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It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection. Significance: Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.

Spread sheet including a single tab per Figure panel conveying each graph's statistical analyses. Note that tabs are named accordingly. The tabs include the comprehensive statistical analyses that were performed for each experiment in which statistical significance was given. Each tab contains the full statistical output for its labeled experiment. The statistical test performed for each experiment is identified within the corresponding tab. Statistical tests were carried out using the software, Prism, version 7.05.

Spreadsheet including the metabolomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.

Supplementary Figure 1. Confirmation that CAFs produce ECMs distinct from NLFs. Supplementary Figure 2. Direct co-culture of CAFs support PDAC cells during nutrient-deprivation. Supplementary Figure 3. Additional CAF cell lines generate sEVs that rescue PDAC cells from nutrient deprivation-induced apoptosis. Supplementary Figure 4. NetG1+ CAF-sEVs support PDAC cell survival in a NGL-1 dependent manner. Supplementary Figure 5. NetG1 expression in CAFs is necessary for sEV-mediated survival of nutrient-deprived PDAC cells. Supplementary Figure 6. NetG1 ablation does not affect the uptake of sEV cargo in PDAC cells. Supplementary Figure 7. sEV supernatant contains DNPs enriched with sub-exosome sized EVs. Supplementary Figure 8. Enriched gene ontology clusters from proteomic and metabolomic analysis comparing sEV and DNP fractions. Supplementary Figure 9. EV cargo requires transfer in intact vesicles to provide tumor-supportive effect.

Spreadsheet including the proteomic analyses corresponding to the experiments indicated by the naming of the assorted tabs. Specific analysis parameters for each experiment are provided within the respective main figure legends.

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEV). The tumor-supportive CAFs possess a distinct phenotypic profile, compared with normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5β1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Furthermore, we show that NetG1 expression in CAFs is required for the prosurvival properties of sEVs. In addition, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5β1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5β1 were detected in sEVs collected from plasma of patients with PDAC, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor–stroma interactions and pathogenic stroma detection.Significance:Results from this study identified two unique types of tumor-supporting CAF EVs, with evidence of these being detected in patients. Thus, this study facilitates a novel avenue to further dissect the subtleties of the tumor–stroma interactions responsible for PDAC homeostasis and progression, as well as the possibility of establishing future means to detect and monitor dynamic stroma staging.

cRGD PC-3

Regulation of SNPH ubiquitination

Anti-glioma activity of MFF peptidomimetic

Supplemental Methods, Figures and Legends

Pathway analysis of SNPH-associated proteins

Syntaphilin (SNPH) inhibits the movement of mitochondria in tumor cells, preventing their accumulation at the cortical cytoskeleton and limiting the bioenergetics of cell motility and invasion. Although this may suppress metastasis, the regulation of the SNPH pathway is not well understood. Using a global proteomics screen, we show that SNPH associates with multiple regulators of ubiquitin-dependent responses and is ubiquitinated by the E3 ligase CHIP (or STUB1) on Lys111 and Lys153 in the microtubule-binding domain. SNPH ubiquitination did not result in protein degradation, but instead anchored SNPH on tubulin to inhibit mitochondrial motility and cycles of organelle fusion and fission, that is dynamics. Expression of ubiquitination-defective SNPH mutant Lys111→Arg or Lys153→Arg increased the speed and distance traveled by mitochondria, repositioned mitochondria to the cortical cytoskeleton, and supported heightened tumor chemotaxis, invasion, and metastasis in vivo. Interference with SNPH ubiquitination activated mitochondrial dynamics, resulting in increased recruitment of the fission regulator dynamin-related protein-1 (Drp1) to mitochondria and Drp1-dependent tumor cell motility. These data uncover nondegradative ubiquitination of SNPH as a key regulator of mitochondrial trafficking and tumor cell motility and invasion. In this way, SNPH may function as a unique, ubiquitination-regulated suppressor of metastasis.Significance: These findings reveal a new mechanism of metastasis suppression by establishing the role of SNPH ubiquitination in inhibiting mitochondrial dynamics, chemotaxis, and metastasis. Cancer Res; 78(15); 4215–28. ©2018 AACR.

Contents of Supplementary Figures ans Table

Detail description of microarray analysis and comparison with cancer stem cell dataset, and TMA of prostate cancer patients

ERK1 and p38 are not activated by alphavbeta6 integrin upon ligand binding.

MFF1 synthetic peptides used in this study

Full-size scans of PSA immunoblots shown in Figure 5.

Morphology of MFF targeting in tumor cells

Full-size scans of PSA and androgen receptor immunoblots shown in Figure 4.

Increased alphavbeta6 integrin and JNK expression and enhanced JNK nuclear localization in castrated mice.

MFF-VDAC1 complex in cancer

alphavbeta6 integrin, but not alphavbeta3 integrin, promotes tumor growth.

The regulators of mitochondrial cell death in cancer have remained elusive, hampering the development of new therapies. Here, we showed that protein isoforms of mitochondrial fission factor (MFF1 and MFF2), a molecule that controls mitochondrial size and shape, that is, mitochondrial dynamics, were overexpressed in patients with non–small cell lung cancer and formed homo- and heterodimeric complexes with the voltage-dependent anion channel-1 (VDAC1), a key regulator of mitochondrial outer membrane permeability. MFF inserted into the interior hole of the VDAC1 ring using Arg225, Arg236, and Gln241 as key contact sites. A cell-permeable MFF Ser223-Leu243 d-enantiomeric peptidomimetic disrupted the MFF–VDAC1 complex, acutely depolarized mitochondria, and triggered cell death in heterogeneous tumor types, including drug-resistant melanoma, but had no effect on normal cells. In preclinical models, treatment with the MFF peptidomimetic was well-tolerated and demonstrated anticancer activity in patient-derived xenografts, primary breast and lung adenocarcinoma 3D organoids, and glioblastoma neurospheres. These data identify the MFF–VDAC1 complex as a novel regulator of mitochondrial cell death and an actionable therapeutic target in cancer.Significance:These findings describe mitochondrial fission regulation using a peptidomimetic agent that disturbs the MFF–VDAC complex and displays anticancer activity in multiple tumor models.See related commentary by Rao, p. 6074

Complete Supplementary Data with Figures embedded

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

Androgen receptor signaling fuels prostate cancer and is a major therapeutic target. However, mechanisms of resistance to therapeutic androgen ablation are not well understood. Here, using a prostate cancer mouse model, Ptenpc−/−, carrying a prostate epithelial-specific Pten deletion, we show that the αvβ6 integrin is required for tumor growth in vivo of castrated as well as of noncastrated mice. We describe a novel signaling pathway that couples the αvβ6 integrin cell surface receptor to androgen receptor via activation of JNK1 and causes increased nuclear localization and activity of androgen receptor. This downstream kinase activation by αvβ6 is specific for JNK1, with no involvement of p38 or ERK kinase. In addition, differential phosphorylation of Akt is not observed under these conditions, nor is cell morphology affected by αvβ6 expression. This pathway, which is specific for αvβ6, because it is not regulated by a different αv-containing integrin, αvβ3, promotes upregulation of survivin, which in turn supports anchorage-independent growth of αvβ6-expressing cells. Consistently, both αvβ6 and survivin are significantly increased in prostatic adenocarcinoma, but are not detected in normal prostatic epithelium. Neither XIAP nor Bcl-2 is affected by αvβ6 expression. In conclusion, we show that αvβ6 expression is required for prostate cancer progression, including castrate-resistant prostate cancer; mechanistically, by promoting activation of JNK1, the αvβ6 integrin causes androgen receptor–increased activity in the absence of androgen and consequent upregulation of survivin. These preclinical results pave the way for further clinical development of αvβ6 antagonists for prostate cancer therapy. Cancer Res; 76(17); 5163–74. ©2016 AACR.

Supplemental Figure 1. Trop2 ICD abundance is maintained by Src kinase. Supplemental Figure 2. Canertinib, a tyrosine kinase inhibitor targeting EGFR reduces cell proliferation but does not affect Trop2 ICD abundance. Supplemental Figure 3. Schematic representation of the mechanism by which Src induction of cyclin D1 enhances cleavage of the membrane associated Trop2 complex to thereby enhance Trop2 ICD. Supplemental Figure 4. Human prostate cancer Tissue Microarray (TMA) IHC staining.

alphavbeta6 integrin promotes prostate cancer growth in castrated athymic nu/nu mice.

Proteomic analysis of castration-resistant prostate cancer demonstrated the enrichment of Src tyrosine kinase activity in approximately 90% of patients. Src is known to induce cyclin D1, and a cyclin D1–regulated gene expression module predicts poor outcome in human prostate cancer. The tumor-associated calcium signal transducer 2 (TACSTD2/Trop2/M1S1) is enriched in the prostate, promoting prostate stem cell self-renewal upon proteolytic activation via a γ-secretase cleavage complex (PS1, PS2) and TACE (ADAM17), which releases the Trop2 intracellular domain (Trop2 ICD). Herein, v-Src transformation of primary murine prostate epithelial cells increased the proportion of prostate cancer stem cells as characterized by gene expression, epitope characteristics, and prostatosphere formation. Cyclin D1 was induced by v-Src, and Src kinase induction of Trop2 ICD nuclear accumulation required cyclin D1. Cyclin D1 induced abundance of the Trop2 proteolytic cleavage activation components (PS2, TACE) and restrained expression of the inhibitory component of the Trop2 proteolytic complex (Numb). Patients with prostate cancer with increased nuclear Trop2 ICD and cyclin D1, and reduced Numb, had reduced recurrence-free survival probability (HR = 4.35). Cyclin D1, therefore, serves as a transducer of v-Src–mediated induction of Trop2 ICD by enhancing abundance of the Trop2 proteolytic activation complex. Cancer Res; 76(22); 6723–34. ©2016 AACR.

alphavbeta6 enhances AR transcriptional activity in LNCaP cells.

PDF file - 180K, The association between Trop-2 and β1 integrins occurs independently on β1 activation or adhesion to ECM ligands.

The molecular circuitries controlling osseous prostate metastasis are known to depend on the activity of multiple pathways, including integrin signaling. Here, we demonstrate that the αvβ6 integrin is upregulated in human prostate cancer bone metastasis. In prostate cancer cells, this integrin is a functionally active receptor for fibronectin and latency-associated peptide-TGF-β1; it mediates attachment and migration upon ligand binding and is localized in focal contacts. Given the propensity of prostate cancer cells to form bone metastatic lesions, we investigated whether the αvβ6 integrin promotes this type of metastasis. We show for the first time that αvβ6 selectively induces matrix metalloproteinase 2 (MMP2) in vitro in multiple prostate cancer cells and promotes osteolysis in vivo in an immunodeficient mouse model of bone metastasis through upregulation of MMP2, but not MMP9. The effect of αvβ6 on MMP2 expression and activity is independent of androgen receptor in the analyzed prostate cancer cells. Increased levels of parathyroid hormone–related protein (PTHrP), known to induce osteoclastogenesis, were also observed in αvβ6-expressing cells. However, by using MMP2 short hairpin RNA, we demonstrate that the αvβ6 effect on bone loss is due to upregulation of soluble MMP2 by the cancer cells, not due to changes in tumor growth rate. Another related αv-containing integrin, αvβ5, fails to show similar responses, underscoring the significance of αvβ6 activity. Overall, these mechanistic studies establish that expression of a single integrin, αvβ6, contributes to the cancer cell—mediated program of osteolysis by inducing matrix degradation through MMP2. Our results open new prospects for molecular therapy for metastatic bone disease. Cancer Res; 74(5); 1598–608. ©2014 AACR.

PDF file - 164K, Surface expression levels of wild type and Δcyto Trop-2 in PC3-2 transfectants.

PDF file - 23K, Contains a detailed version of methods used for the internalization assays shown in Figure S5.

PDF file - 1376K, Trop-2 promotes prostate cancer cell migration on FN.

PDF - 529K, Expression of alphavbeta6 does not affect PC3-2 integrin profile (S1); alphavbeta6 promotes osteolysis in vivo (S2); alphavbeta6 increases osteolytic lesions and osteoclast recruitment in vivo (S3); Downregulation of alphavbeta6 reduces MMP2 levels in RWPE cells (S4); Exogenous expression of alphavbeta6 and alphavbeta3 in C4-2B transfectants (S5); MMP2 is co-expressed with alphavbeta6 in human prostate cancer osteolytic metastases (S6).

PDF file - 30K, PC3-MM2/Trop-2-GFP disseminated in liver parenchyma.

PDF file - 30K, List of Antibodies used in this study.

PDF file - 318K, Surface levels of β1, αv and α5 integrins are not affected by Trop-2.

PDF file - 1575K, Trop-2 co-localizes with α5 and β1 integrin subunits in trafficking vesicles.