Anoikis is an apoptotic cell death program triggered upon detachment from surrounding extracellular structures. The ability to evade cell death by anoikis in the presence of apoptosis-inducing stimuli is necessary for the formation of malignant tumors and progression to metastasis. Our findings indicate that the BRN2 (POU3F2) transcription factor is associated with anoikis resistance in melanoma cells. However, the downstream BRN2 signaling cascade driving anoikis resistance remains unknown. We utilized genome-wide CRISPR screens to validate BRN2 as a driver of anoikis resistance. Small molecule inhibitors targeting the BRN2 protein were employed as probes for quantitative mass spectrometry analysis, and the functional implication of BRN2 inhibition on cell survival, apoptosis and mitochondrial structure and function assessed in vitro experiments. Inhibition of BRN2 with small molecules in melanoma cell lines with acquired anoikis resistance resensitized to death by anoikis in ultra-low attachment conditions. Our quantitative mass spectrometry analysis revealed that BRN2 functionally impacts mitochondrial activity, whereby probes designed to inhibit BRN2 induced apoptosis and mitochondrial fragmentation through increased oxidative phosphorylation and AIFM2 expression. BRN2 inhibition additionally sensitized cells to BRAF-targeted therapy. Our study validated the role of BRN2 in anoikis resistance and identified that BRN2 drives anoikis resistance by promoting mitochondrial dysfunction. For the first time in melanoma models, small molecule inhibitors against BRN2 were demonstrated to reverse anoikis resistance and sensitize cells to BRAF-targeted therapy. Inhibition of BRN2 may prevent the seeding of metastatic disease by reversing anoikis resistance. Role of BRN2 in driving anoikis resistance in melanoma. Upon detachment from the extra-cellular matrix (ECM) melanoma cells must evade cell death by anoikis to seed distant metastases. This study expanded the understanding of the role of the BRN2 transcription factor as a driver of resistance to anoikis in melanoma. The use of small molecule inhibitors targeting BRN2 revealed that the transcription factor drives anoikis resistance via the MAPK and NF-κB signaling pathways, resulting in PPARγ dysregulation and subsequently driving mitochondrial dysfunction. Green boxes = previously published drivers of anoikis resistance in melanoma. Blue box = changes to mitochondrial function following inhibition of BRN2 as determined by proteomics analysis.

Background: Pancreatic ductal adenocarcinoma (PDAC) has a high incidence of perineural invasion (PNI), a pathological feature of the cancer invasion of nerves. PNI is associated with a poor prognosis, local recurrence and cancer pain. It has been suggested that interactions between nerves and the tumor microenvironment (TME) play a role in PDAC tumorigenesis. Methods: Here, we used Nanostring GeoMx Digital Spatial Profiler to analyze the whole transcriptome of both cancer and nerve cells in the microenvironment of PNI and non-PNI foci from 13 PDAC patients. Conclusions: We identified previously reported pathways involved in PNI, including Axonal Guidance and ROBO-SLIT Signaling. Spatial transcriptomics highlighted the role of PNI foci in influencing the immune landscape of the TME and similarities between PNI and nerve injury response. This study revealed that endocannabinoid and polyamine metabolism may contribute to PNI, cancer growth and cancer pain. Key members of these pathways can be targeted, offering potential novel research avenues for exploring new cancer treatment and/or pain management options in PDAC.

Metastatic spread and therapeutic resistance are the principal causes of cancer mortality. For melanoma, these processes rely on the capacity of cells to switch between transcriptional states. Although targeting transcriptional states pharmacologically is promising, the mechanisms by which melanoma cells switch between states—and how these processes differ from melanocytes—remain poorly understood. Here, we isolate distinct melanoma states with unique phenotypes: a MYC-driven state, essential for tumor initiation yet sensitive to BRAF inhibition, and a dedifferentiated, invasive BRN2-high state enriched in therapy-resistant cells but not directly tumorigenic. Transitions between phenotypes occur through intermediate, more differentiated states. Unexpectedly, the BRN2-high state is also present in melanocytes, whereas the MYC state is exclusive to melanoma. Melanoma cells also exhibit an increased frequency of transitions across states. These findings highlight that accelerated phenotypic switching, rather than mere state diversity, is a defining feature of melanoma progression.

Epoxytiglianes are a novel class of diterpene esters. The prototype epoxytigliane, EBC-46 (tigilanol tiglate), is a potent anti-cancer agent in clinical development for local treatment of a range of human and animal tumors. EBC-46 also consistently promotes wound re-epithelialization at the treatment sites, mediated via activation of classical protein kinase C (PKC) isoforms. We have previously shown that epoxytiglianes stimulate proliferative and wound repopulation responses in immortalized human skin keratinocytes (HaCaTs) in vitro, abrogated by pan-PKC inhibitor, bisindolylmaleimide-1. In this study, we further investigate the specific PKC isoforms responsible for inducing such wound healing responses, following HaCaT treatment with 1.51 nM-15.1 mu M EBC46 or analogue, EBC-211. Classical PKC inhibition by GO6976 (1 mu M), significantly attenuated epoxytigliane induced, HaCaT proliferation and wound repopulation at all epoxytigliane concentrations. PKC-(3I/-(3II isoform inhibition by enzastaurin (1 mu M), significantly inhibited HaCaT proliferation and wound repopulation responses induced by both epoxytiglianes, especially at 1.51-151 nM. PKC-alpha inhibitor, Ro 31-8220 mesylate (10 nM), exerted lesser inhibitory effects on HaCaT responses. Epoxytigliane changes in key keratin (KRT17) and cell cycle (cyclin B1, CDKN1A) protein levels were partly attenuated by GO6976 and enzastaurin. GO6976 also inhibited increases in matrix metalloproteinase (MMP-1, MMP-7, MMP-10) activities. Phospho-PKC (p-PKC) studies confirmed that epoxytiglianes transiently activated classical PKC isoforms (p-PKC alpha, p-PKC-(3I/-(3II, p-PKC gamma) in a dose- and time-dependent manner. By identifying how epoxytiglianes stimulate classical PKCs to facilitate keratinocyte healing responses and re-epithelialization, these findings support further epoxytigliane development as topical therapeutics for clinical situations involving impaired re-epithelialization, such as non-healing wounds in skin.

Background Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear.Methods A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed.Results Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade.Conclusions These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.

Pancreatic Ductal Adenocarcinoma (PDAC) has a high incidence of perineural invasion (PNI), a pathological feature of cancer invasion of nerves. PNI is associated with poor prognosis, metastasis, local recurrence and cancer pain. It has been suggested that interactions between nerves and the tumour microenvironment (TME) play a role in the PDAC tumorigenesis, however our ability to study these interactions in patient samples has been technologically limited. Here we used Nanostring GeoMx Digital Spatial Profiler to analyze the whole transcriptome of the cancer and nerve compartments in PNI and non-PNI foci from 13 PDAC patients. We identified previously reported pathways involved in PNI, including Axonal Guidance, ROBO-SLIT Signaling supporting the study approach. Spatial transcriptomics highlighted the potential role of nerve and cancer cells in PNI foci in influencing the immune landscape of the TME and suggested similarities between PNI and nerve injury-response. This study revealed novel pathways, Endocannabinoid and Polyamine metabolism, which may contribute to PNI and cancer pain. These findings require additional in vitro and/or in vivo investigations to dissect the mechanism that Endocannabinoids and Polyamines may play in PNI and cancer growth. These pathways can be targeted providing a potential novel approach to treat PDAC.

Background Tigilanol Tiglate (TT) is a novel small molecule under development for local treatment of solid tumours via intratumoral (I.T.) injection. TT is a protein kinase C (PKC)/C1 domain activator that disrupts tumour vasculature and causes direct oncolysis of tumour cells. Together, these activities lead to haemorrhagic necrosis of injected tumours with enduring ablation of >70% of target tumours in both pre-clinical xenograft/syngeneic mouse models and cutaneous tumours presenting in the veterinary clinic.1–3 TT has completed a Phase I/IIa dose-escalation trial in humans (ACTRN12614000685617), with strong evidence of local anti-cancer efficacy and signs of abscopal effects in some patients.4 However, the underlying mechanism of action (MOA) of TT, together with its immunotherapeutic potential in oncology, is not fully understood. Methods A combination of microscopy, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, LDH release assays and mixed lymphocyte reactions were used to probe the MOA of TT in vitro. TT-mediated damage associated molecular pattern (DAMP) release/externalization was assessed using luciferase (ATP), ELISA (HMGB1), flow cytometry and immunohistochemical (HMGB1, calreticulin) approaches. In vivo experimentation with TT utilized CT-26 and B16-F10 tumor bearing mice. Analysis of DAMP release and immune cell infiltration into TT treated human head and neck tumours (ACTRN12619001407189) was performed by immunohistochemistry. Results Our data reveal that therapeutic concentrations of TT induce the death of cancer and endothelial cell lines via a pathway involving caspase activation and cleavage of the pore forming protein gasdermin E. TT promotes this mechanism of cell death by interacting with ER membranes, causing an ER stress response that results in loss of mitochondrial membrane potential, ATP depletion, organelle swelling and oncosis/pyroptosis. Treatment of cells with TT also led to the release of damage associated molecular patterns (DAMPs), indicative of an immunogenic cell death (ICD) pathway that also resulted in the generation of tumour-specific T cells in CT-26 tumor bearing mice. Whilst the induction of ICD is largely PKC-independent in vitro, PKC/C1 domain signaling appears necessary for efficacious tumour ablation in vivo. Consistent with our pre-clinical data, immunohistochemical analysis of TT-treated head and neck tumors found that drug stimulated DAMP release/externalisation and the recruitment of immune cells, principally CD8+ T cells, into remnant tumour mass. Conclusions These data indicate that TT is an oncolytic small molecule with the potential to ablate target tumours and enhance immunotherapy combinations through promoting immune cell infiltration. TT is currently undergoing Phase II trials in head and neck cancer (NCT05234437) and soft tissue sarcoma (NCT05755113). References Boyle GM, D'Souza MMA, Pierce CJ, Adams RA, Cantor AS, Johns JP, Maslovskaya L, Gordon VA, Reddell PW, Parsons PG. Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models. PLOS ONE 2014;9:e108887. Cullen JK, Boyle GM, Yap PY, Elmlinger S, Simmons JL, Broit N, Johns J, Ferguson B, Maslovskaya LA, Savchenko AI, Mirzayans PM, Porzelle A, Bernhardt PV, Gordon VA, Reddell PW, Pagani A, Appendino G, Parsons PG, Williams CM. Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes. Sci. Rep. 2021;11:207. De Ridder TR, Campbell JE, Burke-Schwarz C, Clegg D, Elliot EL, Geller S, Kozak W, Pittenger ST, Pruitt JB, Riehl J, White J, Wiest ML, Johannes CM, Morton J, Jones PD, Schmidt PF, Gordon VA, Reddell PW. Randomized controlled clinical study evaluating the efficacy and safety of intratumoral treatment of canine mast cell tumors with tigilanol tiglate (EBC-46). J. Vet. Intern. Med. 2021;35:415–429. Panizza BJ, de Souza P, Cooper A, Roohullah A, Karapetis CS, Lickliter JD. Phase I dose-escalation study to determine the safety, tolerability, preliminary efficacy and pharmacokinetics of an intratumoral injection of tigilanol tiglate (EBC-46). EBioMedicine. 2019;50:433–441. Ethics Approval The study obtained ethics approval from the following commitees and boards: Metro South Human Research Ethics Committee, 199 Ipswich Road, Woolloongabba, QLD, 4102, Australia. Ethics approval number: HREC/2019/QMS/54004. Bellberry Limited, 123 Glen Osmond Road, Eastwood, SA 5063, Australia. Ethics approval number: 2019–10-846 (REGIS 2019/ETH13063). Tata Memorial Hospital - Institutional Review Board, IRB Office, Dr. E. Borges Marg, Parel, Mumbai - 400 012, India. Ethics approval number: IEC/1219/3370/001. Tata Medical Center - Institutional Review Board, 14 Major Arterial Road (EW), New Town, Rajarhat, Kolkata - 700 160, India. Ethics approval number: 2019/PHARMA/57/IRB39.

PDF file - 675K, Supplementary Figure 1. Characterization of an ISRE in the human STAT1 gene promoter. Supplementary Figure 2. ChIP assay demonstrates binding of REST to the STAT1 RE-1 element in melanoma cell lines. Supplementary Figure 3. Full-length, uncropped gel image from Fig. 5A of REST mRNA and 18S rRNA levels analysed in MM96 and INS-1 cells by RT-PCR. Supplementary Figure 4. Full-length, uncropped blot images from Fig. 5B of A. REST, and B. α-tubulin protein levels determined in MM96 and INS-1 cells by western blotting. Supplementary Figure 5. Full-length, uncropped gel image from Fig. 7A of REST mRNA and 18S rRNA levels determined by RT-PCR in SK-Mel-28 and MM96 cells. Supplementary Figure 6. Full-length, uncropped images of western blot from Fig. 7B showing REST and STAT1 protein levels detected in SK-Mel-28 and MM96 cells Supplementary Figure 7. The same blot as in Suppl. Fig. 6, stripped and reprobed with α-tubulin antibody. Non-specific background blotch is visible in lane 7. Relevant bands and molecular weights are indicated by arrows in the figure.

The unusual and sterically constrained amino acid, seco-1-azacubane-2-carboxylic acid, was incorporated into a range of bioactive chemical templates, including enalaprilat, perindoprilat, endomorphin-2 and isoniazid, and subjected to biological testing. The endomorphin-2 derivative displayed increased activity at the δ opioid receptor, but a loss in activity was observed in the other cases, although human normal cell line evaluation suggests limited cytotoxic effects.

Supplementary Figure 2 from Kallikrein-Related Peptidase 7 Promotes Multicellular Aggregation via the α5β1 Integrin Pathway and Paclitaxel Chemoresistance in Serous Epithelial Ovarian Carcinoma

Cynanchum viminale subsp. australe, more commonly known as caustic vine, is a leafless succulent that grows in the northern arid zone of Australia. Toxicity toward livestock has been reported for this species, along with use in traditional medicine and its potential anticancer activity. Disclosed herein are novel seco-pregnane aglycones cynavimigenin A (5) and cynaviminoside A (6), together with new pregnane glycosides cynaviminoside B (7) and cynavimigenin B (8). Cynavimigenin B (8) contains an unprecedented 7-oxobicyclo[2.2.1]heptane moiety in the seco-pregnane series, likely arising from a pinacol-type rearrangement. Interestingly, these isolates displayed only limited cytotoxicity in cancer and normal human cell lines, in addition to low activity against acetylcholinesterase and Sarcoptes scabiei bioassays, suggesting that 5-8 are not associated with the reported toxicity of this plant species.

Human skin needs additional protection from damaging ultraviolet radiation (UVR: 280-400 nm). Harmful UVR exposure leads to DNA damage and the development of skin cancer. Available sunscreens offer chemical protection from detrimental sun radiation to a certain extent. However, many synthetic sunscreens do not provide sufficient UVR protection due to the lack of photostability of their UV-absorbing active ingredients and/or the lack of ability to prevent the formation of free radicals, inevitably leading to skin damage. In addition, synthetic sunscreens may negatively affect human skin, causing irritation, accelerating skin aging and even resulting in allergic reactions. Beyond the potential negative effect on human health, some synthetic sunscreens have been shown to have a harmful impact on the environment. Consequently, identifying photostable, biodegradable, non-toxic, and renewable natural UV filters is imperative to address human health needs and provide a sustainable environmental solution. In nature, marine, freshwater, and terrestrial organisms are protected from harmful UVR through several important photoprotective mechanisms, including the synthesis of UV-absorbing compounds such as mycosporine-like amino acids (MAAs). Beyond MAAs, several other promising, natural UV-absorbing products could be considered for the future development of natural sunscreens. This review investigates the damaging impact of UVR on human health and the necessity of using sunscreens for UV protection, specifically UV-absorbing natural products that are more environmentally friendly than synthetic UV filters. Critical challenges and limitations related to using MAAs in sunscreen formulations are also evaluated. Furthermore, we explain how the genetic diversity of MAA biosynthetic pathways may be linked to their bioactivities and assess MAAs' potential for applications in human health.

Supplementary Figures 1, 3, 4, Tables 1-3 from Kallikrein-Related Peptidase 7 Promotes Multicellular Aggregation via the α5β1 Integrin Pathway and Paclitaxel Chemoresistance in Serous Epithelial Ovarian Carcinoma

This file contains Supplementary experimental Procedures, Supplementary Figure Legends, and Table S1. Table S1. Changes to patient CD73 expression following BRAF targeted therapy.

Supplementary Figures 1, 3, 4, Tables 1-3 from Kallikrein-Related Peptidase 7 Promotes Multicellular Aggregation via the α₅β₁ Integrin Pathway and Paclitaxel Chemoresistance in Serous Epithelial Ovarian Carcinoma

The acquisition of resistance to anoikis, the cell death induced by loss of adhesion to the extracellular matrix, is an absolute requirement for the survival of disseminating and circulating tumour cells (CTCs), and for the seeding of metastatic lesions. In melanoma, a range of intracellular signalling cascades have been identified as potential drivers of anoikis resistance, however a full understanding of the process is yet to be attained. Mechanisms of anoikis resistance pose an attractive target for the therapeutic treatment of disseminating and circulating melanoma cells. This review explores the range of small molecule, peptide and antibody inhibitors targeting molecules involved in anoikis resistance in melanoma, and may be repurposed to prevent metastatic melanoma prior to its initiation, potentially improving the prognosis for patients.