Most traditional cancer therapies target either the tumor cells themselves or indiscriminately kill any rapidly dividing cell.
New findings by researchers at University of California San Diego School of Medicine indicate that manipulating macrophages, a type of immune cell found abundantly in the tissues surrounding a tumor, could also be a viable strategy for treating cancer.
The study, published June 10, 2020 in PLoS Biology, is the first to uncover the role a molecule called IRE1α plays in determining whether macrophages promote inflammation in the tissues surrounding cancer cells – a region known as the tumor microenvironment – and throw off the ability of other immune cells to fight cancer.
Inflammation is known to promote tumor growth, making IRE1α an attractive target for future study and drug development.
“We’ve known that it takes a toll on a person’s ability to fight cancer when the tumor microenvironment is not properly regulated, when there’s a mix of pro-and anti-inflammatory macrophages,” said senior author Maurizio Zanetti, MD, professor of medicine at UC San Diego School of Medicine and head of the Laboratory of Immunology at UC San Diego Moores Cancer Center.
“What we discovered here is how that happens, and a potential way to reverse it.”
IRE1α is a key regulator of the unfolded protein response, a cellular process that mammalian cells use to deal with stress.
Life in the tumor microenvironment is stressful for immune and cancer cells, where they may be cut off from oxygen and nutrients. IRE1α and the unfolded protein response can often determine whether a cell survives under these conditions.
In the new study, Zanetti and team show for the first time that IRE1α and the unfolded protein response are also responsible for immune cell malfunction in the tumor microenvironment.
The researchers found that IRE1α regulates macrophage activation, determining whether these abundant immune cells secrete molecules that increase inflammation and at the same time produce signals that suppress the immune system.
They also discovered that IRE1α boosts levels of PD-L1, a molecule that inhibits other immune cells.
To corroborate their findings in mice, Zanetti and team looked for IRE1α patterns in genomic data available in The Cancer Genome Atlas (TCGA), the National Institutes of Health’s database of genomic information from thousands of human tumors.
They found that in human breast and cervical cancers, the presence of macrophage IRE1α predicts the presence of PD-L1.
IRE1α’s newly discovered role in regulating PD-L1 is significant because the interaction between PD-L1 on tumor cells and its receptor on immune cells tells the immune system to leave tumor cells alone.
Checkpoint inhibitors, a type of cancer immunotherapy, treat cancer by blocking that interaction, and thus boosting the immune system’s ability to fight off cancer. Other recent studies have shown that a person’s response to anti-PD-L1 immunotherapy depends on the PD-L1 present on their macrophages, not on their tumor cells.
What this means, Zanetti said, is that a therapeutic drug that inhibits macrophage IRE1α might work indirectly as a checkpoint inhibitor – less IRE1α could mean less PD-L1, removing the brake and allowing a person’s immune system to better attack tumor cells on its own.
To test this approach, the team engineered mice that lack the IRE1α gene in their macrophages. These IRE1α-deficient mice survived melanoma better than control mice.
“The implication for therapy is that, down the line, we might be able to locally inhibit IRE1α to specifically prevent the mis-regulation of the macrophages that infiltrate tumors and thus tip the balance in favor of the immune system rather than the tumor,” Zanetti said. “There is an urgent need to develop IRE1α inhibitors as therapeutics for humans.”
Tumour chemical and cellular microenvironments interact continually to select survival-adapted tumour cell and tumour-associated normal cell populations, and underpins both metastatic progression and therapeutic resistance.
The tumour cellular microenvironment is comprised of “normal” (vascular, stromal and inflammatory cells) and neoplastic components that co-exist within a poorly defined and poorly organized extracellular matrix, characterized by heterogeneous niches created by a highly abnormal vasculature and episodes of microenvironmental hypoxic, nutrient, metabolic and redox stress, which elicit cellular hypoxic, nutrient, oxidative and metabolic stress responses.
Tumour hypoxia promotes glycolytic metabolic adaptation by tumour cellular components, combined with oncogene-promoted metabolic changes, result in the malignant tumour-associated “Warburg” metabo-type [1–3].
The metabo-type, furthermore, promotes an acidic reducing tumour microenvironment, which together with tumour hypoxia, acts as potent driving forces for survival adaptation [4, 5], selecting “normal” and neoplastic tumour cellular components that exhibit increased resistance to programmed cell-death, a pro-angiogenic phenotype, sustained metabolic glycolytic reprogramming, progressive epithelial/mesenchymal (EMT) and stem cell-like de-differentiation, enhanced motile, invasive, scattering and metastatic behaviour, increased genetic instability and enhanced therapeutic resistance [5–13].
Tumour-hypoxia results when tumour cellular components are deprived of oxygen and occurs during all phases of tumour progression, from early initiation through clonal expansion to metastatic progression .
Solid tumours are characterized by heterogenous hypoxic areas adjacent to near normoxic regions and exhibit [pO2] concentrations ≤2.5 mmHg, significantly below those of normal vascular tissues, as a result of an imbalance between oxygen consumption and supply, e.g. [pO2] of 10–16 mmHg in cervical tumour tissues is significantly lower than the [pO2] 40–42 mmHg of normal cervical tissues [9, 10, 15].
Tumour hypoxia arises from a variety of mechanisms. Tumour perfusion-hypoxia is caused by an abnormal disorganized tumour vasculature, characterized by structural, functional and cellular abnormalities and inadequate blood flow, resulting in transient ischemic episodes of varying duration caused by blockage and/or flow stasis.
Tumour diffusion-hypoxia is caused by O2 diffusion distances > 70 μm between tumour tissues and blood vessels, and blood flow countercurrents within the tumour microvascular.
Tumour anemic hypoxia is caused by reduced O2 transport capacity resulting from the tumour itself or by systemic anemia caused by chemotherapy (Fig. 1a). In general, tumour-hypoxia is independent of tumour size, stage, histopathological type and grade, and also independent of patient age, parity, menopausal status and smoking habits [6, 7, 16].
Hypoxia-induced alternative splicing in autonomous neoplastic growth (hallmark 1)
Tumour initiation is determined by a combination of oncogene activation and tumour suppressor inactivation, resulting in the acquisition of autonomous neoplastic growth that is promoted either by autocrine growth factor activity caused by coincidental tumour cell growth factor and growth factor receptor expression or by proliferation-promoting oncogenes damage-activated by oncoviruses, gene amplification, mutation, chromosomal translocation or alternative/aberrant pre-mRNA splicing.
Rapid autonomous neoplastic growth results in tissue hypoxia at O2 diffusion distances > 70 μm, resulting in a pro-angiogenic hypoxic responses, cell-death and an acute inflammatory response, also required for tumour angiogenesis and clonal expansion. During this phase, tumour hypoxia-induced alternative splicing influences oncogenic activity both directly and indirectly, helping to promote and maintain tumour autonomous growth potential (Fig. (Fig.1a)1a) [9–15].
Receptor tyrosine kinase proto-oncogenes  that interact with the hypoxic tumour microenvironment , resulting in oncogenic activation, include the neurotrophin tropomyosin-related tyrosine kinase receptor TrkA that exhibits hypoxia-induced oncogenic alternative TrkAIII splicing in human neuroblastoma, pheochromocytoma, leukemia and medullary thyroid cancer cells. TrkAIII is expressed by advanced stage primary human neuroblastomas, glioblastomas, melanomas and Merkel cell carcinomas, is characterized by cassette exon 6, 7 and 9 skipping, exhibits constitutive activation, transforms NIH3T3 cells, exhibits oncogenic activity in neuroblastoma models and prevents neural-related progenitor cell death induced by the development-regulated NF-YA alternative splice variant NF-YAx, expressed during mouse developmental stages associated with neuroblast culling and neuroblastoma suppression, suggesting potential roles in neuroblastoma initiation and hypoxia-dependent progression [89–92].
Hypoxia also promotes aberrant/alternative splicing of the epithelial growth factor receptor EGFR, resulting in expression of the constitutively active, exon 2–7 skipped EGFRvIII (ΔEx 2–7) isoform, a proliferation promoting driver-oncogene in several tumour-types, including glioblastoma multiforme [93–95], and also induces pro-proliferation Erb4 signaling in mammary epithelial cells .
Hypoxia reduces the KRAS 4A to 4B (exon 4a skipped) alternative splice ratio, helping to explain predominant mutation-activated KRAS4B splice variant oncogene expression in colon tumours and cancer stem cells [36, 97, 98], and induces predominant short form MXIs alternative splicing reducing MIX1 antagonism of Nmyc-dependent proliferation of relevance to aggressive autonomous Nmyc amplified neuroblastoma growth .
In prostate cancer cells, hypoxia induces non-catalytic alternative splicing of the tyrosine-protein phosphatase PTPN13, augmenting tyrosine kinase-dependent signaling and proliferation, induces alternative TTC23 splicing involved in hedgehog signaling and promotes alternative RAP1GDS1 splicing, enhancing GDP/GTP exchange reactions in Rap1a and 1b, RhoA and B and KRas G-proteins, promoting autonomous growth (Fig. 2a) .
n colorectal cancer cells, hypoxia augments the expression and activity of hnRNPA1, Srp55, SF/ASF, Tra-2 beta YB-1 and Sam68 splicing factors, resulting in proliferation-promoting alternative CD44v5 and fibronectin EDA exon splicing; promotes LUCAT1 lncRNA expression and LUCAT/PTBP1 complexing, inducing 63 alternative splicing events (36 skipped and 27 retained exon events) in cell growth, cell cycle and G2/M checkpoint genes that augment tumour cell proliferation and colony formation , and induces alternative CD44v5 splicing, resulting in a novel cytokine and growth factor receptor isoform that promotes autonomous growth .
In breast cancer cells, hypoxia induces alternative APP splicing linked to breast cancer cell proliferation and tumorigenicity  and in non-small cell lung cancer cells, promotes Clk1-dependent Srp55 splicing factor phosphorylation, resulting in alternative VEGFA165b splicing and autonomous growth of VEGFR2 and neuropilin-1 receptor expressing tumour cells [102, 103].
In pancreatic cancer, tumour growth under hypoxic conditions has also been attributed to hypoxia-induced alternative splicing of tissue factor, resulting in as-TF expression, which activates carbonic anhydrase IX implicated in late-stage pancreatic cancer growth under hypoxic conditions (Fig. (Fig.2a)2a) .
Hypoxia-induced alternative splicing also regulates the activity of the HIF-1-target proto-oncogene RON, an epithelial cell-specific c-MET family tyrosine kinase receptor that binds macrophage specific protein (MSP).
RON exhibits hypoxia-induced oncogenic alternative splicing in breast, lung, liver, kidney, bladder, ovarian, colon, pancreatic, gastric and prostate carcinomas and many cancer cell lines and is composed of heterodimers of an extracellular 40kda α chain and 150 kDa β chain that contains extracellular, transmembrane and intracellular tyrosine kinase domains, derived from the same immature pre-protein. RON activation results in intracellular phosphorylation-dependent, SH2-domain adapter protein binding to the β-chain, resulting in IP3K/Akt and MAPK signaling. Alternative RON splicing is complex and results in RONΔ170, Δ165, Δ160, Δ155, Δ110, Δ90 and Δi55 isoforms, several of which exhibit constitutive oncogenic activation, differences in localization, opposing functions and associate with tumour progression and disease stage.
Hypoxia induces oncogenic alternative RONΔ165 splicing by promoting CLK1-mediated, SF2/ASF splice factor phosphorylation-dependent binding to an EES adjacent to an ESS cis-element, resulting in exon 11 skipping. Constitutive RONΔ165 activation promotes RON and β-catenin nuclear translocation, inducing cJun expression and promoting proliferation [99, 104–110].
Furthermore, increased nuclear β-catenin levels, induces TCF4 transcription factor activation, β-catenin/TCF4 complexing and the induction of cMyc, Cyclin D and c-Jun β-catenin/TCF4 target gene expression in gastric cancer cells, promoting proliferation. In addition, complexes between constitutively active RON splice variants and β-catenin also interact with HIF1α to regulate HIF-1-dependent transcription and tumour cell proliferation under hypoxic conditions, confirming a close relationship between hypoxia-induced alternative RON splicing, β-catenin and HIF-target genes in the regulation of autonomous tumour cell growth and tumour progression (Fig. (Fig.2a)2a) [99, 105–111].
In addition to direct oncogene activation, hypoxia-induced alternative splicing also indirectly promotes autonomous growth by activating the unfolded protein response (UPR) in response to ER stress resulting from the accumulation of damaged and misfolded proteins [112, 113].
The UPR is mediated by ER ATF6, PERK and Ire1α proteins, the activation of which results in transient attenuation of protein synthesis, increased protein trafficking through the ER, augmented protein-folding capacity, protein degradation through ERAD and autophagy.
Hypoxia-induced Ire1α activation results in unconventional alternative splicing of a 26 nucleotide intron from the transcription factor XBP1u, resulting in expression of the frame shift XBP1s isoform, that contains a novel transcriptional activating domain and exhibits transcriptional activity.
Both XBP1u and XBP1s isoforms contain leucine zipper DNA binding domains and interact to regulate nuclear translocation and transcription, and XBP1s cooperates with HIF-1α to promote cell survival .
XBP1s binds CRE elements in proliferation, survival and protein-overload response genes, activates NF-κB, AP-1 and Myc oncogenic pathways, up-regulates the expression of 162 proliferation, protein folding and survival genes in human breast cancer cells, augments CD4K, c-Myc and Cyclin D expression to promote proliferation, complexes with and augments the transcriptional activity of c-Myc, promotes PI3K/mTOR-dependent osteosarcoma growth, maintains the autonomous growth potential of multiple myeloma cells , and promotes autocrine/paracrine STAT3-dependent growth of hepatocellular carcinoma cells  (Fig. (Fig.22a).
2. Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science. 2009;324:1029–1033. [PMC free article] [PubMed] [Google Scholar]
4. Romero-Ramirez L, Cao H, Nelson D, Hammond E, Lee A-H, Yoshida H, et al. XBP1 is essential for survival under hypoxic conditions and is required for tumor growth. Cancer Res. 2004;64:5943–5947. [PubMed] [Google Scholar]
5. Daskalaki I, Gkikas I, Tavernarakis N. Hypoxia and selective autophagy in cancer development and therapy. Front Cell Dev Biol. 2018;6:104. doi: 10.3389/cell.2018.00104. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
7. Petrova V, Annicchiarico-Petruzzelli M, Melino G, Amelio I. The hypoxic tumour microenvironment. Oncogenesis. 2018;7:10. doi: 10.1038/s41389-017-0011-9. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
10. Spill F, Reynolds DS, Kamm RD, Zaman MH. Impact of the physical microenvironment on tumor progression and metastasis. Curr Opin Biotechnol. 2016;40:41–48. [PMC free article] [PubMed] [Google Scholar]
13. Philip B, Ito K, Moreno-Sànchez R, Ralph SJ. HIF expression and the role of hypoxic environments within primary tumours as protective sites driving cancer stem cell renewal and metastatic progression. Carcinogenesis. 2013;34:1699–1707. [PubMed] [Google Scholar]
14. Qui G-Z, Jin M-Z, Dai J-X, Sun W, Feng J-H, Jin WL. Reprogramming of the tumor in the hypoxic niche: The emerging concept and associated therapeutic strategies. Trends in Pharmacol Sci. 2017;38:669–686. [PubMed] [Google Scholar]
15. Hockel M, Schlenger K, Aral B, Mitze M, Schaffer U, Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res. 1996;56:4509–4515. [PubMed] [Google Scholar]
16. Monteiro AR, Hill R, Pilkington GJ, Madureira PA. The role of hypoxia in glioblastoma invasion. Cells. 2017;6:45. doi: 10.3390/cells6040045. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
87. Abou-faycal C, Hatat A-S, Gazzeri S, Eymin B. Splice variants of the RTK family: Their role in tumor progression and response to targeted therapy. Int J Mol Sci. 2017;18:383. doi: 10.3390/ijms18020383. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
88. Gong Y, Dong Y, Cui J, Sun Q, Zhen Z, Gao Y, et al. Receptor tyrosine kinase interaction with the tumor microenvironment in malignant progression of human glioblastoma. In Gioma – Contemporary Diagnostic and Therapeutic Approaches Intech Open. 2019. [Google Scholar]
89. Tacconelli A, Farina AR, Cappabianca L, Desantis G, Tessitore A, Vetuschi et al. TrkA alternative splicing: a regulated tumor-promoting switch in human neuroblastoma. Cancer Cell 2004; 6:347–360. [PubMed]
90. Farina AR, Cappabianca L, Ruggeri P, Gneo L, Pellegrini C, Fargnoli MC, et al. The oncogenic neurotrophin receptor tropomyosin-related kinase variant. TrkAIII. J Exp Clin Canc Res. 2018;37:119. doi: 10.1186/s13046. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
91. Cappabianca L, Guadagni S, Maccarone R, Sebastiano M, Chiominto A, Farina AR, et al. A pilot study of alternative splicing in Merkel cell carcinoma: a potential oncogenic mechanism and novel therapeutic target. J Exp Clin Cancer Res. 2019;38:424. doi: 10.1186/s13046-019-1425-3. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
92. Cappabianca L, Farina AR, Di Marcotullio L, Infante P, De Simone D, Sebastiano M, et al. Discovery, characterization and potential roles of a novel NF-YAx splice variant in human neuroblastoma. J Exp Clin Cancer Res. 2019;38:482. doi: 10.1186/s13046-019-8. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
93. Liu Z, Han L, Dong Y, Tan Y, Li Y, Zhao M, et al. EGFRvIII/integrin B3 interaction in hypoxic and vitronectin-enriching microenvironment promote GBM progression and metastasis. Oncotarget. 2015;7:4680–4694. [PMC free article] [PubMed] [Google Scholar]
95. Furnari FB, Cloughesy TF, Cavenee WK, Mischel PS. Heterogeneity of epidermal growth factor receptor signaling networks in glioblastoma. Nat Rev Cancer. 2015;15:302–310. [PMC free article] [PubMed] [Google Scholar]
96. Paatero I, Seagroves TN, Vaparanta K, Han W, Jones FE, Johnson RS, et al. Hypoxia-inducible factor-1a induces Erb4 signaling in the differentiating mammary gland. J Biol Chem. 2014;289:22459–22469. [PMC free article] [PubMed] [Google Scholar]
97. Chen W-C, To MD, Westcott PMK, Delrosario R, Kim IJ, Philips M, et al. Regulation of KRAS4A/B splicing in cancer stem cells by the RBM39 splicing complex. BioRxiv. 2019. 10.1101/646125.
99. Bowler E, Poranzinski S, Uzor S, Thibault P, Durand M, Lappinte E, et al. Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells. BMC Cancer. 2018;18:355. doi: 10.1186/s12885-018-4227-7. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
100. Huan L, Guo T, Wu Y, Xu L, Huang S, Xu Y, et al. Hypoxia induced LUCAT1/PTBP1 axis modulates cancer cell viability and chemotherapy response. Mol Cancer. 2020;19:11. doi: 10.1186/s12943-1122-z.. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
101. Lim S, Yoo BK, Kim H-S, Gilmore HL, Lee Y, Lee HP, et al. Amyloid-b precursor protein promotes cell proliferation and motility of advanced breast cancer. BMC Cancer. 2014;14:928. doi: 10.1186/1471-2407-14-928. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
102. Nowak DG, Woolard J, Amin EM, Konopatskaya O, Saleem MA, Churchill AJ, et al. Expression of pro- and anti-angiogenic isoforms of VEGF is differentially regulated by splicing and growth factors. J Cell Sci. 2008;121:3487–3495. [PMC free article] [PubMed] [Google Scholar]
103. Boudria A, Faycal C, Jia T, Gout S, Keramidas M, Didier C, et al. VEGF 165b, a splice variant of VEGF-A, promotes lung tumor progression and escape from anti-angiogenic therapies through a β1 integrin/VEGFR autocrine loop. Oncogene. 2018;38:1050–1066. [PubMed] [Google Scholar]
104. Ramchandani D, Unruh D, Lewis CS, Bogdanov VY, Weber GF. Activation of carbonic anhydrase IX by alternatively spliced tissue factor under late-stage tumor conditions. Lab Invest. 2016;96:1234–1245. [PMC free article] [PubMed] [Google Scholar]
105. Thangasamy A, Rogge J, Ammanamanchi S. Receptuer d’origine Nantais Tyrosine kinase is a direct target of hypoxia-inducible factor-1α-mediated invasion of breast carcinoma cells. J Biol Chem. 2009;284:14001–14010. [PMC free article] [PubMed] [Google Scholar]
106. Chang H-Y, Liu H-S, Lai M-D, Tsai Y-S, Tzai T-S, Cheng H-L, et al. Hypoxia promotes nuclear translocation and transcriptional function in the oncogenic tyrosine kinase RON. Cancer Res. 2014;74:4549–4562. [PubMed] [Google Scholar]
107. Zhou D, Huang L, Zhou Y, Wei T, Yang L, Li C. RON and RONΔ160 promote gastric cancer cell proliferation, migration, and adaptation to hypoxia via interaction with β-catenin. Aging. 2019;11:2735–2748. [PMC free article] [PubMed] [Google Scholar]
108. Krishnaswamy S, Mohammed AK, Amer OE, Tripathi G, Alokail MS, Al-Daghri NM. Recepteur d’Origine nantais (RON) tyrosine kinase splicing variants lacking exons 18 and 19 occur ubiquitously in lung cancer. Int J Clin Exp Med. 2015;8:20778–20786. [PMC free article] [PubMed] [Google Scholar]
109. Wang M-H, Padhye S, Guin S, Zhou Y-Q. Potential therapeutics specific to c-MET/RON receptor tyrosine kinases for molecular targeting in cancer therapy. Acta Pharmacol Sin. 2010;31:1181–1188. [PMC free article] [PubMed] [Google Scholar]
110. Zhang Y, Kaplan-Lefko PJ, Rex K, Yang Y, Moriguchi J, Osgood T, et al. Identification of a novel recepteur d’Origine nantais/c-Met small molecule kinase inhibitor with antitumor activity in vivo. Cancer Res. 2008;68:6680–6687. [PubMed] [Google Scholar]
111. Ma Q, Zhang K, Guin S, Zhou Y-Q, Wang M-H. Deletion or insertion in the first immunoglobulin-plexin-transcription (IPT) domain differentially regulates expression and tumorigenic activities of RON receptor tyrosine kinase. N Mol Cancer. 2010;9:307. [PMC free article] [PubMed] [Google Scholar]
112. Vandewynckel Y-P, Laukens D, Geerst A, Bogaerts E, Paredaens A, Verhelst X, et al. The paradox of the unfolded protein response in cancer. Anticancer Res. 2013;33:4683–4694. [PubMed] [Google Scholar]
113. Chipurupalli S, Kannan E, Tergaonkar V, D’Andrea R, Robinson N. Hypoxia induces ER stress response as an adaptive mechanism in cancer. Int J Mol Sci. 2019;20:749. doi: 10.3390/ijms20030749. [PMC free article] [PubMed] [CrossRef] [Google Scholar]
114. Xia Z, Wu S, Wei X, Liao Y, Yi P, Liu Y, et al. Hypoxic ER stress suppresses β-catenin expression and promotes cooperation between the transcription factors XBP1 and HIF1α for cell survival. J Biol Chem. 2019;294:13811–13821. [PMC free article] [PubMed] [Google Scholar]
115. Shi W, Chen Z, Li L, Liu H, Zhang R, Cheng Q, et al. Unravel the molecular mechanism of XBP1 in regulating the biology of cancer cells. J Cancer. 2019;10:2035–2046. [PMC free article] [PubMed] [Google Scholar]
More information: Alyssa Batista et al, IRE1α regulates macrophage polarization, PD-L1 expression, and tumor survival, PLOS Biology (2020). DOI: 10.1371/journal.pbio.3000687