TIMP-1 Induces an EMT-Like Phenotypic Conversion in MDCK Cells Independent of Its MMP-Inhibitory Domain

Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) regulate epithelial-mesenchymal transition (EMT) critical for the development of epithelial organs as well as cancer cell invasion. TIMP-1 is frequently overexpressed in several types of human cancers and serves as a prognostic marker. The present study investigates the roles of TIMP-1 on the EMT process and formation of the lumen-like structure in a 3D Matrigel culture of MDCK cells. We show that TIMP-1 overexpression effectively prevents cell polarization and acinar-like structure formation. TIMP-1 induces expression of the developmental EMT transcription factors such as SLUG, TWIST, ZEB1 and ZEB2, leading to downregulation of epithelial marker and upregulation of mesenchymal markers. Importantly, TIMP-1′s ability to induce the EMT-like process is independent of its MMP-inhibitory domain. To our surprise, TIMP-1 induces migratory and invasive properties in MDCK cells. Here, we present a novel finding that TIMP-1 signaling upregulates MT1-MMP and MMP-2 expression, and potentiates MT1-MMP activation of pro-MMP-2, contributing to tumor cell invasion. In spite of the fact that TIMP-1, as opposed to TIMP-2, does not interact with and inhibit MT1-MMP, TIMP-1 may act as a key regulator of MT1-MMP/MMP-2 axis. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule and also as an endogenous inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression

SQSTM1/p62 as a therapeutic target in cancer

Cell survival depends on dynamic interactions among the signaling pathways that control the endoplasmic reticulum (ER) stress response, macroautophagy/autophagy and apoptotic cell death. Our recent study reported an association of cytoplasmic SQSTM1/p62-mediated autophagy with disease progression and therapy resistance in head and neck squamous cell carcinoma (HNSCC). Synthetic small molecule ligands of SQSTM1 activate autophagic flux by binding the SQSTM1 ZZ domain and promoting self-oligomerization. Importantly, we found that the combination of pharmacological activation of SQSTM1 and therapeutic radiation promotes formation of ubiquitinated CASP8 (caspase 8) aggresomes that lead to apoptotic cell death of HNSCC. This finding suggests the potential for the development of a novel therapeutic strategy involving pharmacological activation of SQSTM1 in intrinsically apoptosis-resistant and therapy-resistant cancer cells

Regulation of Tumor Metabolism and Extracellular Acidosis by the TIMP-10–CD63 Axis in Breast Carcinoma

A hallmark of malignant solid tumor is extracellular acidification coupled with metabolic switch to aerobic glycolysis. Using the human MCF10A progression model of breast cancer, we show that glycolytic switch and extracellular acidosis in aggressive cancer cells correlate with increased expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), known to induce intracellular signal transduction through the interaction with its cell surface receptor CD63, independent of its metalloproteinase inhibitory function. We found that, in aggressive breast carcinoma, the TIMP-1–CD63 signaling axis induced a metabolic switch by upregulating the rate of aerobic glycolysis, lowering mitochondrial respiration, preventing intracellular acidification, and inducing extracellular acidosis. Carbonic anhydrase IX (CAIX), a regulator of cellular pH through the hydration of metabolically released pericellular CO2, was identified as a downstream mediator of the TIMP-1–CD63 signaling axis responsible for extracellular acidosis. Consistently with our previous study, the TIMP-1–CD63 signaling promoted survival of breast cancer cells. Interestingly, breast carcinoma cell survival was drastically reduced upon shRNA-mediated knockdown of CAIX expression, demonstrating the significance of CAIX-regulated pH in the TIMP-1–CD63-mediated cancer cell survival. Taken together, the present study demonstrates the functional significance of TIMP-1–CD63–CAXI signaling axis in the regulation of tumor metabolism, extracellular acidosis, and survival of breast carcinoma. We propose that this axis may serve as a novel therapeutic target

Photodynamic Therapy as a Potent Radiosensitizer in Head and Neck Squamous Cell Carcinoma

Despite recent advances in therapeutic modalities such as radiochemotherapy, the long-term prognosis for patients with advanced head and neck squamous cell carcinoma (HNSCC), especially nonviral HNSCC, remains very poor, while survival of patients with human papillomavirus (HPV)-associated HNSCC is greatly improved after radiotherapy. The goal of this study is to develop a mechanism-based treatment protocol for high-risk patients with HPV-negative HNSCC. To achieve our goal, we have investigated molecular mechanisms underlying differential radiation sensitivity between HPV-positive and -negative HNSCC cells. Here, we found that autophagy is associated with radioresistance in HPV-negative HNSCC, whereas apoptosis is associated with radiation sensitive HPV-positive HNSCC. Interestingly, we found that photodynamic therapy (PDT) directed at the endoplasmic reticulum (ER)/mitochondria initially induces paraptosis followed by apoptosis. This led to a substantial increase in radiation responsiveness in HPV-negative HNSCC, while the same PDT treatment had a minimal effect on HPV-positive cells. Here, we provide evidence that the autophagic adaptor p62 mediates signal relay for the induction of apoptosis, promoting ionizing radiation (XRT)-induced cell death in HPV-negative HNSCC. This work proposes that ER/mitochondria-targeted PDT can serve as a radiosensitizer in intrinsically radioresistant HNSCC that exhibits an increased autophagic flux

An oncogenic activity of PDGF-C and its splice variant in human breast cancer

Despite strong evidence for the involvement of PDGF signaling in breast cancer, little is known about the PDGF ligand responsible for PDGFR activation during breast cancer progression. Here, we found PDGF-C to be highly expressed in breast carcinoma cell lines. Immunohistochemical analysis of invasive breast cancer revealed an association between increased PDGF-C expression and lymph node metastases, Ki-67 proliferation index, and poor disease-free survival. We also identified a PDGF-C splice variant encoding truncated PDGF-C (t-PDGF-C) isoform lacking the signal peptide and the N-terminal CUB domain. While t-PDGF C homodimer is retained intracellularly, it can be secreted as a heterodimer with full-length PDGF-C (FL-PDGF-C). PDGF-C downregulation reduced anchorage-independent growth and matrigel invasion of MDA-MB-231 cells. Conversely, ectopic expression of t-PDGF-C enhanced phenotypic transformation and invasion in BT-549 cells expressing endogenous FL-PDGF-C. The present study provides new insights into the functional significance of PDGF-C and its splice variant in human breast cancer

Gene expression analysis of bone metastasis and circulating tumor cells from metastatic castrate-resistant prostate cancer patients

Background: Characterization of genes linked to bone metastasis is critical for identification of novel prognostic or predictive biomarkers and potential therapeutic targets in metastatic castrate-resistant prostate cancer (mCRPC). Although bone marrow core biopsies (BMBx) can be obtained for gene profiling, the procedure itself is invasive and uncommon practice in mCRPC patients. Conversely, circulating tumor cells (CTCs), which are likely to stem from bone metastases, can be isolated from blood. The goals of this exploratory study were to establish a sensitive methodology to analyze gene expression in BMBx and CTCs, and to determine whether the presence or absence of detectable gene expression is concordant in matching samples from mCRPC patients. Methods: The CellSearch® platform was used to enrich and enumerate CTCs. Low numbers of PC3 prostate cancer (PCa) cells were spiked into normal blood to assess cell recovery rate. RNA extracted from recovered PC3 cells was amplified using an Eberwine-based procedure to obtain antisense mRNA (aRNA), and assess the linearity of the RNA amplification method. In this pilot study, RNAs extracted from CTCs and PCa cells microdissected from formalin-fixed paraffin-embedded BMBx, were amplified to obtain aRNA and assess the expression of eight genes functionally relevant to PCa bone metastasis using RT-PCR. Results: RNAs were successfully extracted from as few as 1-5 PCa cells in blood samples. The relative expression levels of reference genes were maintained after RNA amplification. The integrity of the amplified RNA was also demonstrated by RT-PCR analysis using primer sets that target the 5'-end, middle, and 3'-end of reference mRNA. We found that in 21 out of 28 comparisons, the presence or absence of detectable gene expression in CTCs and PCa cells microdissected from single bone lesions of the same patients was concordant. Conclusions: This exploratory analysis suggests that aRNA amplification through in vitro transcription may be useful as a method to detect gene expression in small numbers of CTCs and tumor cells microdissected from bone metastatic lesions. In some cases, gene expression in CTCs and BMBxs was not concordant, raising questions about using CTC gene expression to make clinical decisions.Fil: Cho, Won Jin. Wayne State University; Estados UnidosFil: Oliveira, Daniel S.M.. Wayne State University; Estados UnidosFil: Najy, Abdo J.. Wayne State University; Estados UnidosFil: Mainetti, Leandro Ernesto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Wayne State University; Estados UnidosFil: Aoun, Hussein D.. Wayne State University; Estados UnidosFil: Cher, Michael L.. Wayne State University; Estados UnidosFil: Heath, Elisabeth. Wayne State University; Estados UnidosFil: Kim, Hyeong-Reh C.. Wayne State University; Estados UnidosFil: Bonfil, Ricardo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Wayne State University; Estados Unido