An evaluation of the hepatic proteomic signature in identifying cancer tolerance and resistance mechanisms in a mouse allograft system

Van Niekerk, Gustav (2017-03)

Thesis (PhD)--Stellenbosch University, 2017.

Thesis

ENGLISH ABSTRACT: Background The unfavourable therapeutic index of most treatment modalities has greatly impeded progress in the development of effective cancer therapy. Therefore a need exists for treatment modalities that are less taxing on a patient’s health status (i.e. maintain a patients reserve capacity and thus prolong survival) while additionally not invoking counter evolutionary strategies from tumour cells. Plant biologist have long distinguished between the host’s ability to accommodate pathogen burden, as oppose to its ability to antagonise pathogen load. Hence, the disease trajectory is not only dependent on the hosts’ ability to resist an infection, but also on the capacity to tolerate pathogen burden. This distinction have only recently been applied to animals. A number of observations suggest that this distinction may be of great immunological relevance, including the prevalence of asymptomatic carriers and natural variation in the population with regards to disease progression. Thus, the tolerance/resistance (T/R) paradigm represents a novel approach for understanding disease progression. We hypothesise that similar mechanisms might underlie host-tumour dynamics. Study aims and experimental design The current study aimed to evaluate the application of the T/R framework within an oncological context. A syngeneic mice model system was used to compare tolerance and resistance between two cancer cell lines. C57BL/6 mice were inoculated with either mammary carcinoma cells (EO771) or melanoma cells (B16). In a clinical setting, health status would not only be influenced by tumour load, but also by therapeutic interventions such as cytotoxic therapies, which must also be tolerated. Thus, a second study was performed using chemotherapeutic regimes as a variable to explore the effect of high (5 mg/kg) and low (2 mg/kg) dose doxorubicin (DXR) treatment on tolerance and resistance in mice. In addition, attempts were made to identify mechanisms underlying differences between groups with regards to variation in tolerance and resistance. To this end, a combination of immunoblotting and proteomic analyses were performed. Methodology: quantifying tolerance and resistance Resistance was quantified as the slope of a regression line, with tumour volume as response variable, and time as independent variable. Tolerance was measured similarly, but with body weight as response variable and tumour load as independent variable. Differences in regression slopes was used to compare tolerance and resistance. To confirm tolerance, differences in gastrocnemius muscle cross-sectional area (MCA) were compared between groups. Results Mice inoculated with melanoma (B16) cells showed a significantly lower resistance compared to mice inoculated with breast cancer EO771 cells. With regards to tolerance, B16 cells also exhibited lower tolerance, though tests for homogeneity of regression slopes demonstrated that these differences did not reach significance (p = 0.0856). Similarly, B16 and EO771 groups did not exhibit any difference in MCA. Comparing the effect of high and low dose DXR on mice bearing EO771 revealed that DXR decreases resistance: both low dose and higher dose DXR increased tumour growth as demonstrated by significantly steeper slopes in DXR groups compared to the tumour control group. In order to explain the increase in EO771 tumour growth in mice receiving DXR, the activation of a panel of signalling proteins associated with cell growth and survival (cRaf, ERK, p38 MAPK, JNK, PTEN, PI3Kp85, PDK1, Akt, mTOR, Bcl-2) as well as apoptotic markers (Caspase 3, 8 and 9) in tumour samples were evaluated by western blot analyses. However, the only significant finding include elevated ERK activation in mice receiving DXR, suggesting that extracellular signalling molecules might drive tumour growth. Since the liver plays a critical role in energy homeostasis, as well as in the production and clearance of circulating factors, western blot analyses were performed on liver samples. Markers of autophagy (p62 and LC3B-II) as well as growth signalling proteins (Akt and mTOR) and apoptosis (Caspase 3) were evaluated by western blot analyses. Mice inoculated with B16 demonstrated a marked increase in both p62 and LC3B-II, signifying an increase in autophagosome pool size, most likely due to dysfunctional lysosomal fusion. Surprisingly, other makers in both EO771 and B16 did not significantly differ from control liver samples. Subsequently, liver proteomics were performed making use of a Gene Ontology approach in order to describe biological, functional, structural and other processes that are uniquely altered between groups. Interestingly, a comparison between livers of mice inoculated with B16 melanoma cells and EO771 breast cancer cells also suggested that autophagic activity was not upregulated compared to the control group. DXR groups also did not exhibit differences in autophagic processes, though proteins involved in the proteasomal pathway were upregulated in mice receiving high doses of DXR. An increase expression of enzymes associated with retinoic acid metabolism was observed in the B16 group, which might explain decrease tolerance and resistance in this group. An increase in steroid metabolism was also observed in mice receiving DXR. Since cholesterol form a key component of cell membranes, it is possible that cholesterol synthesis might enable rapidly growing tumours of mice receiving DXR. Finally, concurrent up- and downregulation of certain proteins involved in radical scavenging in DXR mice might suggest a differential free radical scavenging response, thus explaining why anti-oxidant therapies have not proven successful in clinical settings in response to DXR. Collectively, these observations highlight alteration in hepatic activities through which tolerance and resistance mechanism might manifest. In summary, this study have demonstrated the implementation of the T/R framework within an oncological setting. Evidence suggest that defects in hepatic autophagy might contribute to lower tolerance, and possibly also resistance. Autophagy was not significantly upregulated in response to DXR which was associated with lower tolerance. Similar, mice inoculated with B16 tumours exhibited lower tolerance as well as evidence for suppressed lysosomal fusion with autophagosome. These observations suggest that a compromised autophagic apparatus might contribute towards the lower tolerance. Proteomic results are also suggestive of a potential role played by altered liver metabolism, including retinoic acid and steroid metabolism. Future studies evaluating the role of this pathways might identify novel tolerance-promoting pathways.

AFRIKAANSE OPSOMMING: Agtergrond Die ongunstige terapeutiese indeks van behandelingsopsies het die ontwikkeling van effektiewe kankerterapie grootliks vertraag. Dit is dus noodsaaklik dat behandelingsopsies gevind moet word wat minder stremmend op ‘n pasiënt se gesondheidstatus is (dws terapie wat ‘n pasient se reserwekapasiteit en gevolglik oorlewing verleng); terwyl dit ook nie teen-evolusionêre strategieë in kankerselle ontlok nie. Plantbioloë het lankal reeds onderskei tussen die gasheer se vermoë om patogene te akkomodeer teenoor sy vermoë om patogeenlading te onderdruk. Die siekteverloop is dus nie net afhanklik van die gasheer se vermoë om weerstand teen ‘n infeksie te bied nie, maar ook sy vermoë om die patogeen lading te tolereer. Hierdie onderskeiding is eers onlangs op diere toegepas. Uit heelwat navorsing blyk dit dat hierdie onderskeiding van groot immunologiese belang kan wees, insluitend die voorkoms van asimptomatiese draers en natuurlike variasie in die populasie ten opsigte van siekteverloop. Die toleransie/weerstand (T/R) paradigma verteenwoordig dus ‘n nuwe verstaan van siekteverloop. Ons hipotese is dus dat ‘n soortgelyke meganisme gasheer-tumor dinamika onderlê. Studie doelwitte en eksperimentele plan Die doel van hierdie studie is om die toepassing van die T/R raamwerk in ‘n onkologiese konteks toe te pas. ‘n Singeneïese muismodel is gebruik om toleransie en weerstand tussen twee kankersellyne te ondersoek. C57BL/6 muise is ingespuit met borskankerselle (EO771) of met melanoomselle (B16). In ‘n klinese opset word gesondheidstatus nie net beïnvloed deur tumorlading nie, maar ook deur intervensies soos sitotoksiese terapieë wat ook getolereer moet word. ‘n Tweede studie is dus gedoen waar chemoterapie as veranderlike gebruik word om die effek van ‘n hoë (5 mg/kg) en ‘n lae (2 mg/kg) dosis doxorubicin (DXR) behandeling op toleransie en weerstand in muise te bepaal. Daar is verder ook gepoog om meganismes te identifiseer wat onderliggend is aan die verskille tusssen groepe ten opsigte van variasie in toleransie en weerstand. Vir hierdie doel is immunokladtegnieke asook proteoom analises gebruik. Metodologie: kwantifisering van toleransie en weerstand Weerstand is gekwantifiseer as die helling van ‘n regressielyn, met tumorvolume as responsveranderlike en tyd as onafhanklike veranderlike. Toleransie is soorgelyk gemeet, maar met liggaamsgewig as responsveranderlike en tumorlading as onafhanklike veranderlike. Verskille in die regressie hellings is gebruik om toleransie en weerstand te vergelyk. Om toleransie te bevestig, is verskille in die gastrocnemius spier dwarssnit oppervlak (SDO) vergelyk tussen groepe. Resultate Muise wat ingespuit is met melanoom (B16) selle, het ‘n insiggewende laer weerstand getoon teenoor muise wat met EO771 borskankerselle ingespuit is. B16 selle het ook ‘n laer toleransie getoon, alhoewel homogenisiteit ten opsigte van regressie hellings getoon het dat insiggewendheid nie bereik is nie (p = 0.0856). Soortgelyk het B16 en EO771 groepe ook nie insiggewend verskil in die SDO nie. ‘n Vergelyking van die effek van ‘n hoë en ‘n lae dosis DXR in muise met EO771 tumore het getoon dat DXR weerstand laat afneem: beide lae en hoë dosisse DXR het ‘n toename in tumorgroei veroorsaak soos aangetoon is in die insiggewende steiler hellings van die DXR groepe in vergelyking met die tumor kontrole groep. Om die toename in EO771 tumorgroei in muise wat DXR ontvang het, te verklaar, is die aktivering van ‘n paneel seinoordrag proteïene wat geassosieer word met selgroei en oorlewing (cRaf, ERK, p38 MAPK, JNK, PTEN, PI3Kp85, PDK1, Akt, mTOR, Bcl-2) sowel as apoptotiese merkers (Caspase 3, 8 and 9) in tumorweefsel geevalueer deur middel van westelike kladtegnieke. Die enigste insiggewende verandering wat waargeneem is, is ‘n verhoging in ERK aktivering in muise wat DXR ontvang het, wat dus voorstel dat ekstrasellulêre seinoordragmolekules tumorgroei in hierdie muise stimuleer het. Aangesien die lewer ‘n kritiese rol in energie homeostase speel, sowel as in die produksie en verwydering van sirkulerende faktore, is westelike kladanalises ook op lewerweefsel gedoen. Merkers van autofagie (p62 and LC3B-II) sowel as groei seinoordragproteïene (Akt en mTOR) en apoptose merkers (Caspase 3) is geevalueer deur middel van westelike kladtegnieke. Muise wat ingespuit is met B16 selle het ‘n merkbare toename in beide p62 en LC3B-II getoon, wat montlik ‘n aanduiding kan wees van ‘n toename in die aantal autofagosome, heel waarskynlik as gevolg van disfunksionele lisosomale samesmelting. Dit is interessant dat geen ander merkers in beide E0771 en B16 selle insiggewend verskil het van die lewer kontrole weefsel nie. Lewerproteoom analises is gevolglik uitgevoer waar daar gebruik gemaak is van ‘n Geen Ontologie benadering om biologiese, funksionele, strukturele en ander prosesse wat uniek verander is tussen groepe, te bepaal. Interessant, is dat ‘n vergelyking tussen lewers van muise wat met B16 melanoom selle en EO771 borskankerselle ingespuit is, aangedui het dat autofagie aktiwiteit nie opgereguleer is in vergelyking met die kontrolegoep nie. DXR groepe het ook geen verskille in autofagie prosesse getoon nie, alhoewel proteïene in die proteosomale paaie opgereguleer is in muise wat hoë dosisse DXR ontvang het. ‘n Toename in die uitdrukking van ensieme wat geassosieer word met retinoïese suur metabolisme is waargeneem in die B16 groep wat ‘n afname in toleransie en weerstand in hierdie groep kan verklaar. ‘n Toename in steroïedmetabolisme is ook waargeneem in muise wat DXR ontvang het. Aangesien cholesterol ‘n sleutelkomponent van selmembrane is, kan verhoogde cholesterolsintese moontlik verantwoordelik wees vir die vinnige groei van tumorselle in die DXR groep. Gelyktydige op- en afregulering van sekere proteïene wat betrek kan word by vry radikaal opname, kan moontlik ook verklaar waarom antioksidant terapie onsuksesvol in ‘n kliniese konteks in reaksie op DXR is. Hierdie waarnemings plaas klem op veranderinge in hepatiese aktiwiteite waardeur toleransie en weerstand kan manifesteer. Om op te som, hierdie studie demonstreer die implementering van die T/R raamwerk in ‘n onkologiese konteks. Bewyse stel voor dat afwykings in hepatiese autofagie ‘n rol kan speel in laer toleransie, asook moontlik in weerstand. Autofagie is nie insiggewend opgereguleer in reaksie op DXR nie wat moontlik met laer toleransie geassosieer kan word. Soortgelyk, muise met B16 tumore het laer toleransie sowel as onderdrukte lisosomale versmelting met autofagosome getoon. Hierdie bewyse toon dat ‘n foutiewe autofagie apparaat aanleiding kan gee tot laer toleransie. Proteomiese resultate dui ook aan dat ‘n veranderde lewermetabolisme, insluitende retinoïese suur en steroïedmetabolisme, ‘n potensiële rol kan speel. Evaluering van die rol van hierdie paaie kan nuwe toleransie promoverende paaie identifiseer in toekomstige studies.

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