Anika Nagelkerke

Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response

IntroductionThe hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined.MethodsA number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion.ResultsUsing transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids.ConclusionsThus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

Hypoxic Activation of the PERK/eIF2α Arm of the Unfolded Protein Response Promotes Metastasis through Induction of LAMP3

Purpose: Conditions of poor oxygenation (hypoxia) are present in many human tumors, including cervix cancer, and are associated with increased risk of metastasis and poor prognosis. Hypoxia is a potent activator of the PERK/eIF2α signaling pathway, a component of the unfolded protein response (UPR) and an important mediator of hypoxia tolerance and tumor growth. Here, the importance of this pathway in the metastasis of human cervix carcinoma was investigated. Experimental Design: Amplification and expression of LAMP3, a UPR metastasis-associated gene, was examined using FISH and immunofluorescence in a cohort of human cervix tumors from patients who had received oxygen needle electrode tumor oxygenation measurements. To evaluate the importance of this pathway in metastasis in vivo, we constructed a series of inducible cell lines to interfere with PERK signaling during hypoxia and used these in an orthotopic cervix cancer model of hypoxia-driven metastasis. Results: We show that LAMP3 expression in human cervix tumors is augmented both by gene copy number alterations and by hypoxia. Induced disruption of PERK signaling in established orthotopic xenografts resulted in complete inhibition of hypoxia-induced metastasis to the lymph nodes. This is due, in part, to a direct influence of the UPR pathway on hypoxia tolerance. However, we also find that LAMP3 is a key mediator of hypoxia-driven nodal metastasis, through its ability to promote metastatic properties including cell migration. Conclusion: These data suggest that the association between hypoxia, metastasis, and poor prognosis is due, in part, to hypoxic activation of the UPR and expression of LAMP3. Clin Cancer Res; 19(22); 6126–37. ©2013 AACR.

The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response

BACKGROUND AND PURPOSE Lysosome-associated membrane protein 3 (LAMP3) is induced by the PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the unfolded protein response (UPR) during hypoxia. LAMP3 has prognostic value in breast cancer patients treated with radiotherapy. Here, we specifically investigated the role of the PERK/ATF4/LAMP3-arm in the radiation response of breast cancer cells. MATERIAL AND METHODS Radiosensitivity of breast cancer cells was examined after siRNA-mediated knockdown of PERK, ATF4 and LAMP3. Activation of DNA damage repair proteins was evaluated by Western blotting and immunocytochemistry. RESULTS Knockdown of the PERK/ATF4/LAMP3-arm and chemical inhibition of PERK could radiosensitise MDA-MB-231 cells significantly. Western blot analysis of several DNA damage repair proteins showed that LAMP3 knockdowns had an attenuated DNA damage response after radiation compared to controls. γ-H2AX foci analysis revealed that LAMP3 knockdowns had a reduced number of positive cells after irradiation, indicating that their DNA damage repair signalling response is decreased. In addition, the effect of autophagy inhibition was examined and revealed a radiosensitising effect and the presence of residual γ-H2AX foci. CONCLUSIONS The PERK/ATF4/LAMP3-arm causes radioresistance of breast cancer cells by increasing DNA damage repair signalling. Inhibition of PERK and/or autophagy may sensitise tumours to radiotherapy.

Constitutive expression of γ-H2AX has prognostic relevance in triple negative breast cancer.

BACKGROUND AND PURPOSE Constitutive γ-H2AX expression might indicate disruption of the DNA damage repair pathway, genomic instability, or shortened telomeric ends. Here, we quantified expression of endogenous γ-H2AX and its downstream factor 53BP1 in a large number of breast cancer cell lines (n=54) and a node-negative breast cancer cohort that had not received adjuvant systemic treatment (n=122). MATERIALS AND METHODS Formalin fixed paraffin embedded breast cancer cell lines and tumors were immunohistochemically analyzed for γ-H2AX and 53BP1 expression, and related to cell line, patient and tumor characteristics and to disease progression. RESULTS In breast cancer cell lines, γ-H2AX positivity was associated with the triple negative/basal like subgroup (p=0.005), and with BRCA1 (p=0.011) or p53 (p=0.053) mutations. Specifically in triple negative breast cancer patients a high number of γ-H2AX foci indicated a significantly worse prognosis (p=0.006 for triple negative vs. p=0.417 for estrogen receptor (ER), progesterone receptor (PR) or HER2 positive patients). A similar association with disease progression was found for 53BP1. In a multivariate analysis with tumor size, grade, and triple negativity, only the interaction between triple negativity and γ-H2AX remained significant (p=0.002, Hazard Ratio=6.77, 95% CI=2.07-22.2). CONCLUSIONS Constitutive γ-H2AX and 53BP1 staining reveals a subset of patients with triple negative breast tumors that have a significantly poorer prognosis.

Hypoxic regulation and prognostic value of LAMP3 expression in breast cancer.

LAMP3 is a newly described hypoxia regulated gene of potential interest in hypoxia‐induced therapy resistance and metastasis. The prognostic value of LAMP3 in breast cancer was investigated.

LAMP3 is involved in tamoxifen resistance in breast cancer cells through the modulation of autophagy

Lysosome-associated membrane protein 3 (LAMP3) is a member of the LAMP-family of proteins, which are involved in the process of autophagy. Autophagy is induced by tamoxifen in breast cancer cells and may contribute to tamoxifen resistance. In this study, the significance of LAMP3 for tamoxifen resistance in breast cancer was examined. The methods employed included use of clonogenic assays to assess the survival of MCF7 breast cancer cells with LAMP3 knockdown after tamoxifen treatment and of quantitative real-time PCR of LAMP3 to evaluate its predictive value for first-line tamoxifen treatment in patients with advanced breast cancer. Results show that tamoxifen treatment of MCF7 cells induced LAMP3 mRNA expression. LAMP3 knockdown in these cells increased tamoxifen sensitivity. Evaluation of expression of the autophagy markers, LC3B and p62, after LAMP3 knockdown showed increased expression levels, indicating that cells with LAMP3 knockdown have a suppressed ability to complete the autophagic process. In addition, knockdown of autophagy-associated genes resulted in sensitization to tamoxifen. Next, tamoxifen-resistant MCF7 cells were cultured. These cells had a sevenfold higher LAMP3 mRNA expression, showed elevated basal autophagy levels, and could be significantly resensitized to tamoxifen by LAMP3 knockdown. In patients treated with first-line tamoxifen for advanced disease (n=304), high LAMP3 mRNA expression was associated with shorter progression-free survival (P=0.003) and shorter post-relapse overall survival (P=0.040), also in multivariate analysis. Together, these results indicate that LAMP3 contributes to tamoxifen resistance in breast cancer. Tamoxifen-resistant cells are resensitized to tamoxifen by the knockdown of LAMP3. Therefore, LAMP3 may be clinically relevant to countering tamoxifen resistance in breast cancer patients.

Generation of multicellular tumor spheroids of breast cancer cells: how to go three-dimensional

The multicellular tumor spheroid model is a widely used culture system to mimic the three-dimensionality of tumors. Several methods and an even larger number of protocols are available to prepare spheroids from regular monolayers. This paper describes the assessment of several techniques frequently used to culture spheroids of breast cancer cells. We found that some methods produced suboptimal results. Ultimately, an adapted liquid overlay technique generated tight, compact and robust spheroids of the breast cancer cells tested.

Hypoxic regulation of the PERK/ATF4/LAMP3-arm of the unfolded protein response in head and neck squamous cell carcinoma

The purpose of this study was to examine the hypoxic regulation of the PKR‐like endoplasmic reticulum kinase (PERK)/activating transcription factor‐4 (ATF4)/lysosome‐associated membrane protein 3 (LAMP3)‐arm of the unfolded protein response (UPR) in head and neck squamous cell carcinoma (HNSCC).

18S is an appropriate housekeeping gene for in vitro hypoxia experiments.

Sir, We have read with great interest the study by Caradec et al (2010) in British Journal of Cancer into the variability in housekeeping gene expression under different oxygen tensions. We concur with the authors that the choice of normalising gene can have important consequences on the results obtained, and should therefore be validated for the experimental setup, tissue, or cell line under investigation. Earlier, we have carried out a similar investigation of 13 different housekeeping genes in 80 different tumour and normal tissues, trying to compensate for both differences in tumour grade and RNA quality (de Kok et al, 2005). In the study of Caradec et al, however, the housekeeper is not used for compensation in differences in RNA quality, as this is much less of an issue in cell lines than in retrospectively analysed stored tumour tissue. Thus, the housekeeping gene suitable for normalisation of cell-line expression levels under different oxygen tensions should be primarily constant between different levels of hypoxia. For this reason, we have selected 18S to compensate for input, RT, and PCR efficiency for in vitro hypoxia experiments (Mujcic et al, 2009). Although this rRNA species is unsuitable for compensating for RNA degradation, as we have shown earlier (de Kok et al, 2005), and for normalising among cell lines, we have found it to be relatively stable at different oxygen tensions over short periods of time. We would be interested in learning how 18S expression levels compared to the expression levels of the other genes tested by Caradec et al.

Poor prognosis of constitutive γ-H2AX expressing triple-negative breast cancers is associated with telomere length

AIM Here, we set out to establish whether endogenous γ-H2AX is a biomarker in triple-negative breast cancer. METHODS We explored the association of γ-H2AX with mutation status and sensitivity to 139 different anticancer drugs in up to 41 breast cancer cell lines. Further, we correlated γ-H2AX expression in breast cancer tumor tissues with telomere length. RESULTS γ-H2AX positive breast cancer cells exhibit more mutations, and - when p53 mutated - have shorter telomeres. In breast cancer patients γ-H2AX is also related to shorter telomeres, which was in turn associated with poorer prognosis of triple-negative breast cancer patients. CONCLUSION Thus, endogenous γ-H2AX is associated with short telomeres, which might offer a specific target for therapy for triple-negative breast cancer patients.