Martin E. Hemler

The protein-acyltransferase DHHC3 supports a few different tumor malignancies, but mechanisms have been unclear. Here we report that DHHC3-null B16F10 melanoma cells showed markedly elevated oxidative stress and senescence, accompanied by diminished tumor growth within immunocompetent C57/BL6 mice, but not in immunodeficient NOD-SCID mice. These results suggest that absence of DHHC3 enhances innate and/or adaptive anti-melanoma immunity. Consistent with this, DHHC3-null melanomas contained elevated numbers of anti-tumor cells (M1 macrophages, NK, CD4⁺T, CD8⁺T), whereas pro-tumor cells (M2 macrophages, MDSCs) were diminished. Unexpectedly, DHHC3 ablation minimally affected experimental metastasis of cells injected into either immunocompetent C57/BL6 or immunodeficient NOD-SCID mice. We conclude that DHHC3 ablation does not fundamentally alter melanoma cell growth and invasion/metastasis (independent of the immune system) despite its effects on oxidative stress and senescence. However, DHHC3 does control primary melanoma growth by supporting anti-melanoma immunity.

Silencing of DHHC3, an acyltransferase enzyme in the DHHC family, extensively upregulates oxidative stress (OS). Substrates for DHHC3-mediated palmitoylation include several antioxidant proteins and many other redox regulatory proteins. This helps to explain why DHHC3 ablation upregulates OS. DHHC3 also plays a key role in cancer. DHHC3 ablation leads to diminished xenograft growth of multiple cancer cell types, along with diminished metastasis. Furthermore, DHHC3 protein is upregulated on malignant/metastatic cancer samples, and upregulated gene expression correlates with diminished patient survival in several human cancers. Decreased primary tumor growth due to DHHC3 ablation may be partly explained by an elevated OS → senescence → innate immune cell recruitment mechanism. Elevated OS due to DHHC3 ablation may also contribute to adaptive anticancer immunity and impair tumor metastasis. In addition, DHHC3 ablation disrupts antioxidant protection mechanisms, thus enhancing the efficacy of OS-inducing anticancer drugs. A major focus has thus far been on OS regulation by DHHC3. However, remaining to be studied are multiple DHHC3 substrates that may affect tumor behavior independent of OS. Nonetheless, the currently established properties of DHHC3 make it an attractive candidate for therapeutic targeting in situations in which antioxidant protections need to be downmodulated, and also in cancer.

Ablation of protein acyltransferase DHHC3 selectively enhanced the anti-cancer cell activities of several chemotherapeutic agents, but not kinase inhibitors. To understand why this occurs, we used comparative mass spectrometry-based palmitoyl-proteomic analysis of breast and prostate cancer cell lines, ± DHHC3 ablation, to obtain the first comprehensive lists of candidate protein substrates palmitoylated by DHHC3. Putative substrates included 22–28 antioxidant/redox-regulatory proteins, thus predicting that DHHC3 should have antioxidant functions. Consistent with this, DHHC3 ablation elevated oxidative stress. Furthermore, DHHC3 ablation, together with chemotherapeutic drug treatment, (a) elevated oxidative stress, with a greater than additive effect, and (b) enhanced the anti-growth effects of the chemotherapeutic agents. These results suggest that DHHC3 ablation enhances chemotherapeutic drug potency by disabling the antioxidant protections that contribute to drug resistance. Affirming this concept, DHHC3 ablation synergized with another anti-cancer drug, PARP inhibitor PJ-34, to decrease cell proliferation and increase oxidative stress. Hence, DHHC3 targeting can be a useful strategy for selectively enhancing potency of oxidative stress-inducing anti-cancer drugs. Also, comprehensive identification of DHHC3 substrates provides insight into other DHHC3 functions, relevant to in vivo tumor growth modulation.

Tetraspanin protein CD151 has typically been studied as binding partner and functional regulator of laminin-binding integrins. However, we show here that CD151 supports anti-cancer drug resistance independent of integrins. CD151 ablation sensitized multiple tumor cell types to several anti-cancer drugs (e.g., gefitinib and camptothecin), thus increasing apoptosis, as seen using cleaved caspase-3, cleaved PARP (poly (ADP-ribose) polymerase), annexin V, and propidium iodide staining assays. Drug sensitization due to CD151 ablation is integrin-independent, because, (1) effects occurred in cells when integrins were unengaged with ligand, (2) integrin ablation (α3 and α6 subunits) did not mimic effects of CD151 ablation, (3) the CD151^QRD mutant, with diminished integrin association, and CD151^WT (unmutated CD151) similarly reconstituted drug protection, and (4) treatment with anti-cancer drugs selectively upregulated intracellular nonintegrin-associated CD151 (NIA-CD151), consistent with its role in drug resistance. Together, these results suggest that upregulated CD151 expression may support not only typical integrin-dependent functions, but also integrin-independent survival of circulating (and possibly metastatic) cancer cells during anti-cancer drug therapy.

DHHC-type protein acyltransferases may regulate the localization, stability, and/or activity of their substrates. In this study, we show that the protein palmitoyltransferase DHHC3 is upregulated in malignant and metastatic human breast cancer. Elevated expression of DHHC3 correlated with diminished patient survival in breast cancer and six other human cancer types. ZDHHC3 ablation in human MDA-MB-231 mammary tumor cell xenografts reduced the sizes of both the primary tumor and metastatic lung colonies. Gene array data and fluorescence dye assays documented increased oxidative stress and senescence in ZDHHC3-ablated cells. ZDHHC3-ablated tumors also showed enhanced recruitment of innate immune cells (antitumor macrophages, natural killer cells) associated with clearance of senescent tumors. These antitumor effects were reversed upon reconstitution with wild-type, but not enzyme-active site-deficient DHHC3. Concomitant ablation of the upregulated oxidative stress protein TXNIP substantially negated the effects of ZDHHC3 depletion on oxidative stress and senescence. Diminished DHHC3-dependent palmitoylation of ERGIC3 protein likely played a key role in TXNIP upregulation. In conclusion, DHHC3-mediated protein palmitoylation supports breast tumor growth by modulating cellular oxidative stress and senescence. Cancer Res; 77(24); 6880–90. ©2017 AACR.

In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these "present-absent-present" TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is "absent-present-absent" in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 - molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.

Cell surface transmembrane protein IGFS8 (herein called EWI-2) negatively regulates melanoma TGF-β signaling and is well positioned to control the transition in TGF-β signaling from cytostatic (in early melanoma stages) to pro-invasion/metastasis (in later stages). EWI-2 functions by sequestering the tetraspanin proteins CD9 and CD81, thereby making them unavailable to support the association of TGFβ receptor 1 with TGFβ receptor 2.

Ablation of tetraspanin protein TSPAN12 from human MDA-MB-231 cells significantly decreased primary tumor xenograft growth, while increasing tumor apoptosis. Furthermore, TSPAN12 removal markedly enhanced tumor-endothelial interactions and increased metastasis to mouse lungs. TSPAN12 removal from human MDA-MB-231 cells also caused diminished association between FZD4 (a key canonical Wnt pathway receptor) and its co-receptor LRP5. The result likely explains substantially enhanced proteosomal degradation of β-catenin, a key effecter of canonical Wnt signaling. Consistent with disrupted canonical Wnt signaling, TSPAN12 ablation altered expression of LRP5, Naked 1 and 2, DVL2, DVL3, Axin 1, and GSKβ3 proteins. TSPAN12 ablation also altered expression of several genes regulated by β-catenin (e.g. CCNA1, CCNE2, WISP1, ID4, SFN, ME1) that may help to explain altered tumor growth and metastasis. In conclusion, these results provide the first evidence for TSPAN12 playing a role in supporting primary tumor growth and suppressing metastasis. TSPAN12 appears to function by stabilizing FZD4–LRP5 association, in support of canonical Wnt-pathway signaling, leading to enhanced β-catenin expression and function.