression through the G1 phase of the cell cycle. The RB pocket protein family is downstream of p53 and controls S-phase entry. Disruption of actin assembly arrests nontransformed mammalian fibroblasts in G1. We show that this arrest requires intact RB pocket protein function, but surprisingly does not require p53. Thus, mammalian fibroblasts with normal pocket protein function reversibly arrest in G1 on exposure to actin inhibitors regardless of their p53 status. By contrast, pocket protein triple knockout mouse embryo fibroblasts and T antigen-transformed rat embryo fibroblasts lacking both p53 and RB pocket protein function do not arrest in G1. Fibroblasts are very sensitive to actin inhibition in G1 and arrest at drug concentrations that do not affect cell adhesion or cell cleavage. Interestingly, G1 arrest is accompanied by inhibition of surface ruffling and by induction of NF2/merlin. The combination of failure of G1 control and of tetraploid checkpoint control can cause RB pocket protein-suppressed cells to rapidly become aneuploid and die after exposure to actin inhibitors, whereas pocket protein-competent cells are spared. Our results thus establish that RB pocket proteins can be uniquely targeted for tumor chemotherapy.