Juha Klefstrom | University of Helsinki (original) (raw)

Papers by Juha Klefstrom

Cancer Research, 2020

Invasive cancer growth occurs when epithelial tumor cells individually or collectively detach fro... more Invasive cancer growth occurs when epithelial tumor cells individually or collectively detach from the primary tumor and migrate into the adjacent healthy tissue. This process starts a gradual tumor progression that may lead to life-threatening metastatic disease. Hepsin is a type II transmembrane serine protease that is frequently overexpressed in breast, prostate, and other epithelial cancers. It promotes tumor progression through activating growth factors and disrupting cell-cell and cell-basement membrane (BM) junctions. Although hepsin participates in pericellular cancer degradome, the oncogenic upstream mechanisms that activate hepsin in cancer are not well known. We found that oncogenic Ras proteins by activating MAPK pathway signaling alter the ratio of hepsin and HAI-1 to increase the proteolytic activity of hepsin. In 3D epithelial culture models of Ras-driven cancer, depletion of hepsin by shRNA restored desmosomes, BM defects, and glandular morphology but not the apical ...

ABSTRACTEpithelial attachment to the basement membrane (BM) is essential for mammary gland develo... more ABSTRACTEpithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we demonstrate that expression of distinct laminin α-isoforms by luminal and basal mammary epithelial cells enforces lineage identity that is necessary for normal mammary gland growth and function. Laminin α5 (LMα5) is mainly expressed by the luminal epithelial cells, and it is necessary for pubertal mammary gland growth, pregnancy induced gland remodeling, and for alveolar function. Adhesion to LMα5 containing laminin promotes luminal traits in both luminal and basal epithelial cells, and reduces progenitor activity of basal epithelial cells. Mechanistically, we show that Lama5 loss interferes with differentiation of hormone receptor positive luminal cells, which results in reduced Wnt4 expression and defective crosstalk between luminal and basal epithelial cells during gland remodeling. Our results reveal a nove...

Blood, 1997

Gene activation by translocation between an oncogene and an immunoglobulin heavy-chain gene, whic... more Gene activation by translocation between an oncogene and an immunoglobulin heavy-chain gene, which leads to increased expression of the oncoprotein, is a well-known mechanism in the genesis of B-cell lymphomas. In contrast, the role of gene amplification in activation of oncogenes in non-Hodgkin's lymphomas is poorly characterized. To study the BCL2 amplification we performed comparative genomic hybridization (CGH), Southern blot hybridization, Western analysis, immunohistochemistry, metaphase fluorescence in situ hybridization, and chromosome analysis on 26 cases of diffuse large B-cell lymphoma (large noncleaved cell lymphoma). The gain or high-level amplification of 18q was found in eight tumors (31%) by CGH, and Southern analysis revealed BCL2 amplification in these cases, but not in the cases with normal chromosome 18 or t(14; 18)(q32; q21). Western immunoblot analysis and immunohistochemistry revealed a high-level expression of BCL2 protein in the cases with BCL2 amplifica...

The EMBO Journal, 1994

and 'Zentrum fur Molekularbiologie Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, ... more and 'Zentrum fur Molekularbiologie Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany 2Corresponding author Communicated by K.Alitalo lTumor necrosis factor-oc (TNF) is a multifunctional cytokine which is cytotoxic for some tumor cells and transformed cells. The molecular mechanisms which render transformed and tumor cells sensitive to the cytotoxic action of TNF are unclear. We show here that an increased expression of the c-Myc oncoprotein strongly increases cellular sensitivity to TNF cytotoxicity. In RatlA fibroblasts, which are resistant to TNF, the addition of TNF with a concomitant activation of a hormone-inducible c-Myc-estrogen receptor chimera (MycER) resulted in apoptotic cell death. Similarly, c-Myc overexpression enhanced the sensitivity of NIH3T3 fibroblasts to TNF-induced death. The c-Myc and TNF-induced apoptosis was inhibited by ectopic expression of the Bcl2 oncoprotein and by the free oxygen radical scavencing enzyme Mn superoxide dismutase. Furthermore, in highly TNFsensitive fibrosarcoma cells, antisense c-myc oligodeoxynucleotides caused a specific inhibition of TNF cytotoxicity. Our results suggest that the deregulation of c-Myc, which is common in human tumors and tumor cell lines is one reason why these cells are TNF sensitive.

Scientific reports, Jan 5, 2018

In metazoans, epithelial architecture provides a context that dynamically modulates most if not a... more In metazoans, epithelial architecture provides a context that dynamically modulates most if not all epithelial cell responses to intrinsic and extrinsic signals, including growth or survival signalling and transforming oncogene action. Three-dimensional (3D) epithelial culture systems provide tractable models to interrogate the function of human genetic determinants in establishment of context-dependency. We performed an arrayed genetic shRNA screen in mammary epithelial 3D cultures to identify new determinants of epithelial architecture, finding that the key phenotype impacting shRNAs altered not only the data population average but even more noticeably the population distribution. The broad distributions were attributable to sporadic gene silencing actions by shRNA in unselected populations. We employed Maximum Mean Discrepancy concept to capture similar population distribution patterns and demonstrate here the feasibility of the test in identifying an impact of shRNA in populatio...

BioData Mining, 2016

Background: Genomic alterations affecting drug target proteins occur in several tumor types and a... more Background: Genomic alterations affecting drug target proteins occur in several tumor types and are prime candidates for patient-specific tailored treatments. Increasingly, patients likely to benefit from targeted cancer therapy are selected based on molecular alterations. The selection of a precision therapy benefiting most patients is challenging but can be enhanced with integration of multiple types of molecular data. Data integration approaches for drug prioritization have successfully integrated diverse molecular data but do not take full advantage of existing data and literature. Results: We have built a knowledge-base which connects data from public databases with molecular results from over 2200 tumors, signaling pathways and drug-target databases. Moreover, we have developed a data mining algorithm to effectively utilize this heterogeneous knowledge-base. Our algorithm is designed to facilitate retargeting of existing drugs by stratifying samples and prioritizing drug targets. We analyzed 797 primary tumors from The Cancer Genome Atlas breast and ovarian cancer cohorts using our framework. FGFR, CDK and HER2 inhibitors were prioritized in breast and ovarian data sets. Estrogen receptor positive breast tumors showed potential sensitivity to targeted inhibitors of FGFR due to activation of FGFR3. Conclusions: Our results suggest that computational sample stratification selects potentially sensitive samples for targeted therapies and can aid in precision medicine drug repositioning. Source code is available from http://csblcanges.fimm.fi/GOPredict/.

Cell Cycle, 2016

Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppress... more Apoptosis caused by deregulated MYC expression is a prototype example of intrinsic tumor suppression. However, it is still unclear how supraphysiological MYC expression levels engage specific sets of target genes to promote apoptosis. Recently, we demonstrated that repression of SRF target genes by MYC/MIZ1 complexes limits AKT-dependent survival signaling and contributes to apoptosis induction. Here we report that supraphysiological levels of MYC repress gene sets that include markers of basal-like breast cancer cells, but not luminal cancer cells, in a MIZ1-dependent manner. Furthermore, repressed genes are part of a conserved gene signature characterizing the basal subpopulation of both murine and human mammary gland. These repressed genes play a role in epithelium and mammary gland development and overlap with genes mediating cell adhesion and extracellular matrix organization. Strikingly, acute activation of oncogenic MYC in basal mammary epithelial cells is sufficient to induce luminal cell identity markers. We propose that supraphysiological MYC expression impacts on mammary epithelial cell identity by repressing lineage-specific target genes. Such abrupt cell identity switch could interfere with adhesiondependent survival signaling and thus promote apoptosis in pre-malignant epithelial tissue.

Cell Reports, 2015

Highlights d Serine 62-phosphorylated MYC is regulated by CIP2A in Lamin A/C-associated complexes... more Highlights d Serine 62-phosphorylated MYC is regulated by CIP2A in Lamin A/C-associated complexes d CIP2A is required for MYC S62 phosphorylation during proliferation induction in vivo d MYC activity can be systemically targeted without detrimental physiological effects d We identify a phosphorylation switch defining the DNA damage response in vertebrates

The EMBO journal, Jan 3, 2015

Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protec... more Oncogenic levels of Myc expression sensitize cells to multiple apoptotic stimuli, and this protects long-lived organisms from cancer development. How cells discriminate physiological from supraphysiological levels of Myc is largely unknown. Here, we show that induction of apoptosis by Myc in breast epithelial cells requires association of Myc with Miz1. Gene expression and ChIP-Sequencing experiments show that high levels of Myc invade target sites that lack consensus E-boxes in a complex with Miz1 and repress transcription. Myc/Miz1-repressed genes encode proteins involved in cell adhesion and migration and include several integrins. Promoters of repressed genes are enriched for binding sites of the serum-response factor (SRF). Restoring SRF activity antagonizes Myc repression of SRF target genes, attenuates Myc-induced apoptosis, and reverts a Myc-dependent decrease in Akt phosphorylation and activity, a well-characterized suppressor of Myc-induced apoptosis. We propose that high ...

Advances in Cancer Research, 2011

Epithelial architecture is formed in tissues and organs when groups of epithelial cells are organ... more Epithelial architecture is formed in tissues and organs when groups of epithelial cells are organized into polarized structures. The epithelial function and integrity as well as signaling across the epithelial layer is orchestrated by apical junctional complexes (AJCs), which are landmarks for PAR/CRUMBS and lateral SCRIB polarity modules and by dynamic interactions of the cells with underlying basement membrane (BM). These highly organized epithelial architectures are demolished in cancer. In all advanced epithelial cancers, malignant cells have lost polarity and connections to the basement membrane and they have become proliferative, motile, and invasive. Clearly, loss of epithelial integrity associates with tumor progression but does it contribute to tumor development? Evidence from studies in Drosophila and recently also in vertebrate models have suggested that even the oncogene-driven enforced cell proliferation can be conditional, dependant on the influence of cell-cell or cell-microenvironment contacts. Therefore, loss of epithelial integrity may not only be an obligate consequence of unscheduled proliferation of malignant cells but instead, malignant epithelial cells may need to acquire capacity to break free from the constraints of integrity to freely and autonomously proliferate. We discuss how epithelial polarity complexes form and regulate epithelial integrity, highlighting the roles of enzymes Rho GTPases, aPKCs, PI3K, and type II transmembrane serine proteases (TTSPs). We also discuss relevance of these pathways to cancer in light of genetic alterations found in human cancers and review molecular pathways and potential pharmacological strategies to revert or selectively eradicate disorganized tumor epithelium.

Cancer discovery, Jan 25, 2015

Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently... more Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTCs) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with the DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in ten different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by down-regulation of ERα, FOXA1 and GATA3 together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated...

The EMBO Journal, 2007

Oncogenic c-Myc renders cells sensitive to TRAIL-induced apoptosis, and existing data suggest tha... more Oncogenic c-Myc renders cells sensitive to TRAIL-induced apoptosis, and existing data suggest that c-Myc sensitizes cells to apoptosis by promoting activation of the mitochondrial apoptosis pathway. However, the molecular mechanisms linking the mitochondrial effects of c-Myc to the c-Myc-dependent sensitization to TRAIL have remained unresolved. Here, we show that TRAIL induces a weak activation of procaspase-8 but fails to activate mitochondrial proapoptotic effectors Bax and Bak, cytochrome c release or downstream effector caspase-3 in non-transformed human fibroblasts or mammary epithelial cells. Our data is consistent with the model that activation of oncogenic c-Myc primes mitochondria through a mechanism involving activation of Bak and this priming enables weak TRAIL-induced caspase-8 signals to activate Bax. This results in cytochrome c release, activation of downstream caspases and postmitochondrial deathinducing signaling complex-independent augmentation of caspase-8-Bid activity. In conclusion, c-Myc-dependent priming of the mitochondrial pathway is critical for the capacity of TRAIL-induced caspase-8 signals to activate effector caspases and for the establishment of lethal caspase feedback amplification loop in human cells.

Proceedings of the National Academy of Sciences, 2007

Cellular organization into epithelial architecture maintains structural integrity and homeostasis... more Cellular organization into epithelial architecture maintains structural integrity and homeostasis by suppressing cell proliferation and apoptosis. However, it is unclear whether the epithelial organization is sufficient to block induction of cell-autonomous cell cycle progression and apoptotic sensitivity by activated oncogenes. We show that chronic activation of oncogenic c-Myc, starting in the developing 3D organotypic mammary acinar structures, results in hyperproliferation and transformed acinar morphology. Surprisingly, acute c-Myc activation in mature quiescent acini with established epithelial architecture fails to reinitiate the cell cycle or transform these structures. c-Myc does reinitiate the cell cycle in quiescent, but structurally unorganized, acini, which demonstrates that proper epithelial architecture is needed for the proliferation blockade. The capability of c-Myc to reinitiate the cell cycle in acinar structures is also restored by the loss of LKB1, a human homol...

Proceedings of the National Academy of Sciences, 2012

Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly underst... more Although loss of epithelial integrity is a hallmark of advanced cancer, it remains poorly understood whether genetic alterations corrupting this integrity causally facilitate tumorigenesis. We show that conditional deletion of tumor suppressor gene Lkb1 (Par-4) in the mammary gland compromises epithelial integrity manifested by mislocalization of cell polarity markers, lateralization of tight junctions, deterioration of desmosomes and basement membrane (BM), and hyperbranching of the mammary ductal tree. We identify the desmosomal BM remodelling serine protease Hepsin as a key factor mediating Lkb1 loss-induced structural alterations in mammary epithelium and BM fragmentation. Although loss of Lkb1 alone does not promote mammary tumorigenesis, combination of Lkb1 deficiency with oncogenic c-Myc leads to dramatic acceleration in tumor formation. The results coupling Lkb1 loss-mediated epithelial integrity defects to mislocalization of serine protease Hepsin and to oncogenic synergy w...

Proceedings of the National Academy of Sciences, 2013

Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellu... more Oncogenic transcription factor Myc deregulates the cell cycle and simultaneously reprograms cellular metabolism to meet the biosynthetic and bioenergetic needs of proliferation. Myc also sensitizes cells to mitochondria-dependent apoptosis. Although metabolic reprogramming has been circumstantially connected to vulnerability to apoptosis, the connecting molecular pathways have remained poorly defined. Here, we show that Myc-induced altered glutamine metabolism involves ATP depletion and activation of the energy sensor AMP-activated protein kinase (AMPK), which induces stabilizing phosphorylation of p53 at Ser15. Under influence of Myc, AMPK-stabilized tumor suppressor protein p53 accumulates in the mitochondria and interacts with the protein complex comprised of B-cell lymphoma 2 (Bcl-2) antagonist/killer (BAK) and Bcl2-like 1 (Bcl-xL). Mitochondrial p53 induces conformational activation of proapoptotic Bak without disrupting the Bak–Bcl-x L interaction. Further liberation of Bak sp...

Molecular and Cellular Endocrinology, 2010

Of eight million oocytes formed in fetal ovaries, only 400 are ovulated and the rest are degraded... more Of eight million oocytes formed in fetal ovaries, only 400 are ovulated and the rest are degraded via apoptosis. Studies in rodents suggest an important role for Bok and Bcl-X L in ovarian apoptosis, but their expression patterns and roles in human ovaries are not known. Protein expression of Bok and Bcl-X L as well as the death pathway effectors TNF and caspase-3 were determined in an important collection of samples consisting of human fetal and adult ovaries. A penetrant expression of Bok, Bcl-X L , TNF and full length and cleaved caspase-3 were characterized in fetal ovaries, with specific patterns in oocytes and pre-granulosa/granulosa cells. Bok and Bcl-X L were detected also in adult ovaries. Lentiviral shRNA delivery demonstrated that loss of Bok markedly reduces vulnerability to apoptosis and, conversely, loss of Bcl-X L increases apoptosis in human granulosa tumour cell line. The results suggest important roles for Bok and Bcl-X L in human ovarian development, follicle maturation and apoptosis. M a n u s c r i p t 13 independent phospholipase A2 enhances cell migration in non-apoptotic ovarian cancer cells.

Leukemia Research, 1994

The frequencies of major breakpoint region (MBR) and minor cluster region (MCR) breakpoint sites ... more The frequencies of major breakpoint region (MBR) and minor cluster region (MCR) breakpoint sites of t(14;18) were examined by polymerase chain reaction and Southern blotting in 50 non-Hodgkin's lymphomas with cytogenetic evidence of t(14; 18). A translocation breakpoint was detected in 41 cases (82%). The MBR was involved in 66%, and the MCR in 16% of the cases. Most cases in the present series were lymphomas with a follicular or diffuse growth pattern, 38 being of the centroblastic/centrocytic type and eight of the centroblastic type. The series also included four lymphomas of probable non-follicular center cell origin. MBR and/or MCR breakpoints were found in all studied lymphoma subtypes and in the majority of these most of the breakpoints were in the MBR and a minor portion of the breakpoints in the MCR. Our results suggest that a breakpoint site is not related to growth pattern or neoplastic cell type in follicular center cell lymphomas with t(14;18).

Cell Cycle, 2009

Machiavelli wrote, in his famous political treatise Il Principe, about disrupting organization by... more Machiavelli wrote, in his famous political treatise Il Principe, about disrupting organization by planting seeds of dissension or by eliminating necessary support elements. Tumor cells do exactly that by disrupting the organized architecture of epithelial cell layers during progression from contained benign tumor to full-blown invasive cancer. However, it is still unclear whether tumor cells primarily break free by activating oncogenes powerful enough to cause chaos or by eliminating tumor suppressor genes guarding the order of the epithelial organization. Studies in Drosophila have exposed genes that encode key regulators of the epithelial apicobasal polarity and which, upon inactivation, cause disorganization of the epithelial layers and promote unscheduled cell proliferation. These polarity regulator/tumor suppressor proteins, which include products of neoplastic tumor suppressor genes (nTSGs), are carefully positioned in polarized epithelial cells to maintain the order of epithelial structures and to impose a restraint on cell proliferation. In this review, we have explored the presence and prevalence of somatic mutations in the human counterparts of Drosophila polarity regulator/tumor suppressor genes across the human cancers. The screen points out LKB1, which is a causal genetic lesion in Peutz-Jeghers cancer syndrome, a gene mutated in certain sporadic cancers and a human homologue of the fly polarity gene par-4. We review the evidence linking Lkb1 protein to polarity regulation in the scope of our recent results suggesting a coupled role for Lkb1 as an architect of organized acinar structures and a suppressor of oncogenic c-Myc. We finally present models to explain how Lkb1-dependent formation of epithelial architecture is coupled to suppression of normal and oncogene-induced proliferation.

Cell Cycle, 2007

TRAIL ligand induces selectively apoptosis in tumor cells by binding to two death receptors (DR4 ... more TRAIL ligand induces selectively apoptosis in tumor cells by binding to two death receptors (DR4 and DR5) and holds promise as a potential therapeutic agent against cancer. While it has been known for long time that TRAIL receptors are commonly expressed in wide variety of normal tissues, it is not well understood why TRAIL kills tumor cells but leaves normal cells unharmed. The prototypic oncogene c-Myc promotes the cell cycle and simultaneously primes activation of the Bcl-2 family controlled mitochondria apoptosis pathway. A striking reflection of the c-Myc-dependent apoptotic sensitization is the dramatic c-Myc-induced vulnerability of cells to TRAIL and other death receptor ligands. Here we summarize the recent findings regarding the death mechanisms of TRAIL/TRAIL receptor system and the connection of c-Myc to the mitochondrial apoptosis pathway, focusing on our work that couples c-Myc via Bak to the TRAIL death receptor pathway. Finally, we present a mitochondria-priming model to explain how c-Myc-Bak interaction amplifies the TRAIL-induced caspase 8-Bid pathway to induce full-blown apoptosis. We discuss the implications of these findings for understanding the selective cytotoxicity of TRAIL and for the therapeutic exploitation of the death receptor pathway. DeAth receptors: Guilty of MurDer beyonD reAsonAble Doubt? TNF and TRAIL. Tumor necrosis factor-alpha (TNF) is a founding member of large TNF-family of proteins and was once considered in cancer research community as nothing less than phenomenal protein due to its ability to exert cytotoxic effects towards about one third of cancer cell lines without harming non-transformed cell lines. 1,2 However, the systemic administration of recombinant TNF caused severe side effects, including hemorrhagic necrosis, cachexia, fever and shock in laboratory animals and this precluded systemically administered TNF from clinical use. 3,4 The observed side effects were not caused by any rampant cell deaths triggered by the death ligand TNF, but rather they reflected effects of the cytokine TNF, which was found to play a key role in regulation of immunity and inflammation responses. 5 Indeed, inhibitors of TNF show promise in treatment of inflammation-linked disorders such as rheumatoid arthritis. 6 Discovery of TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) and its two TNF receptor 1 related and death domain containing signaling receptors DR4 and DR5 brought death receptors back to attention of cancer field since TRAIL performed well where TNF had failed. TRAIL was demonstrated to exhibit TNF-like selective apoptotic activity towards tumor cells in vitro and in vivo and importantly, unlike TNF, TRAIL has been well tolerated in preclinical toxicity assays. 7,8 These findings quickly prompted several TRAIL based cancer therapies, which are currently in phase I and II clinical trials. 9 TRAIL receptors DR4 and DR5 are constitutively expressed in wide variety of tissues 10,11 (see also NCBI UniGene's EST ProfileViewer) and the ligated receptors can induce apoptosis via DISC mediated activation of caspase-8 (Box 1). However, TRAIL receptors can also stimulate activation of MAP kinase cascades and the anti-apoptotic NF-kB pathway suggesting that not all signals are solely created for the purpose of apoptosis induction. 12-14 In addition, several recently documented TRAIL activities, such as regulation of chemokine production or promotion of metastatic behavior in pancreatic carcinomas do not fit the profile of professional killer. 15,16 Therefore, it is fair to suggest that TRAIL, like TNF or the third major death ligand CD95L 17 has wider array of biological functions than only induction of apoptosis. While the death receptor pathway in context of dying tumor cells is relatively well characterized (Box 1), there are still critical questions