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Most patients who die of cancer do so from its metastasis to other organs. The calcium-binding protein S100A4 can induce cell migration/invasion and metastasis in experimental animals and is overexpressed in most human metastatic cancers. Here, we report that a novel inhibitor of S100A4 can specifically block its increase in cell migration in rat (IC50, 46 µM) and human (56 µM) triple negative breast cancer (TNBC) cells without affecting Western-blotted levels of S100A4. The moderately-weak S100A4-inhibitory compound, US-10113 has been chemically attached to thalidomide to stimulate the proteasomal machinery of a cell. This proteolysis targeting chimera (PROTAC) RGC specifically eliminates S100A4 in the rat (IC50, 8 nM) and human TNBC (IC50, 3.2 nM) cell lines with a near 20,000-fold increase in efficiency over US-10113 at inhibiting cell migration (IC50, 1.6 nM and 3.5 nM, respectively). Knockdown of S100A4 in human TNBC cells abolishes this effect. When PROTAC RGC is injected with mouse TNBC cells into syngeneic Balb/c mice, the incidence of experimental lung metastases or local primary tumour invasion and spontaneous lung metastasis is reduced in the 10–100 nM concentration range (Fisher’s Exact test, p ≤ 0.024). In conclusion, we have established proof of principle that destructive targeting of S100A4 provides the first realistic chemotherapeutic approach to selectively inhibiting metastasis.

Purpose: Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.Experimental Design: Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers.Results: Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing–induced apoptosis. K-Ras–mutated, c-Met–amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways.Conclusion: Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. Clin Cancer Res; 18(2); 380–91. ©2011 AACR.

PDF file - 3.6K, Cancer cells with K-Ras activation mutation (HCT116/Hkh-2 isogenic pair) are more susceptible to Ran silencing-induced apoptosis.

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PDF file - 1.6MB, Percentage of sub-G1 phase apoptotic MCF10a and MDA MB231 cells grown in serum-containing medium after Ran silencing. (A-B) Cells were infected with shRan1 and shRan2. Medium was changed to serum-free medium (DMEM) 24hr post-infection. Cells were harvested at 72hr and 96hr post-infection for (A) cell cycle and (B) Western blot analyses. (A) Significantly more sub-G1 phase cells were observed in MDA MB231 breast cancer cells than in immortalized breast epithelial cells 72 and 96 hr post-infection of the shRNAs. (B) More cleaved PARP, caspase 9 and caspase 3 were observed in MDA MB231 than in MCF10A cells upon Ran silencing, suggesting a higher apoptotic response in MDA MB231 than in MCF10a cells. Levels of Ran knockdown were similar in both cell lines. Actin was used as a loading control. (C-D) Cells were infected with shRan1 and shRan2, and harvested 72hr post-infection for (C) cell cycle and (D) Western blot analyses. (C) More apoptotic cells were observed in MDA MB231 than MCF10a cells upon Ran-silencing 72hr post-infection of shRNAs, when these two cell lines were grown in their normal medium. (D) Higher levels of cleaved PARP, caspase 9 and caspase 3 were observed at 72hr post-infection of shRNAs. Results are plotted as histogram showing the mean � SD from three independent experiments. Key; * represents p < 0.05 and ** represents p

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PDF file - 13.3MB, High level of Ran is correlated with shorter survival time of lung cancer patients.(A) Representative images for IHC staining in lung cancer specimens. Lung cancer specimens stained with different patterns for Ran. Upper panel, 40x magnification; Lower panel, 200x magnification of the same cases. (B) Kaplan-Meier plot of the survival of lung cancer patients using combined nuclear and cytoplasmic expression of Ran; High level, high level expression of both nuclear and cytoplasmc Ran; Intermediate level, high level expression of either nuclear or cytoplasmic Ran; Low level, low level expression of both nuclear and cytoplasmic Ran. Cumulative proportion of lung cancer patients surviving with time is plotted for high, intermediate and low Ran groups. (C) Kaplan-Meier plot of survival of lung cancer patients from seven microarray datasets available in GEO database. Cumulative proportion of lung cancer patients surviving with time is plotted for high (top 25% of patients expressing a high level of Ran) and low (bottom 25% of patients expressing a low level of Ran) Ran groups.

Brain metastases are common and are usually detected by MRI. Diffusion tensor imaging (DTI) is a derivative MRI technique that can detect disruption of white matter tracts in the brain. We have matched preoperative DTI with image-guided sampling of the brain–tumor interface in 26 patients during resection of a brain metastasis and assessed mean diffusivity and fractional anisotropy (FA). The tissue samples were analyzed for vascularity, inflammatory cell infiltration, growth pattern, and tumor expression of proteins associated with growth or local invasion such as Ki67, S100A4, and MMP2, 9, and 13. A lower FA in the peritumoral region indicated more white matter tract disruption and independently predicted longer overall survival times (HR for death = 0.21; 95% confidence interval, 0.06–0.82; P = 0.024). Of all the biological markers studied, only increased density of CD3+ lymphocytes in the same region correlated with decreased FA (Mann–Whitney U, P = 0.037) as well as confounding completely the effect of FA on multivariate survival analyses. We conclude that the T-cell response to brain metastases is not a surrogate of local tumor invasion, primary cancer type, or aggressive phenotype and is associated with patient survival time regardless of these biological factors. Furthermore, it can be assayed by DTI, potentially offering a quick, noninvasive, clinically available method to detect an active immune microenvironment and, in principle, to measure susceptibility to immunotherapy.Significance: These findings show that white matter tract integrity is degraded in areas where T-cell infiltration is highest, providing a noninvasive method to identify immunologically active microenvironments in secondary brain tumors. Cancer Res; 78(3); 610–6. ©2017 AACR.

Fig S1 Whole Mounts, Fig S2 Westerns, Fig S3 Male/Female Bsk, Fig S4 Inhibitors, Fig S5 Breast Tumors, Fig S6 MMP2 + Other, Fig S7 MMP2/S100A4, Supp Methods, Figure 5 Legend.

supplemental materials and methods.

Table S1 MMPs/ S100A4, Table 2 Survival Times, Table S3 Cox Results, Table S4 2x2 Tables, Table S5 Logistic Association , Table S6 Same Area Staining.

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PDF file - 3.5, MTT assay showing that cancer cells with K-Ras activation mutation (HCT116/Hkh-2 and DLD1/DKO3 isogenic pairs) and c-Met (HCC827 parental/GR5 pair) are more susceptible to Ran silencing-induced apoptosis. (A) In HCT116/Hkh-2 colon cancer isogenic pair, HCT116 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 5 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, Hkh-2. (B) In DLD1/DKO3 colon cancer isogenic pair, DLD1 cells, which contain K-Ras activation mutation, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the K-Ras wild-type counterpart, DKO3. (C) In HCC827 parental/GR5 lung cancer pair, the GR5 cells, which contain c-Met amplification, are more susceptible to apoptosis induction by the 2 shRNAs targeting Ran, compared to the wild-type HCC827 cells. Results are plotted as histogram showing the mean � SD from three independent experiments. Key; *, ** and *** represent p < 0.05

Many human glandular cancers metastasize along nerve tracts, but the mechanisms involved are generally poorly understood. The calcium-binding protein S100A4 is expressed at elevated levels in human cancers, where it has been linked to increased invasion and metastasis. Here we report genetic studies in a Drosophila model to define S100A4 effector functions that mediate metastatic dissemination of mutant Ras-induced tumors in the developing nervous system. In flies overexpressing mutant RasVal12 and S100A4, there was a significant increase in activation of the stress kinase JNK and production of the matrix metalloproteinase MMP1. Genetic or chemical blockades of JNK and MMP1 suppressed metastatic dissemination associated with S100A4 elevation, defining required signaling pathway(s) for S100A4 in this setting. In clinical specimens of human breast cancer, elevated levels of the mammalian paralogs MMP2, MMP9, and MMP13 are associated with a 4- to 9-fold relative decrease in patient survival. In individual tumors, levels of MMP2 and MMP13 correlated more closely with levels of S100A4, whereas MMP9 levels correlated more closely with the S100 family member S100P. Overall, our results suggest the existence of evolutionarily conserved pathways used by S100A4 to promote metastatic dissemination, with potential prognostic and therapeutic implications for metastasis by cancers that preferentially exploit nerve tract migration routes. Cancer Res; 77(3); 780–9. ©2016 AACR.

PDF file - 1.8K, Percentage of sub-G1 phase apoptotic cells and apoptotic pathway induced by Ran silencing in different cell lines. (A) Cells were harvested 72 hours post-infection, fixed and stained with Propidium iodide. Significantly fewer Sub-G1 phase apoptotic cells were observed in the immortalized human embryonic kidney 293 (HEK 293) cell line compared to cancer cell lines from breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (HCT116 and DLD-1) upon Ran silencing (p < 0.01). (B) Western blots of apoptosis mediators in immortalized HEK293, breast (MDA MB231), lung (A549), prostate (DU145), esophageal (EC109) and colon (DLD-1 and HCT116) cancer cell lines at 72 hours post-infection with Scr or Ran shRNAs.

PDF file - 1.9MB, Ran silencing induced apoptosis is concentration dependent. Effect of Ran silencing on apoptotic induction was tested using five different shRNAs (shRan1-5) targeting different regions of Ran compared to scramble shRNA (shScr) control in MDA MB231 breast cancer and A549 lung cancer cells. (A) mRNA and (B) protein levels of Ran, and (C) Flow cytometric analysis for DNA content, in MDA MB231 cells infected with different shRNAs targeting Ran. (D) Protein level and (E) Flow analysis for DNA content in A549 cells infected with different shRNAs targeting Ran. Note that the decrease in Ran mRNA level was associated with the decrease in Ran protein levels (r = 0.950, p < 0.01) in MDA MB231 cells infected with shRan1-5. In addition, the percentage of sub-G1 phase apoptotic cells induced by Ran silencing was significantly inversely associated with the levels of Ran in protein levels in MDA MB231 breast cancer cells (r = -0.880, p

supplementary data on IHC stains and image files

PDF file - 4.8, Effect of chemical inhibitors on the activity of signaling pathways in MDA MB231 and A549 cells expressing various levels of Ran. Western blots of phospho- and total Akt, ERK and S6K proteins in MDA MB231 and A549 cells expressing various levels of Ran grown in different growth conditions. Representative blots from two independent experiments are shown.

PDF file - 725K, Ran silencing-induced apoptosis depends on the growth stimulation in MDA MB231 and A549 cells using MTT assay.Percentage of cell survival in (A) MDA MB231 and (B) A549 cells expressing various levels of Ran grown in different conditions as determined by MTT assay. Results are plotted as histograms showing the mean � SD from three independent experiments. Key; *, ** and *** represent p < 0.05

Background: Breast cancer brain metastases (BCBM) are a growing clinical problem and a significant cause of morbidity and mortality. The genomic landscape of BCBM can be divergent from that of the primary breast cancer (BC) and extracranial metastasis. Identifying key and targetable genomic aberrations within BCBM will be key in developing novel therapeutic strategies to treat this area of unmet and to need improve patient outcomes. Identifying rare and low-level mutations due to tumour heterogeneity in cancer samples and/or poor quality DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues requires tools that offer both low frequency detection and efficient use of starting material. This study aims to assess paired primary BC and brain metastasis (BM) for aberrations within known BC drivers: TP53, PIK3CA, ERBB2, ESR1 and AKT utilising the MassARRAY® UltraSEEK™ technology (Agena Bioscience, San Diego, USA), that provides a targeted, multiplexed method for detecting rare events and with sensitivity as low as 0.1%, developed to support DNA isolated from FFPE tissues, plasma and cerebrospinal fluid. Methods: DNA was isolated (Qiagen GeneRead FFPE kit) from of 32 BC and their matched BM cases and screened using the custom UltraSEEK™ BC Panel (Agena Bioscience, San Diego, USA). The Panel tests 44 mutations across 5 oncogenes in 8 multiplex assays; AKT1 (E17K, L52R), ERBB2 (G309E, G309A, S310F, L755R, L755S, L755_T759del, D769H, D769Y, V777L, V777L, L869R), ESR1 (A283V, K303R, E380Q, V392I, S436P, V534E, L536R, L536Q, L536Q, Y537N, Y537S, Y537C, D538G, S576L), PIK3CA (N345K, C420R, E542K, E545K, E545Q, E545A, H1047R, H1047L, E542K), TP53 (R175H, R213*, Y220C, R248W, R248W, R248Q, R273C, R273H). Results: Preliminary data analysis using the automated Typer software v4.0.26.74 (Agena Bioscience) identified more than one mutations in 29/32 (90.6%) primary BCs and 26/32 (81.3%) BM. TP53, ESR1, PIK3CA were mutated in 82.7%, 41.4% and 27.6% respectively of primary cases and in 80.8%, 34.6% and 23.1% respectively of BCBM. Of note, the pattern of mutations was similar in only 13/32 (40.6%) of the paired cases. The commonest mutations identified in both the primary and BCBM were TP53 (R175H, R273H, R248Q, R213X), PIK3CA (H1047L, E545K, E542K). The ESR1 (S576L) was the commonest ESR1 mutation identified in both BC and BM but in only one paired case, whereas the Y537N was present in only 2 BM cases (2/9, 22.2% of BM cases harbouring ESR1 mutations) cases. In addition, the AKT1-E17K was present in 3 patients; a paired case of BC and BM, one BC and one BM. The ERBB2-L755S was present in 1 BC and the ERBB2-L755_T759del only in a BM. As this is a custom BC panel, further analysis and validation is required. Conclusion: This study provides further evidence that the genomic landscape of the BCBM can differ from the primary, that BCBM contain potential actionable mutations and that they harbour ESR1 mutations. Such BCBM specific genomic data will be key to developing rationale therapeutic studies to develop treatments for this area of unmet need. The UltraSEEK™ technology provides a powerful tool to investigate low abundance mutations without compromising analytical accuracy and sensitivity. Citation Format: Athina Giannoudis, Lee Eastoe, Christopher Charlton, Nicholas Hickson, Rasheed Zakaria, Angela Platt-Higgins, Philip S Rudland, Darryl Irwin, Alexander Sartori, Michael D Jenkinson, Carlo Palmieri. UltraSEEK breast cancer panel for low frequency mutation detection in breast cancer brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-09-07.

Purpose Brain metastases (BM) are an increasing clinical problem. This study aimed to assess paired primary breast cancers (BC) and BM for aberrations within TP53, PIK3CA, ESR1, ERBB2 and AKT utilising the MassARRAY® UltraSEEK® technology (Agena Bioscience, San Diego, USA). Methods DNA isolated from 32 paired primary BCs and BMs was screened using the custom UltraSEEK® Breast Cancer Panel. Data acquisition and analysis was performed by the Agena Bioscience Typer software v4.0.26.74. Results Mutations were identified in 91% primary BCs and 88% BM cases. TP53, AKT1, ESR1, PIK3CA and ERBB2 genes were mutated in 68.8%, 37.5%, 31.3%, 28.1% and 3.1% respectively of primary BCs and in 59.4%, 37.5%, 28.1%, 28.1% and 3.1% respectively of BMs. Differences in the mutations within the 5 genes between BC and paired BM were identified in 62.5% of paired cases. In primary BCs, ER-positive/HER2-negative cases harboured the most mutations (70%), followed by ER-positive/HER2-positive (15%) and triple-negatives (13.4%), whereas in BMs, the highest number of mutations was observed in triple-negative (52.5%), followed by ER-positive/HER2-negative (35.6%) and ER-negative/HER2-positive (12%). There was a significant association between the number of mutations in the primary BC and breast-to-brain metastasis-free survival (p = 0.0001) but not with overall survival (p = 0.056). Conclusion These data demonstrate the discordancy between primary BC and BM, as well as the presence of clinically important, actionable mutations in BCBM. The UltraSEEK® Breast Cancer Panel provides a tool for BCBM that can be utilised to direct more tailored treatment decisions and for clinical studies investigating targeted agents.

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. SIMPLE SUMMARY: Ran is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

Immune checkpoint inhibition is an established treatment in programmed death-ligand 1 (PD-L1)-positive metastatic triple-negative (TN) breast cancer (BC). However, the immune landscape of breast cancer brain metastasis (BCBM) remains poorly defined.The tumour-infiltrating lymphocytes (TILs) and the messenger RNA (mRNA) levels of 770 immune-related genes (NanoString™, nCounter™ Immuno-oncology IO360) were assessed in primary BCs and BCBMs. The prognostic role of ARG2 transcripts and protein expression in primary BCs and its association with outcome was determined.There was a significant reduction of TILs in the BCBMs in comparison to primary BCs. 11.5% of BCs presented a high immune infiltrate (hot), 46.2% were altered (immunosuppressed/excluded) and 34.6% were cold (no/low immune infiltrate). 3.8% of BCBMs were hot, 23.1% altered and 73.1% cold. One hundred and twelve immune-related genes including PD-L1 and CTLA4 were decreased in BCBM compared to the primary BCs (false discovery rate0.01, log2 fold-change1.5). These genes are involved in matrix remodelling and metastasis, cytokine-chemokine signalling, lymphoid compartment, antigen presentation and immune cell adhesion and migration. Immuno-modulators such as PD-L1 (CD274), CTLA4, TIGIT and CD276 (B7H3) were decreased in BCBMs. However, PD-L1 and CTLA4 expression was significantly higher in TN BCBMs (P = 0.01), with CTLA4 expression also high in human epidermal growth factor receptor 2-positive (P0.01) compared to estrogen receptor-positive BCBMs. ARG2 was one of four genes up-regulated in BCBMs. High ARG2 mRNA expression in primary BCs was associated with worse distant metastasis-free survival (P = 0.038), while ARG2 protein expression was associated with worse breast-brain metastasis-free (P = 0.027) and overall survival (P = 0.019). High transcript levels of ARG2 correlated to low levels of cytotoxic and T cells in both BC and BCBM (P0.01).This study highlights the immunological differences between primary BCs and BCBMs and the potential importance of ARG2 expression in T-cell depletion and clinical outcome.