Blockade of CCR5 in melanoma: An alternative immune checkpoint modulator.

Melanoma is a deadly tumor, which in recent years has been successfully treated with immune checkpoint inhibitors as PD-1/PD-L1 and CTLA-4 inhibitors and targeted therapy as BRAF and MEK inhibitors. However, immunotherapy poses deleterious side effects and pursuit of new therapeutic targets is warranted. As knowledge of tumor immunology advances, such targets are being recognized. C-motif chemokine receptor-5 (CCR5) is a receptor found on immune cells whose effects impact the immune response both to induce inflammation and to activate suppressor cells causing an anti-inflammatory effect. CCR5 is well known as a target for HIV therapy where its blockade is efficient and safe, it is also known that its mutation CCR5delta32 is for the most part non-pathological to its carriers. In oncology, activation of the CCR5 receptor has been observed in high-stage disease and CCR5 blockade has been associated with an increased immune response. In this letter, we build up the rationale to utilize CCR5 as a therapeutic target for metastatic melanoma.

Cellular Prion Protein Mediates Pancreatic Cancer Cell Survival and Invasion through Association with and Enhanced Signaling of Notch1.

Up-regulation of human prion protein (PrP) in patients with pancreatic ductal adenocarcinoma (PDAC) is associated with a poor prognosis. However, the underlying molecular mechanism of PrP-mediated tumorigenesis is not completely understood. In this study, we found that PDAC cell lines can be divided into either PrP high expresser or PrP low expresser. In addition to filamin A (FLNA), PrP interacts with Notch1, forming a PrP/FLNA/Notch1 complex. Silencing PrP in high-expresser cells decreases Notch1 expression and Notch1 signaling. These cells exhibited decreased proliferation, xenograft growth, and tumor invasion but show increased tumor apoptosis. These phenotypes were rescued by ectopically expressed and activated Notch1. By contrast, overexpression of PrP in low expressers increases Notch1 expression and signaling, enhances proliferation, and increases tumor invasion and xenograft growth that can be blocked by a Notch inhibitor. Our data further suggest that PrP increases Notch1 stability likely through suppression of Notch proteosome degradation. Additionally, we found that targeting PrP combined with anti-Notch is much more effective than singularly targeted therapy in retarding PDAC growth. Finally, we show that coexpression of PrP and Notch1 confers an even poorer prognosis than PrP expression alone. Taken together, our results have unraveled a novel molecular pathway driven by interactions between PrP and Notch1 in the progression of PDAC, supporting a critical tumor-promoting role of Notch1 in PrP-expressing PDAC tumors.

The membrane tethered matrix metalloproteinase MT1-MMP at the forefront of melanoma cell invasion and metastasis.

The Extracellular Matrix (ECM) plays an important role in normal physiological development and functioning of cells, tissues and organs [1]. Under normal physiological conditions degradation of the ECM is a finely regulated process, and altered homeostasis of ECM degradation (excessive or insufficient) is associated with many diseases [2-5] such as cancer, fibrosis, arthritis, nephritis, encephalomyelitis and chronic ulcers. The remodeling of the ECM is carried out by a family of enzymes known as matrix metalloproteinases (MMP). MMPs constitute a large group of multidomain, zinc dependent endopeptidases capable of hydrolyzing all protein components of the ECM [6]. Additional functions of MMPs have also been identified. MMPs, and in particular MT1-MMP, the prototypic membrane-tethered matrix metalloproteinase, are no longer only ECM remodeling enzymes but rather regulators of several cellular functions including growth, migration, invasion and gene expression. Here we will focus on the role of the membrane bound MT1-MMP in melanoma growth, invasion and metastasis. MT1-MMP has in fact emerged as a multifaceted protease capable of influencing melanoma metastasis by canonical means, i.e. ECM degradation, but also via regulation of genes involved in several pro-tumorigenic functions including tumor cell growth and motility.

The thiirane-based selective MT1-MMP/MMP2 inhibitor ND-322 reduces melanoma tumor growth and delays metastatic dissemination.

MT1-MMP and MMP2 have been implicated as pro-tumorigenic and pro-metastatic factors in a wide variety of cancers including melanoma. We have previously demonstrated that MT1-MMP is highly expressed in melanoma where it promotes melanoma cell invasion and metastasis in part through the activation of its target MMP2. Given the accessibility of MMPs, as they are either secreted (e.g. MMP2) or membrane-tethered (e.g. MT1-MMP), they represent ideal targets for specific inhibition via small molecules. Here we show that the novel small-molecule inhibitor ND-322 with high selectivity for MT1-MMP and MMP2, effectively inhibits MT1-MMP and MMP2 activity resulting in reduced in vitro melanoma cell growth, migration and invasion. Importantly, these inhibitory effects lead to significant reduction of melanoma tumor growth and metastasis. We further show that while cell migration and invasion could be similarly hampered by specific inhibition of either MT1-MMP or MMP2 via shRNAs, the growth inhibitory activity of ND-322 could only be mirrored by specific inhibition of MT1-MMP. These data support ND-322 as a novel effective inhibitor capable of counteracting both MT1-MMP and MMP2, two key proteases involved in melanoma growth and metastasis. ND-322 may therefore represent a new inhibitor in the repertoire of treatments against melanoma.

Noncanonical activation of Notch1 protein by membrane type 1 matrix metalloproteinase (MT1-MMP) controls melanoma cell proliferation.

Notch1 is an evolutionarily conserved signaling molecule required for stem cell maintenance that is inappropriately reactivated in several cancers. We have previously shown that melanomas reactivate Notch1 and require its function for growth and survival. However, no Notch1-activating mutations have been observed in melanoma, suggesting the involvement of other activating mechanisms. Notch1 activation requires two cleavage steps: first by a protease and then by γ-secretase, which releases the active intracellular domain (Notch1(NIC)). Interestingly, although ADAM10 and -17 are generally accepted as the proteases responsible of Notch1 cleavage, here we show that MT1-MMP, a membrane-tethered matrix metalloproteinase involved in the pathogenesis of a number of tumors, is a novel protease required for the cleavage of Notch1 in melanoma cells. We find that active Notch1 and MT1-MMP expression correlate significantly in over 70% of melanoma tumors and 80% of melanoma cell lines, whereas such correlation does not exist between Notch1(NIC) and ADAM10 or -17. Modulation of MT1-MMP expression in melanoma cells affects Notch1 cleavage, whereas MT1-MMP expression in ADAM10/17 double knock-out fibroblasts restores the processing of Notch1, indicating that MT1-MMP is sufficient to promote Notch1 activation independently of the canonical proteases. Importantly, we find that MT1-MMP interacts with Notch1 at the cell membrane, supporting a potential direct cleavage mechanism of MT1-MMP on Notch1, and that MT1-MMP-dependent activation of Notch1 sustains melanoma cell growth. Together, the data highlight a novel mechanism of activation of Notch1 in melanoma cells and identify Notch1 as a new MT1-MMP substrate that plays important biological roles in melanoma.

VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration.

A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

The hypoxic microenvironment of the skin contributes to Akt-mediated melanocyte transformation.

Constitutive activation of Akt characterizes a high percentage of human melanomas and represents a poor prognostic factor of the disease. We show that Akt transforms melanocytes only in a hypoxic environment, which is found in normal skin. The synergy between Akt and hypoxia is HIF1alpha mediated. Inhibition of HIF1alpha decreases Akt transformation capacity in hypoxia and tumor growth in vivo, while overexpression of HIF1alpha allows anchorage-independent growth in normoxia and development of more aggressive tumors. Finally, we show that mTOR activity is necessary to maintain the transformed phenotype by sustaining HIF1alpha activity. Taken together, these findings demonstrate that Akt hyperactivation and HIF1alpha induction by normally occurring hypoxia in the skin significantly contribute to melanoma development.

Targeting extracellular matrix remodeling sensitizes glioblastoma to ionizing radiation.

Background: The median survival of Glioblastoma multiforme (GBM) patients is 14+ months due to poor responses to surgery and chemoradiation. Means to counteract radiation resistance are therefore highly desirable. We demonstrate the membrane bound matrix metalloproteinase MT1-MMP promotes resistance of GBM to radiation, and that using a selective and brain permeable MT1-MMP inhibitor, (R)-ND336, improved tumor control can be achieved in preclinical studies. Methods: Public microarray and RNA-sequencing data were used to determine MT1-MMP relevance in GBM patient survival. Glioma stem-like neurospheres (GSCs) were used for both in vitro and in vivo assays. An affinity resin coupled with proteomics was used to quantify active MT1-MMP in brain tissue of GBM patients. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP and inhibition via the MT1-MMP inhibitor (R)-ND336, were used to assess the role of MT1-MMP in radio-resistance. Results: MT1-MMP expression inversely correlated with patient survival. Active MT1-MMP was present in brain tissue of GBM patients but not in normal brain. shRNA- or (R)-ND336-mediated inhibition of MT1-MMP sensitized GSCs to radiation leading to a significant increase in survival of tumor-bearing animals. MT1-MMP depletion reduced invasion via the effector protease MMP2; and increased the cytotoxic response to radiation via induction of replication fork stress and accumulation of double strand breaks (DSBs), making cells more susceptible to genotoxic insult. Conclusions: MT1-MMP is pivotal in maintaining replication fork stability. Disruption of MT1-MMP sensitizes cells to radiation and can counteract invasion. (R)-ND336, which efficiently penetrates the brain, is therefore a novel radio-sensitizer in GBM.

Notch1 is an effector of Akt and hypoxia in melanoma development.

Melanomas are highly aggressive neoplasms resistant to most conventional therapies. These tumors result from the interaction of altered intracellular tumor suppressors and oncogenes with the microenvironment in which these changes occur. We previously demonstrated that physiologic skin hypoxia contributes to melanomagenesis in conjunction with Akt activation. Here we show that Notch1 signaling is elevated in human melanoma samples and cell lines and is required for Akt and hypoxia to transform melanocytes in vitro. Notch1 facilitated melanoma development in a xenograft model by maintaining cell proliferation and by protecting cells from stress-induced cell death. Hyperactivated PI3K/Akt signaling led to upregulation of Notch1 through NF-kappaB activity, while the low oxygen content normally found in skin increased mRNA and protein levels of Notch1 via stabilization of HIF-1alpha. Taken together, these findings demonstrate that Notch1 is a key effector of both Akt and hypoxia in melanoma development and identify the Notch signaling pathway as a potential therapeutic target in melanoma treatment.

Wound Healing Assay for Melanoma Cell Migration.

Cell migration is a critical process involved in morphogenesis, inflammation, and cancer metastasis. Wound healing assay is a simple, non-expensive, and highly reproducible method to study cancer cell migration in vitro. It is based on the observation that cells growing in a monolayer migrate to re-establish cell contacts after the development of an artificial wound. The assay involves creation of a wound in a monolayer, image acquisition during wound closure, and comparison of migrated area at initial and final time points.

MT1-MMP-dependent ECM processing regulates laminB1 stability and mediates replication fork restart.

Radiotherapy remains a mainstay of treatment for a majority of cancer patients. We have previously shown that the membrane bound matrix metalloproteinase MT1-MMP confers radio- and chemotherapy resistance to breast cancer via processing of the ECM and activation of integrinß1/FAK signaling. Here, we further discovered that the nuclear envelope protein laminB1 is a potential target of integrinß1/FAK. FAK interacts with laminB1 contributing to its stability. Stable laminB1 is found at replication forks (RFs) where it is likely to allow the proper positioning of RF protection factors, thus preventing RF degradation. Indeed, restoration of laminB1 expression rescues replication fork stalling and collapse that occurs upon MT1-MMP inhibition, and reduces DNA damage in breast cancer cells. Together, these data highlight a novel mechanism of laminB1 stability and replication fork restart via MT1-MMP dependent extracelluar matrix remodeling.

The biology of human hair greying.

Hair greying (canities) is one of the earliest, most visible ageing-associated phenomena, whose modulation by genetic, psychoemotional, oxidative, senescence-associated, metabolic and nutritional factors has long attracted skin biologists, dermatologists, and industry. Greying is of profound psychological and commercial relevance in increasingly ageing populations. In addition, the onset and perpetuation of defective melanin production in the human anagen hair follicle pigmentary unit (HFPU) provides a superb model for interrogating the molecular mechanisms of ageing in a complex human mini-organ, and greying-associated defects in bulge melanocyte stem cells (MSCs) represent an intriguing system of neural crest-derived stem cell senescence. Here, we emphasize that human greying invariably begins with the gradual decline in melanogenesis, including reduced tyrosinase activity, defective melanosome transfer and apoptosis of HFPU melanocytes, and is thus a primary event of the anagen hair bulb, not the bulge. Eventually, the bulge MSC pool becomes depleted as well, at which stage greying becomes largely irreversible. There is still no universally accepted model of human hair greying, and the extent of genetic contributions to greying remains unclear. However, oxidative damage likely is a crucial driver of greying via its disruption of HFPU melanocyte survival, MSC maintenance, and of the enzymatic apparatus of melanogenesis itself. While neuroendocrine factors [e.g. alpha melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), ß-endorphin, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH)], and micropthalmia-associated transcription factor (MITF) are well-known regulators of human hair follicle melanocytes and melanogenesis, how exactly these and other factors [e.g. thyroid hormones, hepatocyte growth factor (HGF), P-cadherin, peripheral clock activity] modulate greying requires more detailed study. Other important open questions include how HFPU melanocytes age intrinsically, how psychoemotional stress impacts this process, and how current insights into the gerontobiology of the human HFPU can best be translated into retardation or reversal of greying.

Hair(y) Matters in Melanoma Biology.

Melanocyte stem cells (MeSCs), one candidate for the cellular origin of melanoma, reside in the bulge region of the hair follicle (HF), an immune-privileged tissue niche with impaired tumor immunosurveillance. Surprisingly, however, primary melanoma is only very rarely associated with HFs. Here, we explore the hypothesis that this profoundly immunoinhibitory signaling environment deprives both MeSCs and melanocytes of the anagen hair matrix of proinflammatory signals required for full oncogenic transformation. Understanding the cellular and molecular mechanisms for generating a putative antimelanoma tissue habitat, namely in the bulge, could help to recreate a similar melanoma-suppressive signaling environment in melanoma high-risk individuals. We further discuss how mimicking the bulge immune privilege may be an effective melanoma prevention strategy.

Targeting Extracellular Matrix Remodeling Restores BRAF Inhibitor Sensitivity in BRAFi-resistant Melanoma.

PURPOSE: The extracellular matrix (ECM) is an intriguing, yet understudied component of therapy resistance. Here, we investigated the role of ECM remodeling by the collagenase, MT1-MMP, in conferring resistance of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant melanoma to BRAF inhibitor (BRAFi) therapy. EXPERIMENTAL DESIGN: Publicly available RNA-sequencing data and reverse phase protein array were used to determine the relevance of MT1-MMP upregulation in BRAFi-resistant melanoma in patients, patient-derived xenografts, and cell line-derived tumors. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP, inhibition via the selective MT1-MMP/MMP2 inhibitor, ND322, or overexpression of MT1-MMP was used to assess the role of MT1-MMP in mediating resistance to BRAFi. RESULTS: MT1-MMP was consistently upregulated in posttreatment tumor samples derived from patients upon disease progression and in melanoma xenografts and cell lines that acquired resistance to BRAFi. shRNA- or ND322-mediated inhibition of MT1-MMP synergized with BRAFi leading to resensitization of resistant cells and tumors to BRAFi. The resistant phenotype depends on the ability of cells to cleave the ECM. Resistant cells seeded in MT1-MMP uncleavable matrixes were resensitized to BRAFi similarly to MT1-MMP inhibition. This is due to the inability of cells to activate integrinß1 (ITGB1)/FAK signaling, as restoration of ITGB1 activity is sufficient to maintain resistance to BRAFi in the context of MT1-MMP inhibition. Finally, the increase in MT1-MMP in BRAFi-resistant cells is TGFß dependent, as inhibition of TGFß receptors I/II dampens MT1-MMP overexpression and restores sensitivity to BRAF inhibition. CONCLUSIONS: BRAF inhibition results in a selective pressure toward higher expression of MT1-MMP. MT1-MMP is pivotal to an ECM-based signaling pathway that confers resistance to BRAFi therapy.

ErbB3 Phosphorylation as Central Event in Adaptive Resistance to Targeted Therapy in Metastatic Melanoma: Early Detection in CTCs during Therapy and Insights into Regulation by Autocrine Neuregulin.

In recent years the introduction of target therapies with BRAF and MEK inhibitors (MAPKi) and of immunotherapy with anti-CTLA-4 and anti-PD-1 monoclonal antibodies have dramatically improved survival of metastatic melanoma patients. Despite these changes drug resistance remains a major hurdle. Several mechanisms are at the basis of drug resistance. Particular attention has been devoted over the last years to unravel mechanisms at the basis of adaptive/non genetic resistance occurring in BRAF mutated melanomas upon treatment with to MAPKi. In this paper we focus on the involvement of activation of ErbB3 receptor following early exposure of melanoma cells to BRAF or MEK inhibitors, and the following induction of PI3K/AKT pathway. Although different mechanisms have been invoked in the past at the basis of this activation we show here with a combination of approaches that autocrine production of neuregulin by melanoma cells is a major factor responsible for ErbB3 phosphorylation and downstream AKT activation. Interestingly the kinetic of neuregulin production and of the ensuing ErbB3 phosphorylation is different in different melanoma cell lines which underscores the high degree of tumor heterogeneity. Moreover, heterogeneity is further highlighted by the evidence that in different cell lines neuregulin upregulation can occur at the transcriptional or at the post-transcritpional level. Finally we complement our study by showing with a liquid biopsy assay that circulating tumor cells (CTCs) from melanoma patients undergo upregulation of ErbB3 phosphorylation in vivo shortly after initiation of therapy.

The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA damage response that impacts chemo- and radiotherapy responses of breast cancer.

Breast cancer is the second leading cause of death among women in the US. Targeted therapies exist, however resistance is common and patients resort to chemotherapy. Chemotherapy is also a main treatment for triple negative breast cancer (TNBC) patients; while radiation is delivered to patients with advanced disease to counteract metastasis. Yet, resistance to both chemo- and radiotherapy is still frequent, highlighting a need to provide novel sensitizers. We discovered that MT1-MMP modulates DNA damage responses (DDR) in breast cancer. MT1-MMP expression inversely correlates to chemotherapy response of breast cancer patients. Inhibition of MT1-MMP sensitizes TNBC cells to IR and doxorubicin in vitro, and in vivo in an orthotopic breast cancer model. Specifically, depletion of MT1-MMP causes stalling of replication forks and Double Strand Breaks (DBSs), leading to increased sensitivity to additional genotoxic stresses. These effects are mediated by integrinß1, as a constitutive active integrinß1 reverts replication defects and protects cells depleted of MT1-MMP from IR and chemotherapy. These data highlight a novel DNA damage response triggered by MT1-MMP-integrinß1 and provide a new point of therapeutic targeting that may improve breast cancer patient outcomes.

Inhibiting Notch1 enhances immunotherapy efficacy in melanoma by preventing Notch1 dependent immune suppressive properties.

We have previously shown that Notch1 plays a critical role in modulating melanoma tumor cell growth and survival. Here we show that Notch1 also contributes to an immune-suppressive tumor microenvironment (TME). Notch1 inhibition reduces immune suppressive cells (i.e. MDSCs and Tregs) while allowing the recruitment of functional CD8(+) T cells, leading to a decrease in the Tregs/CD8(+) ratio, a key parameter in assessing positive responses to immune-checkpoint inhibitors. Inhibition of Notch1 improves the antitumor activity of nivolumab and ipilimumab, particularly when given in combination. Mechanistically, tumor-associated Notch1 regulates the expression of several chemokines involved in MDSCs and Tregs recruitment. Among them, CCL5, IL6 and IL8, or MIP2 in mouse, were consistently reduced by Notch1 depletion in several human and mouse melanoma cell lines. Notch1 controls the transcription of IL8 and IL6; and the secretion of CCL5 likely by inhibiting the expression of SNAP23, a member of the SNAREs family of proteins involved in cell exocytosis. Inhibition of SNAP23 decreases CCL5 secretion similarly to Notch1 inhibition. Hence, targeting Notch1 would affect both melanoma intrinsic growth/survival properties, and provide an immune-responsive TME, thus improving immune therapy efficacy.

Targeting integrins and PI3K/Akt-mediated signal transduction pathways enhances radiation-induced anti-angiogenesis.

The integrins and PI3K/Akt are important mediators of the signal transduction pathways involved in tumor angiogenesis and cell survival after exposure to ionizing radiation. Selective targeting of either integrins or PI3K/Akt can radiosensitize tumors. In this study, we tested the hypothesis that the combined inhibition of integrin alphanubeta3 by cRGD and PI3K/Akt by LY294002 would significantly enhance radiation-induced inhibition of angiogenesis by vascular endothelial cells. Treatment with cRGD inhibited the adhesion and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibitory effect was further increased when cRGD and LY294002 were applied simultaneously. Both radiation and cRGD induced Akt phosphorylation, up-regulated COX2 expression, and increased PGE2 production in HUVECs. Treatment with LY294002 effectively inhibited radiation- and cRGD-induced Akt phosphorylation and up-regulation of COX2 and increased apoptosis of HUVECs. The combined use of cRGD and LY294002 enhanced radiation-induced cell killing. The clonogenic survival of HUVECs was decreased from 34% with 2 Gy radiation to 4% with these agents combined. These results demonstrate that combined use of ionizing radiation, cRGD and LY294002 inhibited multiple signaling transduction pathways involved in tumor angiogenesis and enhanced radiation-induced effects on vascular endothelial cells.

Inhibition of phosphatidylinositol-3-kinase and mitogen-activated protein kinase kinase 1/2 prevents melanoma development and promotes melanoma regression in the transgenic TPRas mouse model.

A number of human melanomas show hyperactivation of the Ras pathway due to mutations of the molecule or alteration of upstream or downstream effectors. In this study, we evaluated the effect of blocking the two Ras downstream pathways phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase on melanoma development and regression in the TPRas mouse model. The inhibition of these two signaling cascades by topically applied Ly294002 and U0126 significantly delayed melanoma development and significantly decreased the tumor incidence, particularly when the drugs were applied in combination. Treatment with the inhibitors of established melanomas resulted in complete remission in 33% of mice and partial regression in 46% of mice when drugs were delivered in combination. These responses correlated with increased apoptosis and decreased proliferation both in vitro and in vivo and reduced tumor angiogenesis. In conclusion, this study strongly supports the role of the phosphatidylinositol-3-kinase/Akt and Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways in the development and maintenance of Ras-dependent melanomas and supports the notion that specific inhibition of these effectors may represent a very promising avenue for the treatment and prevention of the disease.