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Integrin
Integrin
integrin integrin integrin(1)

INTs consist of an α-subunit and a β-subunit constitute a family of transmembrane receptors that bind extracellularly to the ECM and intracellularly to the cytoskeleton, thereby integrating the extracellular environment with the cell interior.(1) INTs are heterodimeric trans-membrane receptors that function both as cell anchoring and signaling molecules. There are 18 α and 8 β subunits, each of which can bind to several partners giving rise to at least 24 distinct INT heterodimers with different functions and ligand binding activities. INTs serve as anchoring molecules by mediating the adhesion of the cellular cytoskeleton to the ECM.(2)
INT αvβ6 is not expressed in healthy adult epithelia but is upregulated during wound healing and in cancer. αvβ6 has been shown to modulate invasion, inhibit apoptosis, regulate the expression of MMPs and activate TGF-β1. Accumulating evidence indicates that there is extensive crosstalk between INTs and TGF-β signaling. TGF-β affects INT-mediated cell adhesion and migration by regulating the expression of INTs, their ligands and INT-associated proteins.(3) Since αvβ6 is not expressed in normal adult epithelia, but only under special wound healing conditions and in cancers, it lends itself to be a very good molecular target for therapy. However, there is increasing evidence, primarily from in vitro studies, that suggest that αvβ6 may actually promote carcinoma progression.(2)

Drugs/Indications
Trial Drugs/Interactions
Generic Code Old Code Brand Company Indication trials
cilengitide EMD 121974 EMD Serono P3: GBM; P2:HNN, leukemia, melanoma, PC; P1/2: NSCLC, SCCHN trials
E7820 Eisai P2: CRC; P1: solid, lymphoma trials
EMD 525797 DI17E6 EMD Serono P2: PC; P1/2: CRC; P1: ovarian, solid trials
GLPG0187 Galapagos P1: solid trials
Failed Drugs
Generic Code Old Code Brand Company Indication trials
etaracizumab, etaratuzumab MEDI-522, MEDI-523, LM609 Abegrin, Vitaxin Medimmune Last trial started in 2008; P2: PC, melanoma; P1/2: CRC, RCC (terminated); P1: lymphoma, small intestine, solid trials
intetumumab CNTO 95 Centocor Last trial 2009; no current trials ongoing; P2: PC; P1/2: melanoma; P1: solid trials
volociximab M200 Facet Terminated: P2: ovarian, peritoneal, NSCLC, melanoma, pancreatic trials
ATN-161 Ac-PHSCN-NH2 Attenuon Last new trial started in 2006; P2: RCC (terminated); P1/2: brain, CNS trials
natalizumab Biogen Idec terminated; P1/2: MM trials
PF-04605412 Pfizer terminated; P1: solid trials


News
3:15 PM|Current Cancer Drug Targets (Volume 16 - Issue 8)|Labels: INT, CLL
Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of neoplastic cells with the morphological appearance of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. CD49d, the α chain of the α4β1 integrin heterodimer, is one of the main interactors between CLL cells and accessory cells in the microenvironmental sites and one of the main predictors of overall survival. In particular, CD49d is known to play a pivotal role in mediating both cell-cell and cell-matrix interactions in CLL-involved tissues eventually delivering prosurvival signals and protecting CLL cells from drug-induced damages. Treatment strategies targeting the α4β1 integrin could represent an interesting option in CLL. In this context, the recombinant anti-CD49d antibody natalizumab demonstrated the potential to overcome stromal cell-induced resistance of B cell lymphoma cells against cytotoxic drugs and rituximab in vitro. Moreover, a specific interest for the CD49d molecule raises from the clinical activity of the recently proposed inhibitors of kinases downstream the BCR that has been recently related with the inside-out activation of the α4β1 integrin. In the review, we addressed in detail the role of CD49d in CLL cells, including clinical impact, relationship with specific cytogenetic features, and CD49d-dependent interactions in lymph node and bone marrow microenvironment responsible for growth- and survival- supporting signals, eventually influencing CLL prognosis and therapeutic options.
Sunday, September 18, 2016 10:27 AM|Head and Neck Oncology|Labels: INT, thymic, biomarker diagnostic
Although most patients with thyroid carcinoma havegood response to surgery and radioiodine, diagnostic difficulties can lead to the development of more aggressive tumors characterized by lymphatic and distant metastasis. Thus, it is urgent to identify new molecular markers to improve diagnostic accuracy and to predict the metastatic potential of thyroid carcinoma.We examined the expression of integrin αvβ6 in thyroid tissue specimens by immunohistochemistry and evaluated the relationship between αvβ6 expression and clinicopathological features of 150 thyroid carcinoma patients.Integrin αvβ6 expression was consistently negative in normal thyroid tissue, and occasional/weak in thyroid adenoma. In contrast, thyroid carcinoma, metastatic lymph nodes, thyroid nodular goiter, and thyroiditis tissues showed a positive αvβ6 expression. Overall, αvβ6 expression distinguished thyroid carcinoma from non-malignant tissues witha sensitivity of 78.9% and specificity of 62%. We also observed significant association of αvβ6 expression with age and lymph node status.No significant difference. In conclusion, the detection of αvβ6 expression may help to improve diagnostic accuracy and predict metastatic potential for thyroid carcinomas.
Thursday, September 15, 2016 6:45 AM|Pang, M.-F., Siedlik, M. J., Han, S., Stallings-Mann, M., Radisky, D. C., Nelson, C. M.|Cancer Research recent issues|Labels: INT, breast cancer
Breast tumors are stiffer and more hypoxic than nonmalignant breast tissue. Here we report that stiff and hypoxic microenvironments promote the development of breast cancer stem-like cells (CSC) through modulation of the integrin-linked kinase ILK. Depleting ILK blocked stiffness and hypoxia-dependent acquisition of CSC marker expression and behavior, whereas ectopic expression of ILK stimulated CSC development under softer or normoxic conditions. Stiff microenvironments also promoted tumor formation and metastasis in ovo, where depleting ILK significantly abrogated the tumorigenic and metastatic potential of invasive breast cancer cells. We further found that the ILK-mediated phenotypes induced by stiff and hypoxic microenvironments are regulated by PI3K/Akt. Analysis of human breast cancer specimens revealed an association between substratum stiffness, ILK, and CSC markers, insofar as ILK and CD44 were expressed in cancer cells located in tumor regions predicted to be stiff. Our results define ILK as a key mechanotransducer in modulating breast CSC development in response to tissue mechanics and oxygen tension. Cancer Res; 76(18); 5277–87. ©2016 AACR.
Tuesday, August 30, 2016 6:00 PM|Pere Puigserver|Nature - Issue - nature.com science feeds|Labels: INT, melanoma

A PGC1α-mediated transcriptional axis suppresses melanoma metastasis

Nature 537, 7620 (2016). doi:10.1038/nature19347

Authors: Chi Luo, Ji-Hong Lim, Yoonjin Lee, Scott R. Granter, Ajith Thomas, Francisca Vazquez, Hans R. Widlund & Pere Puigserver

Melanoma is the deadliest form of commonly encountered skin cancer because of its rapid progression towards metastasis. Although metabolic reprogramming is tightly associated with tumour progression, the effect of metabolic regulatory circuits on metastatic processes is poorly understood. PGC1α is a transcriptional coactivator that promotes mitochondrial biogenesis, protects against oxidative stress and reprograms melanoma metabolism to influence drug sensitivity and survival. Here, we provide data indicating that PGC1α suppresses melanoma metastasis, acting through a pathway distinct from that of its bioenergetic functions. Elevated PGC1α expression inversely correlates with vertical growth in human melanoma specimens. PGC1α silencing makes poorly metastatic melanoma cells highly invasive and, conversely, PGC1α reconstitution suppresses metastasis. Within populations of melanoma cells, there is a marked heterogeneity in PGC1α levels, which predicts their inherent high or low metastatic capacity. Mechanistically, PGC1α directly increases transcription of ID2, which in turn binds to and inactivates the transcription factor TCF4. Inactive TCF4 causes downregulation of metastasis-related genes, including integrins that are known to influence invasion and metastasis. Inhibition of BRAFV600E using vemurafenib, independently of its cytostatic effects, suppresses metastasis by acting on the PGC1α–ID2–TCF4–integrin axis. Together, our findings reveal that PGC1α maintains mitochondrial energetic metabolism and suppresses metastasis through direct regulation of parallel acting transcriptional programs. Consequently, components of these circuits define new therapeutic opportunities that may help to curb melanoma metastasis.

Wednesday, August 10, 2016 1:56 AM|Kroes, R. A., Moskal, J. R.|Glycobiology - Advance Access|Labels: INT, brain cancer

The mechanism of transcriptional silencing of ST6Gal1 in gliomas has not yet been elucidated. Multiple independent promoters govern the expression of the ST6Gal I gene. Here, we investigated whether epigenetic abnormalities involving DNA methylation affect ST6Gal1 expression. Transcript-specific qRT-PCR following exposure of glioma cell lines to 5-aza-2’-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, resulted in the re-expression of the normally quiescent ST6Gal1 mRNA driven exclusively by the P3 promoter sequence. The P3 promoter-specific transcription start site (TSS) was delineated by primer extension and core promoter sequences and associated functional transcription elements identified by deletion analysis utilizing chloramphenicol acetyltransferase reporter constructs. Minimal promoter activity was found to reside within the first 100 bp of the TSS and maximal activity was controlled by functional AP2 binding sites residing between 400 and 500 bp upstream of the initiation site. As altered AP2 binding was not directly associated with AP2 availability, these analyses demonstrate that ST6Gal1 transcription is regulated by DNA methylation within core promoter regions, ultimately by determining critical transcription factor accessibility within these regions. Transcriptional reactivation of ST6Gal1 expression by 5-aza-dC resulted in increased cell surface α2,6 sialoglycoconjugate expression, increased α2,6 sialylation of β1 integrin, and decreased adhesion to fibronectin substrate: functional correlates of decreased invasivity. The effects of global hypomethylation are not glycome-wide. Focused glycotranscriptomic analyses of three invasive glioma cell lines following 5-aza-dC treatment demonstrated the modulation of select glycogene transcripts. Taken together, these results demonstrate that epigenetic modulation of ST6Gal1 expression plays a key role in the glioma phenotype in vitro and that that therapeutic approaches targeting elements of the epigenetic machinery for the treatment of human glioblastoma are warranted.

Monday, August 1, 2016 1:00 AM|Leisner, T. M., Freeman, T. C., Black, J. L., Parise, L. V.|The FASEB Journal recent issues|Labels: INT

Calcium- and integrin-binding protein 1 (CIB1) is a small, ubiquitously expressed protein that was first identified as an intracellular binding partner of a platelet-specific α-integrin cytoplasmic tail. Although early studies revealed a role for CIB1 in regulating platelet integrin activity, recent studies have indicated a more diverse role for CIB1 in many different cell types and processes, including calcium signaling, migration, adhesion, proliferation, and survival. Increasing evidence also points to a novel role for CIB1 in cancer and cardiovascular disease. In addition, an array of CIB1 binding partners has been identified that provide important insight into how CIB1 may regulate these processes. Some of these binding partners include the serine/threonine kinases, p21-activated kinase 1 (PAK1), apoptosis signal-regulating kinase 1 (ASK1), and polo-like kinase 3 (PLK3). Structural and mutational studies indicate that CIB1 binds most or all of its partners via a well-defined hydrophobic cleft. Although CIB1 itself lacks known enzymatic activity, it supports the PI3K/AKT and MEK/ERK oncogenic signaling pathways, in part, by directly modulating enzymes in these pathways. In this review, we discuss our current understanding of CIB1 and key questions regarding structure and function and how this seemingly diminutive protein impacts important signaling pathways and cellular processes in human health and disease.—Leisner, T. M., Freeman, T. C., Black, J. L., Parise, L. V. CIB1: a small protein with big ambitions.

Sunday, July 31, 2016 10:05 PM|Cormerais, Y., Giuliano, S., LeFloch, R., Front, B., Durivault, J., Tambutte, E., Massard, P., de la Ballina, L. R., Endou, H., Wempe, M. F., Palacin, M., Parks, S. K., Pouyssegur, J.|Cancer Research recent issues|Labels: INT, mTOR
The CD98/LAT1 complex is overexpressed in aggressive human cancers and is thereby described as a potential therapeutic target. This complex promotes tumorigenesis with CD98 (4F2hc) engaging β-integrin signaling while LAT1 (SLC7A5) imports essential amino acids (EAA) and promotes mTORC1 activity. However, it is unclear as to which member of the heterodimer carries the most prevalent protumoral action. To answer this question, we explored the tumoral potential of each member by gene disruption of CD98, LAT1, or both and by inhibition of LAT1 with the selective inhibitor (JPH203) in six human cancer cell lines from colon, lung, and kidney. Each knockout respectively ablated 90% (CD98KO) and 100% (LAT1KO) of Na+-independent leucine transport activity. LAT1KO or JPH203-treated cells presented an amino acid stress response with ATF4, GCN2 activation, mTORC1 inhibition, and severe in vitro and in vivo tumor growth arrest. We show that this severe growth phenotype is independent of the level of expression of CD98 in the six tumor cell lines. Surprisingly, CD98KO cells with only 10% EAA transport activity displayed a normal growth phenotype, with mTORC1 activity and tumor growth rate undistinguishable from wild-type cells. However, CD98KO cells became extremely sensitive to inhibition or genetic disruption of LAT1 (CD98KO/LAT1KO). This finding demonstrates that the tumoral potential of CD98KO cells is due to residual LAT1 transport activity. Therefore, these findings clearly establish that LAT1 transport activity is the key growth-limiting step of the heterodimer and advocate the pharmacology development of LAT1 transporter inhibitors as a very promising anticancer target. Cancer Res; 76(15); 4481–92. ©2016 AACR.
Thursday, July 28, 2016 7:56 AM|Hoye, A. M., Tolstrup, S., Cox, T. R., Mauldin, J. P., Frankel, A. E., Erler, J. T.|Cancer Research recent issues|Labels: INT, CRC, preclinical
Tumor Endothelial Marker 8 (TEM8) is an integrin-like cell-surface receptor originally identified being specific to tumor endothelial cells in colorectal cancer (CRC) blood vessels. Further investigation showed upregulated TEM8 expression in tumor vessels of various tumor types, as well as in certain tumor cells. We observe an increase in TEM8 expression in metastatic cancer cell lines compared to their non-metastatic counterpart in both colon and breast cancer cell lines.In order to investigate whether TEM8 is required for tumor growth and metastasis we have developed a humanized anti-TEM8-toxin-conjugated antibody. The antibody shows preclinical effectiveness in a CRC model in vivo. We have also utilized the CRISPR-Cas9 technology to create TEM8 knockout cell lines, which show impaired proliferation and invasion in vitro and reduced tumor growth in vivo compared to control cell lines.Intriguingly, microarray analysis showed a significant increase in TEM8 mRNA levels in breast cancer cells seeded on collagen modified by the crosslinking enzyme lysyl oxidase (LOX). We have previously shown that LOX plays a role in tumor progression, metastasis, and pre-metastatic niche formation through its ability to modulate matrix stiffness. Importantly, both high TEM8 and LOX expression is correlated with decreased survival in ER- breast cancer patients.Collagen VI, a TEM8 ligand, and their interaction have been implicated in being important for migration and for stimulating Wnt-signaling in breast cancer stem cells. Using global quantitative mass spectrometry we observe an increase in collagen VI in the extracellular matrix of CRC tumors overexpressing catalytically active, but not catalytically inactive, LOX compared to low-LOX expressing tumors. Furthermore, using publicly available databases TEM8 and collagen VI are found to be co-expressed in CRC patient datasets.Thus, we speculate that another possible mechanism of LOX mediated tumor and metastasis progression is through TEM8. Further, that combined targeting of TEM8 and LOX may inhibit primary and metastatic tumor growth.Citation Format: Anette M. Høye, Sofie Tolstrup, Thomas R. Cox, Jeremy P. Mauldin, Arthur E. Frankel, Janine T. Erler. Disrupting tumor growth through targeting TEM8. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B17.
Thursday, July 28, 2016 7:56 AM|Eruslanov, E., Bhojnagarwala, P., Quatromoni, J., O'Brien, S., Moon, E., Stephen, T., Rao, A., Garfall, A., Hancock, W., Conejo-Garcia, J., Deshpande, C., Feldman, M., Singhal, S., Albelda, S.|Cancer Research recent issues|Labels: INT, lung cancer
To date there has been an increasing focus on the interactions between inflammatory myeloid cells and T cells in the tumor microenvironment because cytotoxic anti-tumoral T cells represent the chief effector mechanism of anti-tumoral immunity. Tumor-associated neutrophils (TANs) represent a significant portion of inflammatory cells in lung tumors; however, whether specialized neutrophil subpopulations capable of regulating T cell responses exist in human cancers is unknown. Our goal was to identify subsets of TANs and determine their specific roles in the regulation of T cell responses in patients with early stage lung cancer.To address this question, freshly isolated tumors from Non-Small Cell Lung Cancer (NSCLC) patients with Stage I-II squamous cell and adenocarcinoma histology were studied. An extensive phenotypic analysis of 55 early-stage human lung tumors revealed that TANs, defined as CD11b+Arg1+MPO+CD66b+CD15+ cells, contained two major sub-populations. One subset of canonical TANs expressed classic neutrophil markers. A second subset of TANs displayed a combination of neutrophil markers plus markers (CD14+HLA-DR+CCR7+CD86+) normally expressed on antigen-presenting cells (APC). This subset of TANs was found in lung tumor tissue but not in adjacent uninvolved lung. We termed this unique neutrophil population “APC-like hybrid TANs”. The frequency of these hybrid TANs varied widely within lung cancers and ranged from 0.5-25% of all TANs. Interestingly, the frequency of this hybrid population declined as tumors enlarged, and they were almost completely absent in tumors greater than 5 cm in diameter.Mechanistically, we determined that low doses of IFN-γ and GM-CSF in the tumors were required for the development of APC-like hybrid neutrophils. The high proportion of hybrid TANs (>10% of all TANs) directly correlated with the presence of IFN-γ and GM-CSF in the autologous tumor tissue. Using bone marrow-derived immature granulocytes, which were found to have prolonged survival in vitro, we discovered that these APC-like hybrid neutrophils originate from CD11b+CD15+CD10-CD16-/low/int neutrophil progenitors in the presence of IFN-γ and GM-CSF or in tumor-conditioned media. By contrast, mature CD11b+CD15+CD10+CD16hi neutrophils did not acquire the phenotype of hybrid TANs, when cultured under these conditions. In addition, we determined that IFN-γ and GM-CSF synergistically exerted APC promoting effects on immature neutrophils via the downregulation of the transcription factor, Ikaros. However, the development of APC-like hybrid neutrophils was profoundly inhibited under hypoxic conditions.Functionally, the APC-like hybrid neutrophils are superior to canonical neutrophils in their ability to: 1) produce APC cytokines such as TNF and IL-12 after stimulation, 2) stimulate antigen non-specific autologous T cell responses induced by plate-bound anti-CD3 antibodies 3) directly stimulate antigen-specific autologous memory T cell responses to virus-derived antigens, 4) augment NY-ESO-1 specific effector T cell responses by providing a co-stimulatory signals through the OX40L, 4-1BBL CD86, CD54 molecules, and 5) cross present tumor-associated antigen as IgG-immune complex.In summary, we provide the first evidence of two subsets of TANs in lung cancer. All TANs had an activated phenotype and could support (rather than inhibit) T cell functions to some degree. However, we identified a subset of TAN in early-stage lung tumors that can undergo a unique differentiation process resulting in formation of specialized subset of APC-like hybrid neutrophils. These hybrid TANs had enhanced ability to trigger and support T cell responses in direct cell-cell interactions. This property of hybrid neutrophils may provide new opportunities to boost the efficacy of vaccines based on cytotoxic T lymphocyte induction.Citation Format: Evgeniy Eruslanov, Pratik Bhojnagarwala, Jon Quatromoni, Shaun O'Brien, Edmund Moon, Tom Stephen, Abhishek Rao, Alfred Garfall, Wayne Hancock, Jose Conejo-Garcia, Charuhas Deshpande, Michael Feldman, Sunil Singhal, Steven Albelda. The origin and role of APC-like hybrid tumor-associated neutrophils in early-stage human lung cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A02.
Thursday, July 28, 2016 7:56 AM|Khan, S. Q., Faridi, M. H., Ruffell, B., Lopez, A. M. S., Jehangir, S., Barbosa, A. J., Torroella-Kouri, M., Coussens, L. M., Varner, J., Gupta, V.|Cancer Research recent issues|Labels: INT, breast cancer
Breast cancer remains the second highest cancer-related cause of death in women today. Neoadjuvant therapies reduce tumor size in patients but long-term survival remains poor. Tumor microenvironment shows a significant infiltration of CD11b+ tumor associated macrophages (TAMs) that promote tumor growth and metastases, while suppressing T-cell mediated anti-tumor immunity, suggesting that reducing infiltration of CD11b+ TAMs could have a therapeutic benefit. We recently developed a novel small molecule that targets CD11b+ cells and reduces their migration and tissue recruitment. Unlike previously described strategies, our methodology involves allosteric activation of CD11b, which induces transient increase in adhesion of CD11b+ cells to the vascular endothelium, thereby reducing the ability of the myeloid cells to transmigrate out of circulation and suppressing their recruitment into tumor and inflamed tissues. However, it is currently not known as to whether small molecule mediated CD11b activation can reduce TAM infiltration in tumors and whether that will have a therapeutic benefit. Here, we present results from studies with our lead compound leukadherin1 (LA1) in murine models of breast cancer. Our studies show that treatment of established syngeneic Cl66 orthotopic breast tumors in Balb/c mice with LA1 significantly reduces tumor growth and increases survival. LA1 treatment significantly reduced infiltration of CD11b+ cells into the tumor stroma, without affecting the levels of cells in circulation. LA1 treatment also significantly reduced expression of molecules that promote angiogenesis and tumor growth including MMP9, S100A8 and ARG1 that coincided with decreased CD31 and α-SMA, suggesting tumor vasculature disruption. Immune cell profiling revealed that LA1 treatment further promotes anti-tumor immunity by increasing the influx of cytotoxic CD8+ T cells into tumors. These data provide a rationale for using allosteric CD11b agonists to target TAM recruitment as a novel therapeutic approach for breast cancer.Citation Format: Samia Q. Khan, Mohd H. Faridi, Brian Ruffell, Ana M. S. Lopez, Shehryar Jehangir, Antonio J. Barbosa, Marta Torroella-Kouri, Lisa M. Coussens, Judith Varner, Vineet Gupta. An integrin agonist suppresses breast cancer by reducing CD11b+ leukocytes, promoting anti-tumor immunity. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A17.
Sunday, February 21, 2016 4:00 PM|Angiogenesis|MedWorm: Carcinoma in Situ|Comments|Labels: INT, VEGF, breast cancer
In conclusion, total α v ß 3 integrin expression is the best discriminator between breast cancer, fibroadenoma and normal breast tissue. With respect to vascular targeting and molecular imaging of angiogenesis, endothelial VEGFR-2 appeared to be slightly superior to endothelial α v ß 3 for differentiating benign from cancerous lesions. (Source: Angiogenesis)
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References

1. Margadant C SA. Integrin-TGF-beta crosstalk in fibrosis, cancer and wound healing. EMBO Rep. 2010;11(2):97-105. PMCID: 20075988.

2. Bandyopadhyay A RS. Defining the role of integrin alphavbeta6 in cancer. Curr Drug Targets. 2009;10(7):645-52. PMCID: 19601768.

3. Cortez V NB, Chakravarty D, Vadlamudi RK. Integrin-linked kinase 1: role in hormonal cancer progression. Front Biosci (Schol Ed). 2010;3:788-96. PMCID: 21196412.