Integrin alpha5 in human breast cancer is a mediator of bone metastasis and a therapeutic target for the treatment of osteolytic lesions.

Bone metastasis remains a major cause of mortality and morbidity in breast cancer. Therefore, there is an urgent need to better select high-risk patients in order to adapt patient's treatment and prevent bone recurrence. Here, we found that integrin alpha5 (ITGA5) was highly expressed in bone metastases, compared to lung, liver, or brain metastases. High ITGA5 expression in primary tumors correlated with the presence of disseminated tumor cells in bone marrow aspirates from early stage breast cancer patients (n = 268; p = 0.039). ITGA5 was also predictive of poor bone metastasis-free survival in two separate clinical data sets (n = 855, HR = 1.36, p = 0.018 and n = 427, HR = 1.62, p = 0.024). This prognostic value remained significant in multivariate analysis (p = 0.028). Experimentally, ITGA5 silencing impaired tumor cell adhesion to fibronectin, migration, and survival. ITGA5 silencing also reduced tumor cell colonization of the bone marrow and formation of osteolytic lesions in vivo. Conversely, ITGA5 overexpression promoted bone metastasis. Pharmacological inhibition of ITGA5 with humanized monoclonal antibody M200 (volociximab) recapitulated inhibitory effects of ITGA5 silencing on tumor cell functions in vitro and tumor cell colonization of the bone marrow in vivo. M200 also markedly reduced tumor outgrowth in experimental models of bone metastasis or tumorigenesis, and blunted cancer-associated bone destruction. ITGA5 was not only expressed by tumor cells but also osteoclasts. In this respect, M200 decreased human osteoclast-mediated bone resorption in vitro. Overall, this study identifies ITGA5 as a mediator of breast-to-bone metastasis and raises the possibility that volociximab/M200 could be repurposed for the treatment of ITGA5-positive breast cancer patients with bone metastases.

[Biology of metastases and molecular mechanisms of their formation]. / Biologie des métastases et mécanismes moléculaires de leur formation.

Metastasis in cancer patients is often associated with a poor prognosis. However, we still have limited knowledge of the underlying molecular mechanisms, due to the great complexity of the biological processes involved in the formation of metastases. During tumor progression, the metastatic cells acquire genetic and epigenetic modifications allowing them to adapt to the various environments they will encounter (in the circulation and the host microenvironment) and to resist to the antitumor therapeutic agents. In this review, we expose the current knowledge on the biology of metastases. We summarize the different signaling pathways involved in the successive steps of the metastatic cascade, highlighting recent advances in the field to better understand the molecular mechanisms leading to metastasis formation. In addition, our understanding of metastatic progression has made great progress with the recent advances in high throughput sequencing techniques. We expose data from genomic analyzes of metastases. These studies allowed the identification of alterations acquired exclusively in distant metastases. They highlight the emergence of alterations offering new targeted therapeutic options for cancer patients and they provide new insight into the mechanisms of treatment resistance at the origin of metastatic relapses. Finally, we present latest clinical trials based on the genomic profiles of metastases, initiated in recent years, and we discuss their potential impact in personalized medicine.

Distinct expression profiles and functions of Kindlins in breast cancer.

BACKGROUND: Kindlin-1, - 2, and - 3 are the three members of the Kindlin family. They are best known as regulators of integrin functions, contributing to fundamental biological processes such as cell survival, adhesion and migration. Their deregulation leads to diverse pathologies including a broad range of cancers in which both, tumor-promoting and tumor-inhibiting functions have been described. METHODS: To better characterize Kindlins implication in breast cancer, in vitro experiments were performed in a series of cancer cell lines. We first assessed their expression profiles and subcellular distributions. Then, their involvement in breast cancer cell morphology, migration and invasion was verified by examining phenotypic changes induced by the depletion of either isoforms using RNA interference. An expression study was performed in a series of breast cancer patient derived xenografts (n = 58) to define the epithelial and stromal contribution of each Kindlin. Finally, we analyzed the expression levels of the three Kindlins in a large series of human breast tumors, at the RNA (n = 438) and protein (n = 129) levels and we evaluated their correlation with the clinical outcome. RESULTS: We determined that Kindlin-1 and Kindlin-2, but not Kindlin-3, were expressed in breast tumor cells. We uncovered the compensatory roles of Kindlin-1 and -2 in focal adhesion dynamics and cell motility. Remarkably, Kindlin-2 had a predominant effect on cell spreading and Kindlin-1 on cell invasion. In line with these experimental observations, Kindlin-1 overexpression was associated with a worse patients' outcome. Notably, Kindlin-3, expressed by tumor infiltrating leukocytes, also correlated with a poor prognosis of breast cancer patients. CONCLUSION: This study demonstrates that each one of the Kindlin family members has a different expression profile emphasizing their redundant and complementary roles in breast tumor cells. We highlight the specific link between Kindlin-1 and breast cancer progression. In addition, Kindlin-3 overexpression in the tumor microenvironment is associated with more aggressive breast tumors. These results suggest that Kindlins play distinctive roles in breast cancer. Kindlins may be useful in identifying breast cancer patients with a worst prognosis and may offer new avenues for therapeutic intervention against cancer progression.

VOPP1 promotes breast tumorigenesis by interacting with the tumor suppressor WWOX.

BACKGROUND: The WW domain-containing oxidoreductase (WWOX) gene, frequently altered in breast cancer, encodes a tumor suppressor whose function is mediated through its interactions with cancer-related proteins, such as the pro-apoptotic protein p73α. RESULTS: To better understand the involvement of WWOX in breast tumorigenesis, we performed a yeast two-hybrid screen and co-immunoprecipitation assays to identify novel partners of this protein. We characterized the vesicular overexpressed in cancer pro-survival protein 1 (VOPP1) as a new regulator of WWOX. In breast cancer cells, VOPP1 sequestrates WWOX in lysosomes, impairs its ability to associate with p73α, and inhibits WWOX-dependent apoptosis. Overexpressed VOPP1 potentiates cellular transformation and enhances the growth of transplanted tumors in vivo. VOPP1 is overexpressed in breast tumors, especially in tumors that retain WWOX. Moreover, increased expression of VOPP1 is associated with reduced survival of patients with WWOX-positive, but not with WWOX-negative, tumors. CONCLUSIONS: These findings emphasize the importance of the sequestration of WWOX by VOPP1 in addition to WWOX loss in breast tumors and define VOPP1 as a novel oncogene promoting breast carcinogenesis by inhibiting the anti-tumoral effect of WWOX.

The iron chelator deferasirox synergises with chemotherapy to treat triple-negative breast cancers.

To ensure their high proliferation rate, tumor cells have an iron metabolic disorder causing them to have increased iron needs, making them more susceptible to iron deprivation. This vulnerability could be a therapeutic target. In breast cancers, the development of new therapeutic approaches is urgently needed for patients with triple-negative tumors, which frequently relapse after chemotherapy and suffer from a lack of targeted therapies. In this study, we demonstrated that deferasirox (DFX) synergises with standard chemotherapeutic agents such as doxorubicin, cisplatin and carboplatin to inhibit cell proliferation and induce apoptosis and autophagy in triple-negative breast cancer (TNBC) cells. Moreover, the combination of DFX with doxorubicin and cyclophosphamide delayed recurrences in breast cancer patient-derived xenografts without increasing the side-effects of chemotherapies alone or altering the global iron storage of mice. Antitumor synergy of DFX and doxorubicin seems to involve downregulation of the phosphoinositide 3-kinase and nuclear factor-κB pathways. Iron deprivation in combination with chemotherapy could thus help to improve the effectiveness of chemotherapy in TNBC patients without increasing toxicity. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT-PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple-negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanib's main targets.

A methodological approach to unravel organ-specific breast cancer metastasis.

Breast cancer is the most commonly diagnosed and the second highest cause of cancer-related mortality. Although major breakthroughs have emerged during the past decades concerning the characterization of major malignant tumors hallmarks, little is known about the molecular process that sustains the most deadly feature of cancer: metastasis to distant organs. In fact, this colonization of tumor cells to secondary sites is not random but rather orientated, and depends on several signalling events that are not fully elucidated yet. Understanding the precise molecular and cellular mechanisms accountable for the specific invasion of tissues by breast cancer cells is likely to be important for developing new therapeutic strategies to effectively prevent metastasis in patients diagnosed with early cancer lesions. Here, we briefly describe a multidisciplinary approach based on the molecular profiling of breast cancer metastases, the elaboration of prognostic gene signatures, the clinical validation and the experimental confirmation using cell and animal models to better address breast cancer metastasis. This methodology can be considered as a useful workflow to identify and validate the genes that trigger and support organ tropism of breast cancer cells during metastasis.

Role of the focal adhesion protein kindlin-1 in breast cancer growth and lung metastasis.

BACKGROUND: Fermitin family member 1 (FERMT1, Kindlin-1) is an epithelial-specific regulator of integrin functions and is associated with Kindler syndrome, a genetic disorder characterized by skin blistering, atrophy, and photosensitivity. However, the possible role of kindlin-1 in cancer remains unknown. METHODS: Kindlin-1 expression was quantified in several human cancers using quantitative real-time polymerase chain reaction and published microarray datasets. The association between kindlin-1 expression and patient metastasis-free survival (N = 516) was assessed with Kaplan-Meier analyses. Effects of ectopic expression or silencing of kindlin-1 on cell signaling, migration, and invasion were assessed in human breast cancer cell lines using western blotting, immunofluorescence, wound healing assays, and invasion on Matrigel or type I collagen substrates. Breast tumor growth and lung metastasis were evaluated in 12-week-old female BALB/c mice (10 controls and six Kindlin-1-knockdown mice). All statistical tests were two-sided. RESULTS: Kindlin-1 expression was consistently higher in tumors than in normal tissues in various cancer types metastasizing to the lungs, including colon and bladder cancer. Kindlin-1 expression was associated with metastasis-free survival in both breast and lung adenocarcinoma (breast cancer: hazard ratio of lung metastasis = 2.55, 95% confidence intervals [CI] = 1.39 to 4.69, P = .001; lung cancer: hazard ratio of metastasis = 1.96, 95% CI = 1.25 to 3.07, P = .001). Overexpression of kindlin-1 induced changes indicating epithelial-mesenchymal transition and transforming growth factor beta (TGFß) signaling, constitutive activation of cell motility, and invasion (number of migrating cells, Kindlin-1 cells vs control, mean = 164.66 vs. 19.00, difference = 145.6, 95% CI = 79.1 to 212.2, P = .004; invasion rate, Kindlin-1-cells vs control = 9.65% vs. 1.92%, difference = 7.73%, 95% CI = 4.75 to 10.70, P < .001). Finally, Kindlin-1 depletion in an orthotopic mouse model statistically significantly inhibited breast tumor growth (P < .001) and lung metastasis (P = .003). CONCLUSION: These results suggest a role for kindlin-1 in breast cancer lung metastasis and lung tumorigenesis and advance our understanding of kindlin-1 as a regulator of TGFß signaling, offering new avenues for therapeutic intervention against cancer progression.

Confounding effects in "a six-gene signature predicting breast cancer lung metastasis": reply.

Molecular signatures have begun to elucidate the biological and molecular mechanisms underlying the phenotypic diversity of breast tumors. Breast tumors are characterized by five different molecular subtypes that are associated with distinct clinical outcomes in terms of prognosis, treatment response, and site of relapse. In particular, the basal-like and luminal B subtypes of tumors are more aggressive and have a higher tendency to metastasize to the lung than do the other subtypes. Given this difference in metastatic profiles of breast tumors, the six-gene signature (6GS) that we showed to be predictive of lung relapse was reexamined in the context of the tumor subtypes. This first analysis suggested that the 6GS is a surrogate for molecular subtype, discriminating basal-like tumors rather than tumors that metastasize to the lung. Here, we show that the 6GS discriminates the two overlapping features, the basal-like subtype and the tendency to metastasize to the lung. Nevertheless, the 6GS predicts lung metastases of breast tumors independent of the molecular subtypes.

GATA3 is a master regulator of the transcriptional response to low-dose ionizing radiation in human keratinocytes.

BACKGROUND: The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown. RESULTS: The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knock-down was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide microarrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGFbeta signalling and stress response. CONCLUSION: Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation.