Six-year experience of Australia's first dedicated cancer of unknown primary clinic.

BACKGROUND: Diagnosis and management of cancers of unknown primary (CUP) remain challenging. This study examines the referral patterns, management and outcomes of patients referred to Australia's first dedicated CUP clinic. METHODS: Retrospective medical record review was conducted for patients seen at the Peter MacCallum Cancer Centre CUP clinic between July 2014 and August 2020. Overall survival (OS) was analysed for patients with a CUP diagnosis where treatment information was available. RESULTS: Of 361 patients referred, fewer than half had completed diagnostic work-up at the time of referral. A diagnosis of CUP was established in 137 (38%), malignancy other than CUP in 177 (49%) and benign pathology in 36 (10%) patients. Genomic testing was successfully completed in 62% of patients with initial provisional CUP and impacted management in 32% by identifying a tissue of origin or actionable genomic alteration. The use of site-specific, targeted therapy or immunotherapy was independently associated with longer OS compared to empirical chemotherapy. CONCLUSION: Our specialised CUP clinic facilitated diagnostic work-up among patients with suspected malignancy and provided access to genomic testing and clinical trials for patients with a CUP diagnosis, all of which are important to improve outcomes in this patient population.

UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma.

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2's K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.

INPP4B promotes PI3Kα-dependent late endosome formation and Wnt/ß-catenin signaling in breast cancer.

INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER+ breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER+ breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3ß lysosomal degradation and activation of Wnt/ß-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER+ breast cancer via PI(3,4)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/ß-catenin therapies.

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.

The molecular origin and taxonomy of mucinous ovarian carcinoma.

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases.

Calcium-Dependent Enhancement by Extracellular Acidity of the Cytotoxicity of Mitochondrial Inhibitors against Melanoma.

Extracellular acidity is a hallmark of cancers and is independent of hypoxia. Because acidity potentiates malignant phenotypes, therapeutic strategies that enhance the targeting of oncogenic mechanisms in an acidic microenvironment should be effective. We report here that drugs which abrogate mitochondrial respiration show enhanced cytotoxicity against melanoma cells in a normoxic but acidic extracellular pH, independent from P53 mutations, BRAF (V600E) mutations, and/or resistance against BRAF inhibitors. Conversely, the cytotoxicity against melanoma cells of mitochondrial inhibitors is impaired by a neutral or alkaline extracellular pH, and in vivo systemic alkalinization with NaHCO3 enhanced subcutaneous tumor growth and lung metastasis of B16F10 cells in mice treated with the mitochondrial inhibitor phenformin. Intracellular calcium (Ca2+) was significantly increased in melanoma cells treated with mitochondrial inhibitors at an acidic extracellular pH and an intracellular Ca2+ chelator, BAPTA/AM, inhibited cytoplasmic Ca2+ as well as melanoma cell death. Surprisingly, ROS scavengers synergized with increased apoptosis in cells treated with mitochondrial inhibitors, suggesting that ROS contributes to cell survival in this context. Notably, the cytotoxic enhancement of mitochondrial inhibitors by acidity was distinct from PGC1alpha-driven mitochondrial addiction, from therapy-induced senescence, and from slow, JARID1B-high-associated cell cycling, all of which have been shown to promote vulnerability to mitochondrial inhibition. These data indicate that extracellular pH profoundly modulates the cytotoxicity of mitochondrial inhibitors against cancer cells. Mol Cancer Ther; 16(5); 936-47. ©2017 AACR.

Desmoglein 2 promotes vasculogenic mimicry in melanoma and is associated with poor clinical outcome.

Tumors can develop a blood supply not only by promoting angiogenesis but also by forming vessel-like structures directly from tumor cells, known as vasculogenic mimicry (VM). Understanding mechanisms that regulate VM is important, as these might be exploitable to inhibit tumor progression. Here, we reveal the adhesion molecule desmoglein 2 (DSG2) as a novel mediator of VM in melanoma. Analysis of patient-derived melanoma cell lines and tumor tissues, and interrogation of The Cancer Genome Atlas (TCGA) data, revealed that DSG2 is frequently overexpressed in primary and metastatic melanomas compared to normal melanocytes. Notably, this overexpression was associated with poor clinical outcome. DSG2+ melanoma cells self-organized into tube-like structures on Matrigel, indicative of VM activity, which was inhibited by DSG2 knockdown or treatment with a DSG2-blocking peptide. Mechanistic studies revealed that DSG2 regulates adhesion and cell-cell interactions during tube formation, but does not control melanoma cell viability, proliferation or motility. Finally, analysis of patient tumors revealed a correlation between DSG2 expression, VM network density and expression of VM-associated genes. These studies identify DSG2 as a key regulator of VM activity in human melanoma and suggest this molecule might be therapeutically targeted to reduce tumor blood supply and metastatic spread.

CD271 Expression on Patient Melanoma Cells Is Unstable and Unlinked to Tumorigenicity.

The stability of markers that identify cancer cells that propagate disease is important to the outcomes of targeted therapy strategies. In human melanoma, conflicting data exist as to whether hierarchical expression of CD271/p75/NGFR (nerve growth factor receptor) marks cells with enriched tumorigenicity, which would compel their specific targeting in therapy. To test whether these discrepancies relate to differences among groups in assay approaches, we undertook side-by-side testing of published methods of patient-derived melanoma xenografting (PDX), including comparisons of tissue digestion procedures or coinjected Matrigel formulations. We found that CD271(-) and CD271(+) melanoma cells from each of seven patients were similarly tumorigenic, regardless of assay variations. Surprisingly variable CD271 expression patterns were observed in the analyses of sibling PDX tumors (n = 68) grown in the same experiments from either CD271(-) or CD271(+) cells obtained from patients. This indicates unstable intratumoral lineage relationships between CD271(-) and CD271(+) melanoma cells that are inconsistent with classical, epigenetically based theories of disease progression, such as the cancer stem cell and plasticity models. SNP genotyping of pairs of sibling PDX tumors grown from phenotypically identical CD271(-) or CD271(+) cells showed large pairwise differences in copy number (28%-48%). Differences were also apparent in the copy number profiles of CD271(-) and CD271(+) cells purified directly from each of the four melanomas (1.4%-23%). Thus, CD271 expression in patient melanomas is unstable, not consistently linked to increased tumorigenicity and associated with genetic heterogeneity, undermining its use as a marker in clinical studies. Cancer Res; 76(13); 3965-77. ©2016 AACR.

The Inositol Polyphosphate 5-Phosphatase PIPP Regulates AKT1-Dependent Breast Cancer Growth and Metastasis.

Metastasis is the major cause of breast cancer mortality. Phosphoinositide 3-kinase (PI3K) generated PtdIns(3,4,5)P3 activates AKT, which promotes breast cancer cell proliferation and regulates migration. To date, none of the inositol polyphosphate 5-phosphatases that inhibit PI3K/AKT signaling have been reported as tumor suppressors in breast cancer. Here, we show depletion of the inositol polyphosphate 5-phosphatase PIPP (INPP5J) increases breast cancer cell transformation, but reduces cell migration and invasion. Pipp ablation accelerates oncogene-driven breast cancer tumor growth in vivo, but paradoxically reduces metastasis by regulating AKT1-dependent tumor cell migration. PIPP mRNA expression is reduced in human ER-negative breast cancers associated with reduced long-term outcome. Collectively, our findings identify PIPP as a suppressor of oncogenic PI3K/AKT signaling in breast cancer.

INPP4B is highly expressed in prostate intermediate cells and its loss of expression in prostate carcinoma predicts for recurrence and poor long term survival.

BACKGROUND: Phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in prostate carcinoma due to the loss of tumor suppressor PTEN, which leads to increased Akt activity. Expression of INPP4B, another negative regulator of the PI3K/Akt pathway, is also reduced in prostate carcinoma. However, uncertainty exists regarding the association of INPP4B expression and biochemical and clinical relapse of prostate carcinoma. METHODS: INPP4B expression in benign prostate acini was analyzed by co-immunofluorescence with cytokeratins (CK) 5, 8, 19, androgen receptor (AR), c-MET, chromogranin A and Ki67. INPP4B expression in prostate carcinoma was analyzed in two independent cohorts (n = 406). The association of INPP4B with biochemical and clinical prostate carcinoma relapse was assessed by Kaplan-Meier and Cox proportional hazards modeling. RESULTS: INPP4B was expressed in luminal epithelium within benign ducts, and was highly expressed in CK5+/CK8+/CK19+/AR-/c-MET+/Ki67- intermediate cells in proliferative inflammatory atrophic acini. Overall, INPP4B expression was reduced in prostate carcinoma compared to benign epithelium. Absent/low INPP4B expression was associated with reduced biochemical relapse-free survival (P = 0.01) and increased risk of clinical relapse (P = 0.01). Absence of INPP4B expression was an independent predictor of clinical relapse free survival (P = 0.004) when modeled with Gleason score (P = 0.027) and pathologic stage (P = 0.07). CONCLUSIONS: INPP4B is highly expressed in intermediate cells within proliferative inflammatory atrophic ducts, and expression is reduced in prostate carcinoma. Absence of INPP4B expression is associated with poor outcome following radical prostatectomy, and represents an independent prognostic marker of prostate carcinoma clinical recurrence.

Navigating the challenge of tumor heterogeneity in cancer therapy.

The future of cancer treatment lies in personalized strategies designed to specifically target tumorigenic cell populations present in an individual. Although recent advances in directed therapies have greatly improved patient outcomes in some cancers, intuitive drug design is proving more difficult than expected owing largely to the complexity of human cancers. Intratumoral heterogeneity, the presence of multiple genotypically and/or phenotypically distinct cell subpopulations within a single tumor, is a likely cause of drug resistance. Advances in systems biology are helping to unravel the mysteries of cancer progression. In this issue of Cancer Discovery, Zhao and colleagues define a path for functional validation of computational modeling in the context of heterogeneous tumor populations and their potential for drug response and resistance.

Regulation of the transcriptional coactivator FHL2 licenses activation of the androgen receptor in castrate-resistant prostate cancer.

It is now clear that progression from localized prostate cancer to incurable castrate-resistant prostate cancer (CRPC) is driven by continued androgen receptor (AR), signaling independently of androgen. Thus, there remains a strong rationale to suppress AR activity as the single most important therapeutic goal in CRPC treatment. Although the expression of ligand-independent AR splice variants confers resistance to AR-targeted therapy and progression to lethal castrate-resistant cancer, the molecular regulators of AR activity in CRPC remain unclear, in particular those pathways that potentiate the function of mutant AR in CRPC. Here, we identify FHL2 as a novel coactivator of ligand-independent AR variants that are important in CRPC. We show that the nuclear localization of FHL2 and coactivation of the AR is driven by calpain cleavage of the cytoskeletal protein filamin, a pathway that shows differential activation in prostate epithelial versus prostate cancer cell lines. We further identify a novel FHL2-AR-filamin transcription complex, revealing how deregulation of this axis promotes the constitutive, ligand-independent activation of AR variants, which are present in CRPC. Critically, the calpain-cleaved filamin fragment and FHL2 are present in the nucleus only in CRPC and not benign prostate tissue or localized prostate cancer. Thus, our work provides mechanistic insight into the enhanced AR activation, most notably of the recently identified AR variants, including AR-V7 that drives CRPC progression. Furthermore, our results identify the first disease-specific mechanism for deregulation of FHL2 nuclear localization during cancer progression. These results offer general import beyond prostate cancer, given that nuclear FHL2 is characteristic of other human cancers where oncogenic transcription factors that drive disease are activated like the AR in prostate cancer.

MDM4 is a key therapeutic target in cutaneous melanoma.

The inactivation of the p53 tumor suppressor pathway, which often occurs through mutations in TP53 (encoding tumor protein 53) is a common step in human cancer. However, in melanoma-a highly chemotherapy-resistant disease-TP53 mutations are rare, raising the possibility that this cancer uses alternative ways to overcome p53-mediated tumor suppression. Here we show that Mdm4 p53 binding protein homolog (MDM4), a negative regulator of p53, is upregulated in a substantial proportion (∼65%) of stage I-IV human melanomas and that melanocyte-specific Mdm4 overexpression enhanced tumorigenesis in a mouse model of melanoma induced by the oncogene Nras. MDM4 promotes the survival of human metastatic melanoma by antagonizing p53 proapoptotic function. Notably, inhibition of the MDM4-p53 interaction restored p53 function in melanoma cells, resulting in increased sensitivity to cytotoxic chemotherapy and to inhibitors of the BRAF (V600E) oncogene. Our results identify MDM4 as a key determinant of impaired p53 function in human melanoma and designate MDM4 as a promising target for antimelanoma combination therapy.

Phosphoinositide phosphatases: just as important as the kinases.

Phosphoinositide phosphatases comprise several large enzyme families with over 35 mammalian enzymes identified to date that degrade many phosphoinositide signals. Growth factor or insulin stimulation activates the phosphoinositide 3-kinase that phosphorylates phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] to form phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)], which is rapidly dephosphorylated either by PTEN (phosphatase and tensin homologue deleted on chromosome 10) to PtdIns(4,5)P(2), or by the 5-phosphatases (inositol polyphosphate 5-phosphatases), generating PtdIns(3,4)P(2). 5-phosphatases also hydrolyze PtdIns(4,5)P(2) forming PtdIns(4)P. Ten mammalian 5-phosphatases have been identified, which regulate hematopoietic cell proliferation, synaptic vesicle recycling, insulin signaling, and embryonic development. Two 5-phosphatase genes, OCRL and INPP5E are mutated in Lowe and Joubert syndrome respectively. SHIP [SH2 (Src homology 2)-domain inositol phosphatase] 2, and SKIP (skeletal muscle- and kidney-enriched inositol phosphatase) negatively regulate insulin signaling and glucose homeostasis. SHIP2 polymorphisms are associated with a predisposition to insulin resistance. SHIP1 controls hematopoietic cell proliferation and is mutated in some leukemias. The inositol polyphosphate 4-phosphatases, INPP4A and INPP4B degrade PtdIns(3,4)P(2) to PtdIns(3)P and regulate neuroexcitatory cell death, or act as a tumor suppressor in breast cancer respectively. The Sac phosphatases degrade multiple phosphoinositides, such as PtdIns(3)P, PtdIns(4)P, PtdIns(5)P and PtdIns(3,5)P(2) to form PtdIns. Mutation in the Sac phosphatase gene, FIG4, leads to a degenerative neuropathy. Therefore the phosphatases, like the lipid kinases, play major roles in regulating cellular functions and their mutation or altered expression leads to many human diseases.

The PTEN and Myotubularin phosphoinositide 3-phosphatases: linking lipid signalling to human disease.

Two classes of lipid phosphatases selectively dephosphorylate the 3 position of the inositol ring of phosphoinositide signaling molecules: the PTEN and the Myotubularin families. PTEN dephosphorylates PtdIns(3,4,5)P(3), acting in direct opposition to the Class I PI3K enzymes in the regulation of cell growth, proliferation and polarity and is an important tumor suppressor. Although there are several PTEN-related proteins encoded by the human genome, none of these appear to fulfill the same functions. In contrast, the Myotubularins dephosphorylate both PtdIns(3)P and PtdIns(3,5)P(2), making them antagonists of the Class II and Class III PI 3-kinases and regulators of membrane traffic. Both phosphatase groups were originally identified through their causal mutation in human disease. Mutations in specific myotubularins result in myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy; and loss of PTEN function through mutation and other mechanisms is evident in as many as a third of all human tumors. This chapter will discuss these two classes of phosphatases, covering what is known about their biochemistry, their functions at the cellular and whole body level and their influence on human health.

Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers.

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas.

Regulation of phosphoinositide signaling by the inositol polyphosphate 5-phosphatases.

Phosphoinositide signaling molecules control cellular growth, proliferation and differentiation, intracellular vesicle trafficking, and cytoskeletal rearrangement. The inositol polyphosphate 5-phosphatase family remove the D-5 position phosphate from PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3,5)P2 forming PtdIns(3,4)P2, PtdIns(4)P and PtdIns(3)P respectively. This enzyme family, comprising ten mammalian members, exhibit seemingly non-redundant functions including the regulation of synaptic vesicle recycling, hematopoietic cell function and insulin signaling. Here we highlight recently established insights into the functions of two well characterized 5-phosphatases OCRL and SHIP2, which have been the subject of extensive functional studies, and the characterization of recently identified members, SKIP and PIPP, in order to highlight the diverse and complex functions of this enzyme family.

The inositol polyphosphate 5-phosphatase, PIPP, Is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation.

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P3), which localizes and facilitates Akt activation and stimulates GSK-3beta inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P3 signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) and/or PtdIns(3,4,5)P3. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P3 forming PtdIns(3,4)P2, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3beta, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P3 at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3beta signaling.